Tetrandrine inhibits the occurrence and development of frozen shoulder by inhibiting inflammation, angiogenesis, and fibrosis.

BACKGROUND: Frozen shoulders (FS) is a major clinical concern, where chronic synovial inflammation, abnormal angiogenesis, and fibrosis represent the critical pathologies in the glenohumeral capsule. However, no pharmacotherapy has been introduced to treat this pathology. Tetrandrine (TET) has been proposed as a treatment for many diseases due to its strong anti-inflammatory, anti-angiogenic, and anti-fibrotic effects. PURPOSE: To study the anti-inflammatory, anti-angiogenic, and anti-fibrotic effects of TET on FS, and identify whether TET can prevent the development of FS in rats. STUDY DESIGN: A controlled laboratory study. METHODS: Forty-eight male Sprague-Dawley (SD) rats were randomly divided into control, TET, and FS groups. The TET group was intraperitoneally injected with TET every 2 days. TET and saline treatment were started on the day of FS surgery. After 8 weeks, the animals were sacrificed, and samples were collected for X-ray examination, glenohumeral range of motion (ROM) evaluation, histology and immunohistochemistry analysis, transmission electron microscopy (TEM) observation, and profibrogenic factors as well as proinflammatory cytokines measurements. RESULTS: No significant difference in shoulder ROM was observed between the TET and control groups, but a significant difference was noted between these groups and the FS group (P < 0.01). Immunohistochemical staining showed no abnormal angiogenesis or fibrosis in the TET group or the control group. However, significant angiogenesis, collagen remodeling, and fibrosis were observed in the FS group, and the expression and proportion of type I and type III collagen in the FS group were significantly higher than those in the TET group or the control group (P < 0.01). TEM observation showed that TET protected the ultrastructure of collagen fibrous reticular arrangement of the articular capsule and prevented the formation of scar-like fibrotic structures, which are unique to FS. The significantly increased expression of Smad7 and the suppressed expression of Smad 2 in the TET group compared with that of the FS group indicated that TET also significantly inhibited the TGF-ß1 intracellular signal pathway. The expression of profibrogenic factors and proinflammatory cytokines in the TET group and the control group was significantly lower than that in the TET group (P < 0.01). CONCLUSION: The results demonstrated that TET protected the normal reticular structure of the capsule during the freezing period and prevented the development of FS by inhibiting inflammation, angiogenesis, and fibrosis in a rat FS model. CLINICAL RELEVANCE: TET may be a safe and effective clinical medication for preventing and treating FS.

Bioactive Injectable Hydrogels Containing Desferrioxamine and Bioglass for Diabetic Wound Healing.

Diabetic wound is hard to heal mainly because of the difficulty in vascularization in the wound area. Accumulating results have shown that desferrioxamine (DFO) can promote secretion of hypoxia inducible factor-1 (HIF-1α), thereby upregulating the expression of angiogenic growth factors and facilitating revascularization. Our preliminary study has demonstrated that Si ions in bioglass (BG) can upregulate vascular endothelial growth factor (VEGF) expression, thus promoting revascularization. It is hypothesized that the combined use of BG and DFO may have a synergistic effect in promoting VEGF expression and revascularization. To prove this, we first determined DFO concentration range that had no apparent cytotoxicity on human umbilical vein endothelial cells (HUVECs). Then, the optimal concentration of DFO promoting tube formation of HUVECs was determined by cell migration and tube formation assays. In addition, we demonstrated that combination use of BG and DFO improved the migration and tube formation of HUVECs as compared with the use of either BG or DFO alone as BG and DFO could synergistically upregulate VEGF expression. Furthermore, a sodium alginate hydrogel containing both BG and DFO was developed, and this hydrogel better facilitated diabetic skin wound healing than the use of either BG or DFO alone as BG and DFO in the hydrogels worked synergistically in promoting HIF-1α and VEGF expression and subsequently vascularization in the wound sites. Therefore, in this study, the synergistic effect in promoting revascularization between BG and DFO was first demonstrated and an injectable hydrogel simultaneously containing BG and DFO was developed for enhancing repair of diabetic chronic skin defects by taking advantages of the synergistic effects of BG and DFO in promoting revascularization. The study opens up a new prospect for the development of skin repair-promoting biomaterials.

Evaluation of zinc-doped mesoporous hydroxyapatite microspheres for the construction of a novel biomimetic scaffold optimized for bone augmentation.

