Strontium doped mesoporous silica nanoparticles accelerate osteogenesis and angiogenesis in distraction osteogenesis by activation of Wnt pathway.

Distraction osteogenesis (DO) is a powerful method to reconstruct segmented bone defects in the extremities. However, the main shortcoming of DO is the time-consuming consolidation period. To shorten the consolidation process, two biocompatible inorganic ions, strontium and silicone, were applied to design a biocompatible material to enhance bone mineralization ability during DO. In the present study, we integrated strontium into a one-pot synthesis of mesoporous silica nanoparticles to obtain strontium-doped mesoporous silica nanoparticles characterized by a homogeneous spherical morphology and uniform ion-releasing dynamics. This dual-ion releasing osteogenic and angiogenic drug delivery system was investigated to accelerate mineralization in DO. Osteogenesis was promoted by activation of the Wnt/ß-catenin pathway, while bone resorption was inhibited by reduction of the osteoclastogenic factor RANKL/OPG. In addition, angiogenesis may have been enhanced indirectly by secretion of vascular endothelial growth factor (VEGF) from bone marrow stem cells. Therefore, strontium-doped mesoporous silica nanoparticles could be a potential biomaterial candidate for accelerating consolidation during DO.

IL-1ß Impaired Diabetic Wound Healing by Regulating MMP-2 and MMP-9 through the p38 Pathway.

Diabetes mellitus is one of the most prominent metabolic disorders in the world, and insulin resistance in diabetic patients leads to several complications including increased inflammation and delayed wound healing. Fibroblast migration and reepithelialization play a significant role in wound healing. In this study, we explored the effects of IL-1ß signaling on proliferation and migration of human fibroblasts from diabetic wound tissues. We observed elevated levels of IL-1ß in samples from diabetic patients when compared to normal wound tissues. At high concentrations, IL-1ß inhibited cell proliferation and migration in ex vivo fibroblast cultures. Moreover, expression of matrix metalloproteinases (MMPs) was upregulated, and tissue inhibitor of metalloproteinases (TIMPs) was downregulated in diabetic wound tissues and cells. These effects were regulated by levels of IL-1ß. Furthermore, IL-1ß induced p38 phosphorylation thereby activating the p38 MAPK pathway that in turn regulated the expression of MMPs and TIMPs. Together, our study identifies a novel mechanism behind delayed wound closure in diabetes mellitus that involves IL-1ß-dependent regulation of cell proliferation and migration.

Changes in Temporal and Spatial Patterns of Intrinsic Brain Activity and Functional Connectivity in Upper-Limb Amputees: An fMRI Study.

Background: Amputation in adults is a serious procedure or traumatic outcome, one that leads to a possible "remapping" of limb representations (somatotopy) in the motor and sensory cortex. The temporal and spatial extent underlying reorganization of somatotopy is unclear. The aim of this study was to better understand how local and global structural plasticity in sensory-motor cortical networks changes temporally and spatially after upper-limb amputation. Methods: We studied 8 healthy nonamputee control subjects and 16 complete upper-limb amputees. Resting-state MRI (rs-fMRI) was used to measure local and large-scale relative differences (compared to controls) in both the amplitude of low-frequency fluctuations (ALFF) and degree of centrality (DC) at 2 months, 6 months, and 12 months after traumatic amputation. Results: In amputees, rs-fMRI scans revealed differences in spatial patterns of ALFF and DC among brain regions over time. Significant relative increases in ALFF and DC were detected not only in the sensory and motor cortex but also in related cortical regions believed to be involved in cognition and motor planning. We observed changes in the magnitude of ALFFs in the pre- and postcentral gyrus and primary sensory cortex, as well as in the anterior cingulate, parahippocampal gyrus, and hippocampus, 2 months after the amputation. The regional distribution of increases/decreases in ALFFs and DC documented at 2-month postamputation was very different from those at 6 and 12-month postamputation. Conclusion: Local and wide-spread changes in ALFFs in the sensorimotor cortex and cognitive-related brain regions after upper-limb amputation may imply dysfunction not only in sensory and motor function but also in areas responsible for sensorimotor integration and motor planning. These results suggest that cortical reorganization after upper extremity deafferentation is temporally and spatially more complicated than previously appreciated, affecting DC in widespread regions.

Role of Arterial Perfusion in Early Survival of Arterialized Venous Flaps: A Mechanism Study and Clinical Application.

BACKGROUND: The arterialized venous flap (AVF) is appropriate as a flap for hand and foot resurfacing meet the aesthetic demands in the same time. However, the inconsistency of survival rate limited its popularization in clinical settings. The purpose of this study was to investigate the role played by the caliber and location of the artery. METHODS: Arterialized venous flaps were designed on the abdomen of New Zealand rabbits, and the animals were randomized into 3 groups and 2 groups in experiment 1 and 2, respectively. In experiment 1, the artery flow was restricted with vascular staplers of different calibers. In experiment 2, the artery was anastomosed with the afferent vein in the center or at the margin of the flap. Blood perfusion state, water content, epidermal metabolite levels, and flap survival status were observed in both experiments. Furthermore, outcomes of 12 patients received AVF to resurface soft tissue defects in the digits, hands, and feet between January 2016 and February 2018 were analyzed. RESULTS: In experiment 1, compared with the control group, groups with restricted artery showed poor results regarding blood perfusion state, water content, epidermal metabolite levels, and flap survival status. In experiment 2, group with the afferent vein in the center of the flap showed better results mentioned previously. All the flaps survived uneventfully in this study. Two flaps partially failed (20% of the flap area) because of insufficient perfusion. Generally, larger caliber and center-located vein helped the survival of AVF. CONCLUSIONS: Experimental findings suggested that increased arterial perfusion and center-located vein are beneficial for the survival of AVF. Clinical series proved the findings previously. The problem of inconsistency of AVF can be partially solved by increasing arterial perfusion and dissecting afferent vein into the center of flap, and still, further studies are needed to shed light on the mechanism behind.

Superficial Peroneal Neurocutaneous Flap for Coverage of Donor Site Defect After the Combined Transfer of Toe and Dorsal Foot Flap.

BACKGROUND: Reconstruction of digital loss with soft tissue defects remains a tough challenge. Although a combined flap of toe and dorsal foot skin provides a good option for "like-for-like" hand reconstruction, the disappointed donor site morbidity prevents it from popularity. In this study, we presented experiences of the superficial peroneal neurocutaneous (SPNC) flap for donor site closure after the combined toe and dorsal foot flap transfer. METHODS: Superficial peroneal neurocutaneous flaps were used to cover foot donor site defects in 9 patients. The flaps harvested from feet including 3 cases of wrap-around flap with dorsal foot flap, 4 cases of 2nd toe flap with dorsal foot flap, 2 cases of 2nd and 3rd toe flap with dorsal foot flap. The flap size, operation time, and complications were documented, and the donor sites were evaluated by the subjective outcome measure, the foot evaluation questionnaire, and the Vancouver Scar Scale. RESULTS: All flaps but one survived completely without complications. Marginal necrosis occurred in the distal part of the flap in one case, which was treated by daily dressings. The skin grafts on the lower leg healed uneventfully. The average operation time of flap transfer was 40 minutes. Follow-up ranged from 9 to 16 months, and patients were content with the results of the foot donor site according to the outcome measures. All the patients were able to wear normal shoes walking and running with a normal gait, and none sustained complications of skin erosion or ulceration. Protective sensibility was obtained in all the flaps. Two patients complained of cold intolerance and 2 could not wear a thong sandal. The donor site scars on the lower leg were measured 3.2 on average on the Vancouver Scar Scale. CONCLUSIONS: The SPNC flap is a practical procedure for donor site closure on the foot, especially when extra dorsal foot skin is elevated with a toe flap for hand reconstruction.

Postconditioning With Red-Blue Light Therapy Improves Survival of Random Skin Flaps in a Rat Model.