Biomaterials with high osteogenic activity are desirable for sufficient healing of bone defects resulting from trauma, tumor, infection, and congenital abnormalities. Synthetic materials mimicking the structure and composition of human trabecular bone are of considerable potential in bone augmentation. In the present study, a zinc (Zn)-doped mesoporous hydroxyapatite microspheres (Zn-MHMs)/collagen scaffold (Zn-MHMs/Coll) was developed through a lyophilization fabrication process and designed to mimic the trabecular bone. The Zn-MHMs were synthesized through a microwave-hydrothermal method by using creatine phosphate as an organic phosphorus source. Zn-MHMs that consist of hydroxyapatite nanosheets showed relatively uniform spherical morphology, mesoporous hollow structure, high specific surface area, and homogeneous Zn distribution. They were additionally investigated as a drug nanocarrier, which was efficient in drug delivery and presented a pH-responsive drug release behavior. Furthermore, they were incorporated into the collagen matrix to construct a biomimetic scaffold optimized for bone tissue regeneration. The Zn-MHMs/Coll scaffolds showed an interconnected pore structure in the range of 100-300 µm and a sustained release of Zn ions. More importantly, the Zn-MHMs/Coll scaffolds could enhance the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. Finally, the bone defect repair results of critical-sized femoral condyle defect rat model demonstrated that the Zn-MHMs/Coll scaffolds could enhance bone regeneration compared with the Coll or MHMs/Coll scaffolds. The results suggest that the biomimetic Zn-MHMs/Coll scaffolds may be of enormous potential in bone repair and regeneration.

Enhanced osteogenesis and angiogenesis by mesoporous hydroxyapatite microspheres-derived simvastatin sustained release system for superior bone regeneration.

Biomaterials with both excellent osteogenic and angiogenic activities are desirable to repair massive bone defects. In this study, simvastatin with both osteogenic and angiogenic activities was incorporated into the mesoporous hydroxyapatite microspheres (MHMs) synthesized through a microwave-assisted hydrothermal method using fructose 1,6-bisphosphate trisodium salt (FBP) as an organic phosphorous source. The effects of the simvastatin-loaded MHMs (S-MHMs) on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) and angiogenesis in EA.hy926 cells were investigated. The results showed that the S-MHMs not only enhanced the expression of osteogenic markers in rBMSCs but also promoted the migration and tube formation of EA.hy926 cells. Furthermore, the S-MHMs were incorporated into collagen matrix to construct a novel S-MHMs/collagen composite scaffold. With the aid of MHMs, the water-insoluble simvastatin was homogenously incorporated into the hydrophilic collagen matrix and presented a sustained release profile. In vivo experiments showed that the S-MHMs/collagen scaffolds enhanced the bone regeneration and neovascularization simultaneously. These results demonstrated that the water-insoluble simvastatin could be incorporated into the MHMs and maintained its biological activities, more importantly, the S-MHMs/collagen scaffolds fabricated in this study are of immense potential in bone defect repair by enhancing osteogenesis and angiogenesis simultaneously.

Sex-determining region Y-box protein 3 induces epithelial-mesenchymal transition in osteosarcoma cells via transcriptional activation of Snail1.

BACKGROUND: The transcription factor sex-determining region Y-box protein 3 (SOX3) plays important roles in various types of cancer. However, its expression and function have not yet been elucidated in osteosarcoma (OS). METHODS: The expression levels of SOX3 in OS tissues and OS cell lines were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. The effects of SOX3 expression on OS cell biological traits were investigated by overexpressing and downregulating SOX3 protein. The expression of epithelial-mesenchymal transition (EMT) markers and transcription factors associated with EMT (EMT-TFs), were detected simultaneously. The mechanism underlying SOX3-mediated Snail1 expression was further investigated. RESULTS: SOX3 was upregulated in human OS tissues. SOX3 overexpression promoted the EMT, migration and invasion in OS cells. The downregulation of SOX3 resulted in opposing effects. Furthermore, SOX3 upregulation enhanced the expression of the transcriptional repressor Snail1 by binding to its promoter region. Additionally, a positive correlation among the expression of SOX3, Snail1, and E-cadherin was demonstrated in human OS tissues. CONCLUSIONS: SOX3 promotes migration, invasiveness, and EMT in OS cells via transcriptional activation of Snail1 expression, suggesting that SOX3 is a novel regulator of EMT in OS and may serve as a therapeutic target for the treatment of OS metastasis.

Strontium-Doped Amorphous Calcium Phosphate Porous Microspheres Synthesized through a Microwave-Hydrothermal Method Using Fructose 1,6-Bisphosphate as an Organic Phosphorus Source: Application in Drug Delivery and Enhanced Bone Regeneration.