BACKGROUND: Random skin flap ischemic necrosis is a serious challenge in reconstructive surgery. Photobiomodulation is a noninvasive effective technique to improve microcirculation and neovascularization. Photobiomodulation with red or blue light has been separately proven to partially prevent skin flap necrosis, but the synergistic effect of red and blue light not been elucidated. Our experiment evaluated the impact of postconditioning with red-blue light therapy on the viability of random flaps. METHODS: Thirty Sprague-Dawley male rats (male, 12 weeks) with a cranially based random pattern skin flap (3 × 8 cm) were divided into 3 groups: control group, red light group, and red-blue light group. On postoperative day 7, flap survival was observed and recorded using transparent graph paper, flaps were obtained and stained with hematoxylin and eosin, and microvessel density was measured. Micro-computed tomography was used to measure vascular volume and vascular length. On days 0, 3, and 7 after surgery, blood flow was measured by laser Doppler. To investigate the underlying mechanisms, the amount of nitric oxide (NO) metabolites in the flap tissue was assessed on days 3, 5, and 7 after surgery. RESULTS: The mean percentage of skin flap survival was 59 ± 10% for the control group, 69 ± 7% for the red light group, and 79 ± 9% for the red-blue light group (P < 0.01). The microvessel density was 12.3 ± 1.2/mm2 for the control group, 31.3 ± 1.3/mm2 for the red light group, and 36.5 ± 1.4/mm2 for the red-blue light group (P < 0.01). Both vascular volume and total length in the red-blue light group showed significantly increased compared with the red light and control group (P < 0.01). Blood flow in the red-blue light treated flap showed significantly increased at postsurgery days 3 and 7 compared with the red light and control group (P < 0.01). The level of the NO metabolites was significantly increased in flap tissues belonging to the red-blue light group compared with the other 2 groups (P < 0.01). CONCLUSIONS: This study showed that postconditioning with red-blue light therapy can enhance the survival of random skin flap by improving angiogenesis and NO releasing.

Free extended posterior tibial artery perforator flap with the neurovascular plexus of a saphenous nerve branch for large soft tissue and sensory reconstruction: Anatomic study and clinical application.

BACKGROUND: The posterior tibial artery perforator (PTAP) flap is a useful tool for reconstruction of soft tissue defects in the leg. However, the size and reliability of the flap largely depends on the quality of the perforator by which the flap is supplied, and the sensory recovery of the flap is limited. In this study, the anatomy of the saphenous nerve branches and their accompanying vessels was investigated, and a free extended PTAP flap with the neurovascular plexus of a saphenous nerve branch was designed for large soft tissue and sensory reconstruction in a series of clinical cases. METHODS: Sixteen adult cadaveric legs perfused with red latex in the femoral artery were dissected. The number and location of the saphenous nerve branches and the features of their accompanying vessels were dissected and studied. From January 2016 to December 2017, six patients with soft tissue defects ranged from 8 × 2.5 cm to 21 × 4 cm were repaired by the free extended PTAP flap. The patients' average age was 48 years. The causes of the defects included machine injuries in three patients and traffic injuries in the other three. The defects located at the hand in three cases, foot in two cases, and ankle in one case. The flap was designed based on the perforators of the posterior tibial artery and included a branch of saphenous nerve. The perforator pedicle and the nerve branch were connected to the vessels and nerve in the recipient site, respectively. RESULTS: The saphenous nerve gave off 5.8 ± 1.1 branches, with a relatively constant one issuing 8.1 ± 0.7 cm distal to the medial femoral condyle. Every nerve branch had an accompanying vessel, which connected with the PTAPs and supplied the skin. The size of the flap ranged from 10 × 3.5 cm to 23 × 5 cm. All of the flaps survived completely without complications. Follow-up varied from 6 to 12 months. All the patients obtained cold/hot sensation and pain sensation. The results of Semmes-Weinstein monofilament test ranged from 4.31(2 g) to 5.46 (26 g), and the 2-point discrimination test varied from 20 to 35 mm. CONCLUSION: The free extended PTAP flap, containing the saphenous nerve branch and its accompanying vessels, may be an alternative for large soft tissue reconstruction with improved sensation recovery.

Exosomes Secreted by Young Mesenchymal Stem Cells Promote New Bone Formation During Distraction Osteogenesis in Older Rats.

Distraction osteogenesis (DO) is a clinically effective procedure to regenerate large bone defects. However, the treatment duration is undesirably lengthy, especially in elderly patients. Exosomes derived from mesenchymal stem cells (MSC-Exos) could exert the beneficial effects while avoiding the possible complications of stem cell transplantation. This study aimed to evaluate the effects of MSC-Exos on bone regeneration during DO in older rats. Exosomes were isolated from the supernatants of young bone marrow mesenchymal stem cells (BMSCs) through ultra-centrifugation, and characterized using transmission electron microscopy, western blot, and tunable resistive pulse sensing analysis. The effects of MSC-Exos on the proliferation and differentiation of older BMSCs were evaluated using CCK-8 assay, ALP and ARS staining, and qRT-PCR. Unilateral tibial DO model was established on older Sprague-Dawley rats and MSC-Exos or phosphate buffer saline was locally injected into the distraction gaps after distraction weekly. Bone regeneration were evaluated using X-ray, Micro-CT, mechanical test, and histological staining. The MSC-Exos were round or cup-shaped vesicles ranging from 60 to 130 nm in diameter and expressed markers including CD9, CD63, and TSG101. The in vitro results indicated that MSC-Exos could enhance the proliferation and osteogenic differentiation of older BMSCs. Bone regeneration was markedly accelerated in rats treated with MSC-Exos according to the results of X-ray, micro-CT, and histological analysis. The distracted tibias from the MSC-Exos group also demonstrated better mechanical properties. These results suggest that MSC-Exos promote DO-mediated bone regeneration in older rats through enhancing the proliferation and osteogenic capacity of BMSCs.

Is EDTA Irrigation Effective in Reducing Bacterial Infection in a Rat Model of Contaminated Intra-articular Knee Implants?

BACKGROUND: To mitigate the possibility of infection after arthroplasty, intraoperative irrigation is essential to remove contaminating bacteria. Previous studies have demonstrated that irrigation with an EDTA solution before wound closure is superior to irrigation with normal saline in removing contaminating bacteria in a rat model of open fractures. However, the effectiveness of an EDTA solution in a model with a contaminated intra-articular implant remains unclear. QUESTIONS/PURPOSES: (1) Does irrigation with an EDTA solution decrease the proportion of culture-positive joints compared with normal saline, benzalkonium chloride, and povidone iodine? (2) Is an EDTA solution toxic to cells resident in joints including chondrocytes, osteoblasts, and synovial fibroblasts? (3) Does irrigation with an EDTA solution have adverse effects including arthrofibrosis and hypocalcemia? METHODS: We first established a model of contaminated intra-articular implants. Female Sprague-Dawley rats (n = 30 for each treatment group) underwent knee arthrotomy and implantation of a femoral intramedullary wire with 1 mm of intra-articular communication. To simulate bacterial contamination, the inserted wire was inoculated with either Staphylococcus aureus or Escherichia coli. After 1 hour, the wound and implant were irrigated with normal saline, benzalkonium chloride, povidone iodine, or an EDTA solution (1 mM). The animals were euthanized 1 week later, and the distal femur, knee capsule, and implanted wire were harvested for bacterial culture using standard techniques. In this study, we used a well-established animal model of an intra-articular implant and inoculated the implant to simulate the clinical setting of intraoperative contamination. The proportion of culture-positive joints in normal saline, benzalkonium chloride, povidone-iodine, and EDTA groups were compared. The viable cell numbers (chondrocytes, osteoblasts, and synovial fibroblasts) were counted and compared after treatment with either solution. Measurement of blood calcium level and histological examination of the joint were performed to rule out hypocalcemia and arthrofibrosis after EDTA irrigation. RESULTS: With S. aureus inoculation, EDTA irrigation resulted in fewer culture-positive joints than normal saline (37% [11 of 30] versus 70% [21 of 30]; p = 0.019), benzalkonium chloride (83% [25 of 30]; p < 0.001), and povidone iodine (83% [25 of 30]; p < 0.001) irrigation. Likewise, infection rates for implant inoculation with E. coli were also lower in the EDTA irrigation group (13% [four of 30]) than in the normal saline (60% [18 of 30]; p < 0.001), benzalkonium chloride (77% [23 of 30]; p < 0.001), and povidone iodine (80% [24 of 30]; p < 0.001) groups. Between normal saline control and EDTA, there were no differences in cell viability in chondrocytes (normal saline: 98% ± 18%; EDTA: 105% ± 18%; p = 0.127), osteoblasts (normal saline: 102 ± 19%, EDTA: 103 ± 14%; p = 0.835), and synovial fibroblasts (normal saline: 101% ± 21%, EDTA: 110% ± 13%; p = 0.073). EDTA irrigation did not result in hypocalcemia (before irrigation: 2.21 ± 0.32 mmol/L, after irrigation: 2.23 ± 0.34 mmol/L; p = 0.822); and we observed no arthrofibrosis in 30 histologic samples. CONCLUSIONS: In a rat model of a bacteria-contaminated intra-articular implants, intraoperative irrigation with 1 mmol/L of an EDTA solution was superior to normal saline, 0.03% benzalkonium chloride, and 0.3% povidone iodine in preventing surgical-site infection and caused no adverse effects including death of resident cells, arthrofibrosis, and hypocalcemia. Future studies should seek to replicate our findings in other animal models, perhaps such as dog and goat. CLINICAL RELEVANCE: If other animal models substantiate the efficacy and safety of the EDTA solution, clinical trials would be warranted to determine whether the use of an EDTA irrigation solution might reduce the risk of periprosthetic joint infections in patients compared with traditional irrigation solutions.