Nanostructured calcium phosphate porous microspheres are of great potential in drug delivery and bone regeneration due to their large specific surface area, biocompatibility, and similarity to inorganic component of osseous tissue. In this work, strontium (Sr)-doped amorphous calcium phosphate porous microspheres (SrAPMs) were synthesized through a microwave-hydrothermal method using fructose 1,6-bisphosphate trisodium salt as the source of phosphate ions. The SrAPMs showed a mesoporous structure and a relatively high specific area. Compared with the hydroxyapatite nanorods prepared by using Na2HPO4·12H2O as the phosphorus source, the SrAPMs with a higher specific surface area were more effective in drug loading using vancomycin as the antiobiotics of choice and consequently having a higher antibacterial efficiency both on agar plates and in broths. Furthermore, to assess the potential application of SrAPMs in bone defect repair, a novel biomimetic bone tissue-engineering scaffold consisting of collagen (Coll) and SrAPMs was constructed using a freeze-drying fabrication process. Incorporation of the SrAPMs not only improved the mechanical properties, but also enhanced the osteogenesis of rat bone marrow mesenchymal stem cells. The in vivo experiments demonstrated that the SrAPMs/Coll scaffolds remarkably enhanced new bone formation compared with the Coll and APMs/Coll scaffolds in a rat critical-sized calvarial defect model at 8 weeks postimplantation. In summary, SrAPMs developed in this work are promising as antibiotic carriers and may encourage bone formation when combined with collagen.

Effects of Tumor Necrosis Factor Inhibitor on Stress-Shielded Tendons.

Mechanical stress plays an important role in preserving the integrity of bone and ligament. Stress shielding reduces mechanical load on bone or tendons, resulting in tissue degradation. Previous studies showed that deterioration of the tendon structure during stress shielding is associated with elevated expression of tumor necrosis factor (TNF)-α. This study examined the therapeutic potential of the TNF inhibitor etanercept in preventing morphologic deterioration of the Achilles tendon after stress shielding. Rats (N=48) were exposed to stress shielding of the left Achilles tendon and treated with etanercept or phosphate-buffered saline for 2 or 4 weeks. The right Achilles tendons were used as controls. After 2 or 4 weeks, stress-shielded tendons appeared less smooth than control tendons, and the stress-shielded tendons formed adhesions with surrounding tissues. Transmission electron microscopy also showed disarray of the collagen fibrils and a significant increase in the number of small-diameter collagen fibrils. These changes were associated with increased expression of TNF-α, matrix metalloproteinase (MMP)-13, MMP-3, collagen I, and collagen III. Treatment with 2 weeks of etanercept injection reduced morphologic changes in collagen organization and structure induced by stress shielding. Etanercept treatment also attenuated upregulation of MMP-13, MMP-3, and collagen III levels. However, no significant difference was observed between the etanercept group and the phosphate-buffered saline group after 4 weeks of treatment. The current findings show that TNF-α inhibition can protect against the early stages of tendon tissue remodeling induced by stress shielding, but additional interventions may be necessary to prevent tendon degeneration with long-term stress shielding. [Orthopedics. 2017; 40(1):49-55.].

In vitro and in vivo evaluation of MgF2 coated AZ31 magnesium alloy porous scaffolds for bone regeneration.

Porous magnesium scaffolds are attracting increasing attention because of their degradability and good mechanical property. In this work, a porous and degradable AZ31 magnesium alloy scaffold was fabricated using laser perforation technique. To enhance the corrosion resistance and cytocompatibility of the AZ31 scaffolds, a fluoride treatment was used to acquire the MgF2 coating. Enhanced corrosion resistance was confirmed by immersion and electrochemical tests. Due to the protection provided by the MgF2 coating, the magnesium release and pH increase resulting from the degradation of the FAZ31 scaffolds were controllable. Moreover, in vitro studies revealed that the MgF2 coated AZ31 (FAZ31) scaffolds enhanced the proliferation and attachment of rat bone marrow stromal cells (rBMSCs) compared with the AZ31 scaffolds. In addition, our present data indicated that the extract of the FAZ31 scaffold could enhance the osteogenic differentiation of rBMSCs. To compare the in vivo bone regenerative capacity of the AZ31 and FAZ31 scaffolds, a rabbit femoral condyle defect model was used. Micro-computed tomography (micro-CT) and histological examination were performed to evaluate the degradation of the scaffolds and bone volume changes. In addition to the enhanced the corrosion resistance, the FAZ31 scaffolds were more biocompatible and induced significantly more new bone formation in vivo. Conversely, bone resorption was observed from the AZ31 scaffolds. These promising results suggest potential clinical applications of the fluoride pretreated AZ31 scaffold for bone tissue repair and regeneration.

Copper-doped mesoporous hydroxyapatite microspheres synthesized by a microwave-hydrothermal method using creatine phosphate as an organic phosphorus source: application in drug delivery and enhanced bone regeneration.