Thumb reconstruction with combination of the wrap-around flap prefabricated by medialis pedis perforator flap with nail bed and phalanx banked from the amputated thumb: A case report.

Thumb reconstruction has been a very challenging issue for hand surgeons. In this report, we present a case of thumb reconstruction with combination of the wrap-around flap prefabricated by the medialis pedis perforator flap with phalanx and nail bed banked from the amputated thumb. A 22-year-old man suffered from the left thumb amputation as well as large soft tissue defect of hand and comminuted fracture in wrist due to a crush accident. The distal phalanx and nail bed of left thumb were exposed and no suitable vessels for microsurgical anastomosis could be found, resulting in the great difficulty of replantation. After debridement, nail bed of the amputated thumb was dissected and banked on the medial side of foot, while the distal phalanx was buried in the abdominal subcutaneous tissue. The fracture was fixed with an external fixation and the soft tissue defect was covered with a free anterolateral flap. Wound and bone healing was achieved 6 months after the initial treatment. Thumb was reconstructed with combination of the banked phalanx and a wrap-around flap prefabricated by the medialis pedis perforator flap and the banked nail bed. The postoperative course was uneventful with complications from both reconstruction and donor sites. The nail of the reconstructed thumb grew normally. Thumb oppositional function was rebuilt. The patient was satisfied with the aesthetic and functional outcome at 5-year postoperative follow-up. We propose that tissue banked from the nonreplantable amputated thumb could be used for secondary reconstruction with the technique of flap prefabrication.

Ferulic acid improves self-renewal and differentiation of human tendon-derived stem cells by upregulating early growth response 1 through hypoxia.

We aimed to investigate the potential beneficial effect of ferulic acid (FA) on stemness of human tendon-derived stem cells (hTSCs) in vitro and to elucidate the underlying molecular mechanism. The self-renewal ability of hTSCs was evaluated by colony formation and cell proliferation was determined by CCK-8 kit. Adipogenesis, osteogenesis, and chondrogenesis were determined by Oil Red O, Alizarin Red, and Alcian Blue stainings, respectively. Relative mRNA levels of PPARγ, Col2A1, Acan, Runx2, HIF1α, and EGR1 were measured with real-time PCR. Protein levels of HIF1α and EGR1 were detected by western blot. Direct binding of HIF1α with EGR1 promoter was analyzed by ChIP assay. Hypoxia-induced expression of EGR1 was interrogated by luciferase reporter assay. We demonstrated that FA treatment improved both self-renewal ability and multi-differentiation potential of hTSCs. FA induced hypoxia which in turn upregulated EGR1 expression via direct association with its hypoxia response element consensus sequence. Furthermore, we showed that both HIF1α and EGR1 were required for the enhancing effects of FA on hTSC self-renewal and differentiation. We hereby characterize the beneficial effect of FA on the stemness of hTSCs and highlight the critical role of HIF1α-EGR1 axis in this process.

Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells.

Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic development. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based neuroectoderm developmental model, in which pre-patterned geometrical confinement induces emergent patterning of neuroepithelial and neural plate border cells, mimicking neuroectoderm regionalization during early neurulation in vivo. In this hPS-cell-based neuroectoderm patterning model, two tissue-scale morphogenetic signals-cell shape and cytoskeletal contractile force-instruct neuroepithelial/neural plate border patterning via BMP-SMAD signalling. We further show that ectopic mechanical activation and exogenous BMP signalling modulation are sufficient to perturb neuroepithelial/neural plate border patterning. This study provides a useful microengineered, hPS-cell-based model with which to understand the biomechanical principles that guide neuroectoderm patterning and hence to study neural development and disease.

MicroRNA-296-5p promotes healing of diabetic wound by targeting sodium-glucose transporter 2 (SGLT2).

BACKGROUND: Diabetic wounds are refractory and very difficult to heal. We aimed to use miRNA to identify novel and specific molecular markers for diabetes mellitus (DM) diagnosis and treatment. METHODS: The expression level of miR-296-5p was determined in tissue samples of 12 DM patients. The effect of miR-296-5p on proliferation of ß-cells was examined using Cell Counting Kit-8 (CCK-8) and colony formation assay. The effect of miR-296-5p on cell cycle progression was analysed using flow cytometry. The target gene was verified using luciferase reporter assay. A rat diabetes model was used to assess the effect of miR-296-5p in vivo. RESULTS: Overexpression of miR-296-5p suppressed cell proliferation, arrested cell cycle progression, and increased the healing rate of diabetic wounds both in vivo and in vitro. TargetScan analysis results showed that miR-296-5p is a direct regulator of SGLT2. CONCLUSIONS: miR-296-5p can increase the healing rate of diabetic wounds and may be an effective molecular tool in DM diagnosis and therapy.

All-Trans Retinoic Acid Promotes Osteogenic Differentiation and Bone Consolidation in a Rat Distraction Osteogenesis Model.

Distraction osteogenesis (DO) is used to treat specific disorders associated with growth abnormalities and/or loss of bone stock secondary to trauma or disease. However, a high rate of complications and discomfort hamper its further application in clinical practice. Here, we investigated the effects of all-trans retinoic acid (ATRA) on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs) and bone consolidation in a rat DO model. Different doses of ATRA were used to treat rBMSCs. Cell viability and osteogenic differentiation were assessed using CCK-8 and alkaline phosphatase staining, respectively. The mRNA expression of osteogenic differentiation-genes (including ALP, Runx2, OCN, OPN, OSX, and BMP2) and angiogenic genes (including VEGF, HIF-1, FLK-2, ANG-2, and ANG-4) were determined by quantitative real-time PCR analysis. Further, we locally injected ATRA or PBS into the gap in the rat DO model every 3 days until termination. X-rays, micro-computed tomography (Micro-CT), mechanical testing, and immunohistochemistry stains were used to evaluate the quality of the regenerates. ATRA promoted osteogenic differentiation of rBMSCs. Moreover, ATRA elevated the mRNA expression levels of osteogenic differentiation-genes and angiogenic genes. In the rat model, new bone properties of bone volume/total tissue volume and mechanical strength were significantly higher in the ATRA-treatment group. Micro-CT examination showed more mineralized bone after the ATRA-treatment, and immunohistochemistry demonstrated more new bone formation after ATRA-treatment than that in the PBS group. In conclusion, as a readily available and very cost effective bio-source, ATRA may be a novel therapeutic method to enhance bone consolidation in the clinical setting.

Roxadustat promotes angiogenesis through HIF-1α/VEGF/VEGFR2 signaling and accelerates cutaneous wound healing in diabetic rats.

Diabetic foot ulcers are a major health-care burden worldwide. One primary cause of the delayed wound healing in diabetic patients is impaired function of the hypoxia-inducible factor-1α/vascular endothelial growth factor (HIF-1α/VEGF) axis, which results in compromised neovascularization in response to hypoxia. In the present study, we aimed to investigate the effect of roxadustat, a novel HIF prolyl-4-hydroxylase inhibitor, on angiogenesis and its therapeutic effect on cutaneous wound healing in diabetic rats. In vitro, we found that roxadustat could promote the angiogenic activity of human umbilical vein endothelial cells, accompanied by up-regulation of HIF-1α/VEGF/VEGFR2 signaling. Next, we demonstrated that Ki8751, a VEGFR2-specific inhibitor, could inhibit the increased angiogenic activity of human umbilical vein endothelial cells induced by roxadustat. In vivo, we performed a Matrigel plug assay and demonstrated that roxadustat induced vascularization of the Matrigel plugs, and this effect could be partially inhibited by Ki8751. Finally, we utilized a streptozotocin-induced diabetic rat model and found that roxadustat could accelerate cutaneous wound healing and promote angiogenesis in the wound sites. In conclusion, roxadustat promotes angiogenesis via activation of the HIF-1α/VEGF/VEGFR2 pathway and exhibits therapeutic effects on diabetic wound healing by increasing angiogenesis. Our findings suggest that roxadustat can be a promising strategy to promote diabetic cutaneous wound healing.