The development of multifunctional biomaterials with drug delivery ability, and pro-osteogenic and pro-angiogenic activities has garnered increasing interest in the field of regenerative medicine. In the present study, hypoxia-mimicking copper (Cu)-doped mesoporous hydroxyapatite (HAP) microspheres (Cu-MHMs) were successfully synthesized through a microwave-hydrothermal method by using creatine phosphate as an organic phosphorus source. The Cu-MHMs doped with 0.2, 0.5 and 1 mol% Cu were prepared. The Cu-MHMs consisting of HAP nanorods or nanosheets exhibited a hierarchically mesoporous hollow structure and a high specific surface area. Then the Cu-MHMs were investigated as a drug nanocarrier using doxorubicin hydrochloride (DOX) as a model drug. The Cu-MHMs showed a relatively high drug-loading capacity and a pH-responsive drug release behavior. Furthermore, the Cu-MHMs were incorporated into a chitosan (CS) matrix to construct a biomimetic scaffold optimized for bone regeneration. The Cu-MHM/CS composite scaffolds maintained high degrees of porosity and showed a sustained release of Cu ions. More importantly, the Cu-MHM/CS scaffolds not only enhanced the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs) but also promoted the migration and tube formation of EA.hy926 cells. When implanted in rat critical-sized calvarial defects, the Cu-MHM/CS scaffolds significantly enhanced bone regeneration accompanied by more new blood vessel formation at 8 weeks post-operation compared with the MHM/CS scaffolds. These results suggest that the hypoxia-mimicking Cu-MHM/CS scaffolds could encourage bone regeneration by enhancing osteogenesis and angiogenesis simultaneously, which bodes well for the reconstruction of vascularized tissue-engineered bone.

Platelet-Rich Plasma-Loaded Poly(d,l-lactide)-Poly(ethylene glycol)-Poly(d,l-lactide) Hydrogel Dressing Promotes Full-Thickness Skin Wound Healing in a Rodent Model.

Traditional therapeutic methods for skin wounds have many disadvantages, and new wound dressings that can facilitate the healing process are thus urgently needed. Platelet-rich plasma (PRP) contains multiple growth factors (GFs) and shows a significant capacity to heal soft tissue wounds. However, these GFs have a short half-life and deactivate rapidly; we therefore need a sustained delivery system to overcome this shortcoming. In this study, poly(d,l-lactide)-poly(ethylene glycol)-poly(d,l-lactide) (PDLLA-PEG-PDLLA: PLEL) hydrogel was successfully created as delivery vehicle for PRP GFs and was evaluated systematically. PLEL hydrogel was injectable at room temperature and exhibited a smart thermosensitive in situ gel-formation behavior at body temperature. In vitro cell culture showed PRP-loaded PLEL hydrogel (PRP/PLEL) had little cytotoxicity, and promoted EaHy926 proliferation, migration and tube formation; the factor release assay additionally indicated that PLEL realized the controlled release of PRP GFs for as long as 14 days. When employed to treat rodents' full-thickness skin defects, PRP/PLEL showed a significantly better ability to raise the number of both newly formed and mature blood vessels compared to the control, PLEL and PRP groups. Furthermore, the PRP/PLEL-treated group displayed faster wound closure, better reepithelialization and collagen formation. Taken together, PRP/PLEL provides a promising strategy for promoting angiogenesis and skin wound healing, which extends the potential of this dressing for clinical application.

Effects of interleukin-1 receptor antagonist on collagen and matrix metalloproteinases in stress-shielded achilles tendons of rats.

Based on previous studies showing that interleukin-1 (IL-1) significantly increased after stress shielding, this article reports further research into the possible therapeutic applications of IL-1 receptor antagonist (IL-1Ra). Forty rats whose left Achilles tendons were denervated and completely stress shielded were divided into 5 groups: 2-week phosphate-buffered saline (PBS); 4-week PBS; 2-week IL-1Ra; 4-week IL-1Ra; and normal control. The Achilles tendons were tested morphologically, and the changes in collagen I and III, matrix metalloproteinases (MMP)-1 and -3, and tissue inhibitors of metalloproteinase (TIMP)-1 were determined. The collagen fibrils in the IL-1Ra groups were morphologically more similar to those in the control group than to those in the PBS groups. The collagen I levels increased in the 2-week groups. Significant differences existed between the PBS and IL-1Ra groups at 4 weeks. The MMP-1 level increased dramatically after stress shielding and increased less in the 2-week IL-1Ra group than in the 2-week PBS group. The degree of decrease of MMP-3 in the IL-1Ra groups was significantly less than that in the PBS groups. The collagen III and TIMP-1 levels continued to increase, and no difference was found between the PBS and IL-1Ra groups. Interleukin-1 receptor antagonist prevented morphological deterioration and collagen metabolism of the denervated Achilles tendons after stress shielding, likely by inhibiting the decline of MMP-3 and increasing MMP-1 levels at an early stage.