Advanced Glycation End Products (AGEs) Induce Apoptosis of Fibroblasts by Activation of NLRP3 Inflammasome via Reactive Oxygen Species (ROS) Signaling Pathway.

BACKGROUND Type 2 diabetes impairs the healing process and induces apoptosis of fibroblasts, which are thought to be involved in this process. We investigated the possible mechanisms involved in AGEs-induced apoptosis of human dermal fibroblasts. MATERIAL AND METHODS We examined the expression of apoptosis-related proteins in fibroblasts isolated from human diabetic wounds. Human dermal fibroblasts exposed to AGEs were used to study the links among apoptosis, ROS, and NLRP3 inflammasome activation. Signaling mechanisms were evaluated by preincubating the cells with appropriate inhibitors. Cleaved caspase-8, cleaved caspase-3, BAX, Bcl-2, and NLRP3 inflammasome expression were measured by Western blot analysis. ROS generation, cell viability, and cell apoptosis were assessed. RESULTS We observed a higher level of cleaved caspase-8 and cleaved caspase-3 expression in fibroblasts isolated from human diabetic wounds compared with controls. AGEs decreased the proliferation of cells in a concentration-dependent and time-dependent manner. The exposure of fibroblasts to AGEs significantly increased the number of cells in early and late apoptosis stages. AGES-induced human dermal fibroblasts showed high expressions of cleaved caspase3, cleaved caspase8, and Bax. Treatment with AGEs induced the expression of NLRP3, caspase-1, and ASC. AGES-induced apoptosis was blocked by BAY 11-7082, an inhibitor of the NLRP3 inflammasome. AGEs increased the production of ROS in fibroblasts, and its apoptogenic effect was blocked by NAC. CONCLUSIONS AGEs cause apoptosis of fibroblasts by inducing the generation of ROS and activating the NLRP3 inflammasome. In vivo experiments are needed to confirm these results.

Regeneration of large bone defects using mesoporous silica coated magnetic nanoparticles during distraction osteogenesis.

Distraction osteogenesis (DO) represents an effective but undesirably lengthy treatment for large bone defects. Both magnetic nanoparticles and silicon have been shown to induce osteogenic differentiation of mesenchymal stem cells (MSCs), the key participant in bone regeneration. We herein synthesized mesoporous silica coated magnetic (Fe3O4) nanoparticles (M-MSNs) and evaluated its potential for acceleration of bone regeneration in a rat DO model. The M-MSNs exhibited good biocompatibility and remarkable capability in promoting the osteogenic differentiation of MSCs via the canonical Wnt/ß-catenin pathway in vitro. More importantly, local injection of M-MSNs dramatically accelerated bone regeneration in a rat DO model according to the results of X-ray imaging, micro-CT, mechanical testing, histological examination, and immunochemical analysis. This study demonstrates the notable potential of M-MSNs in promoting bone regeneration during DO by enhancing the osteogenic differentiation of MSCs, paving the way for clinical translation of M-MSNs in DO to repair large bone defects.

Bifocal osteosynthesis to treat radial shortening deformity with dislocation of the inferior radioulnar joint.

BACKGROUND: Several methods have been reported to correct deformity and shortening of the distal radius. However, the results are not entirely satisfactory. The results of bifocal osteosynthesis were retrospectively analyzed in this study. METHODS: Eight patients treated with bifocal osteosynthesis were evaluated retrospectively. Pre-operative and post-operative clinical and radiographic examinations were performed. Subjective symptoms and objective joint function were assessed. Radiographic data of the extent of radial lengthening and distal radial articular angle were collected. RESULTS: The mean follow-up period was 46 months (37-68 months). Satisfactory wrist appearance and radial lengthening was achieved in all patients. All patients were satisfied with the wrist appearance and willing to undergo the same treatment again. The range of motion (ROM) of the forearm and wrist was significantly improved. Pin-track infections occurred in two patients, for which they received wound care and oral antibiotics. Complications such as fixation device failure, tendon rupture, fracture of regenerated bone or nerve impairment did not occur. The duration of lengthening depended on the shortening of the radius. Delayed union in the docking site was observed in two patients and union was achieved after bone grafting. CONCLUSIONS: Bifocal osteosynthesis using the Ilizarov method provides a useful method for correction of radial shortening deformity with dislocation of the inferior radioulnar joint. Despite the fact that we did not validate pre-and post-operation functional outcome scores, all patients were satisfied with the wrist appearance and function.

The Microsurgical Calcaneus Osteocutaneous Fillet Flap After Traumatic Amputation in Lower Extremities: Flap Design and Harvest Technique.

BACKGROUND: Attempts to salvage upper and lower extremities have performed more frequently in recent decades, although there are clear cases that cannot be salvaged. The purpose of this retrospective study was to present our experience in using free calcaneus osteocutaneous fillet flap for preserving below-knee amputation stump after traumatic amputations or functional preserving after nonsalvageable lower extremities. METHODS: Between January 2012 and May 2017, 11 free calcaneus osteocutaneous fillet flap were used to preserving or lengthening below-knee amputation stump secondary to amputation on 8 males and 3 females. Patients' information and postoperative data were collected, including age of patient, sex, amputation site, flap survival, sensation recovery, and number of complications. RESULT: All amputations were trauma related and secondary to motor vehicle accidents (n = 8) and industrial accidents (n = 3). The age of the patients ranged from 16 to 59 years, with a mean of 34.4 years. Free calcaneus osteocutaneous fillet flap were designed and harvested from all patients. All flaps survived and 2 complications developed in 2 patients. Nine of 11 patients obtained protective sensory recovery during the period of follow-up. CONCLUSIONS: The free calcaneus osteocutaneous fillet flap harvested from the amputated limb provides reliable and robust tissue for reconstruction of large defects of the residual limb without additional donor-site morbidity.

Von Hippel-Lindau (VHL) protein antagonist, VH298, promotes functional activities of tendon-derived stem cells and accelerates healing of entheses in rats by inhibiting ubiquitination of hydroxy-HIF-1α.

Enthesis is the region where a tendon attaches to a bone. It is a relatively vulnerable position, and in most cases surgical treatment is required upon rupture. The reconstructed enthesis is usually weaker compared to the original, and is prone to rupture again. Hypoxia-inducible factor-1 α (HIF-1α) is known to be involved in extensive activities in cells. It is inhibited under normoxic conditions, and undergoes two essential processes, hydroxylation and ubiquitination, the latter of which has been largely unexplored. Herein, we measured the levels of HIF-1α and hydroxy-HIF-1α in VH298-treated rat tendon-derived stem cells (TDSCs) by immunoblotting. We also detected the proliferation of TDSCs using CCK-8 assay and the mRNA levels of related genes by quantitative RT-PCR. The TDSCs were observed to be induced and the chondrogenic differentiation related genes were found to be enhanced. We also simulated in-vitro wounding in a scratch test and reconstructed the enthesis in a rat model of Achilles tendon by classical surgery followed by administration of phosphate buffer saline (PBS) injection or VH298 injection. We observed that HIF-1α and hydroxy-HIF-1α levels were increased in VH298-treated TDSCs in a dose- and time-dependent manner. Thirty micromolar VH298 could significantly increase cell proliferation, migration, and expression of collagen-1α, collagen-3α, decorin, tenomodulin, tenascin C genes, and chondrogenic differentiation-related genes, collagen-2α, SRY-box9, aggrecan. VH298-treated enthesis could tolerate more load-to-failure, had a better healing pattern, and activation of HIF signaling pathway. VH298 can thus enhance the functional activities of TDSCs, enhance their chondrogenic differentiation potential, and accelerate enthesis healing by inhibiting the ubiquitination of hydroxy-HIF-1α.