Knockdown of MicroRNA-21 Promotes Neurological Recovery After Acute Spinal Cord Injury.

To assess the therapeutic effects of microRNA-21 (miR-21) knockdown (KD) for acute thoracic spinal cord contusion using a mouse model. Forty C57/BL6 mice were randomly divided into four groups: mice in the sham-operated (Sham) group received surgical procedure without spinal cord contusion; the spinal cord injury (SCI) group mice underwent spinal cord contusion without treatment; mice in the miR-21 KD group underwent spinal cord contusion followed by a single dose subdural injection of miR-21 KD vectors (1 × 107 TU); and the negative control (NC) group mice were given subdural injection of comparable amount of NC vectors (1 × 107 TU) after spinal cord contusion. The Basso Mouse Scale (BMS) was employed to assess hindlimb motor functions. Hematoxylin-eosin and Luxol fast blue staining were performed to evaluate pathologic changes in spinal cord tissues. Peripheral blood serum levels of tumor necrosis factor α (TNFα), transforming growth factor ß (TGF-ß) and interleukin-1ß (IL-1ß) were determined by the enzyme-linked immunosorbent assay, and mRNA expression of Brain derived neurotrophic factor (BDNF) was examined by reverse transcription-polymerase chain reaction (RT-PCR). Western blotting was performed to analyze the AKT signaling pathway. KD of miRNA-21 effectively improved the BMS scores at day 14 post-surgery compared with the SCI group (p < 0.01). The spinal cord tissue in the miR-21 KD group displayed the most overt histologic signs of recovery, with axonal regeneration and the recovery of neuronal morphology at day 14 post-surgery. Significantly alleviation of TGF-ß1, TNF-α and IL-1ß was also found in sera from the miR-21 inhibition group in comparison to others, whereas BDNF gene expression was upregulated following miR-21 KD (p < 0.01). Further, significantly decreased AKT phosphorylation activity was illustrated in the miR-21 KD group (p < 0.001). The data suggest that miR-21 KD significantly reduces the inflammatory response at the damaged spinal cord site and promotes motor functional recovery. The treatment also elevated expression of BDNF, a neurotrophin participating in nerve regeneration.

Macrophage Polarization in IL-10 Treatment of Particle-Induced Inflammation and Osteolysis.

This study investigated the therapeutic influence and potential mechanism of IL-10 in ameliorating orthopedic debris particle-induced inflammation and osteolysis. A murine air pouch with bone implantation and polyethylene particles was also used to evaluate the therapeutic effects of IL-10. The data suggested that the particle challenges significantly promoted macrophage activation and osteoclastogenesis, with dramatically increased macrophage infiltration into the pouch membranes and elevated tartrate-resistant acid phosphatase-positive cell deposition. Immunohistochemical stains revealed a significantly higher ratio of induced nitric oxide synthase-expressing cells in the particle-challenged group; treatment with IL-10 resulted in marked switching to CD163(+) cells. Also, IL-10 effectively reduced tartrate-resistant acid phosphatase-positive stained cells in the pouch membranes, and minimized the bone mineral density loss compared with untreated samples. Real-time PCR and Western blot examination indicated that IL-10 treatment significantly diminished the particle-induced IL-1ß expression but promoted expression of CD163, transforming growth factor-ß1, and CCR2. Furthermore, IL-10 significantly inhibited the ultra-high-molecular-weight polyethylene particle-elevated phospho-STAT1 and phospho-NF-κB p65 productions, and promoted phospho-STAT3 expression. Overall, the data indicate the pivotal effects of IL-10 on macrophage polarization. The effects of IL-10 in ameliorating local inflammation and osteolysis may be associated with macrophage polarization through the up-regulation of the Janus activating kinase/STAT3 signaling pathway, and the down-regulation of NF-κB and Janus activating kinase/STAT1 expression.

Cardamonin inhibits osteogenic differentiation by downregulating Wnt/beta-catenin signaling and alleviates subchondral osteosclerosis in osteoarthritic mice.

Osteoarthritis (OA) is a common degenerative joint disease, and subchondral osteosclerosis is an important pathological change that occurs in its late stages. Cardamonin (CD) is a natural flavonoid isolated from Alpinia katsumadai that has anti-inflammatory activity. The objectives of this study were to investigate the therapeutic effects and potential mechanism of CD in regulating OA subchondral osteosclerosis at in vivo and in vitro settings. Eight-week-old male C57BL/6J mice were randomly divided into four groups: sham operation, anterior cruciate ligament transection (ACLT)-induced OA model, low-dose and high-dose CD treated ACLT-OA model groups. Histological assessment and immunohistochemical examinations for chondrocyte metabolism-related markers metalloproteinase-13, ADAMTS-4, Col II, and Sox-9 were performed. Microcomputed tomography was used to assess the sclerosis indicators in subchondral bone. Further, MC3T3-E1 (a mouse calvarial preosteoblast cell line) cells were treated with various concentrations of CD to reveal the influence and potential molecular pathways of CD in osteogenic differentiations. Animal studies suggested that CD alleviated the pathological changes in OA mice such as maintaining integrity and increasing the thickness of hyaline cartilage, decreasing the thickness of calcified cartilage, decreasing the Osteoarthritis Research Society International score, regulating articular cartilage metabolism, and inhibiting subchondral osteosclerosis. In vitro investigation indicated that CD inhibited alkaline phosphatase expression and production of calcium nodules during osteogenic differentiation of MC3T3-E1 cells. In addition, CD inhibited the expression of osteogenic differentiation-related indicators and Wnt/ß-catenin pathway-related proteins. In conclusion, CD inhibits osteogenic differentiation by downregulating Wnt/ß-catenin signaling and alleviating subchondral osteosclerosis in a mouse model of OA.

Local gene transfer of OPG prevents joint damage and disease progression in collagen-induced arthritis.

This study examined the influence of osteoprotegerin (OPG) gene transfer on a murine collagen-induced arthritis model. A single periarticular injection of AAV-OPG or AAV-LacZ on the arthritic paw successfully incorporated the exogenous gene to the local tissue and resulted in marked transgene expression in the joint homogenate for at least three weeks. Clinical disease scores were significantly improved in OPG treated mice starting at 28-day post-treatment (P < 0.05). Histological assessment demonstrated that OPG gene transfer dramatically protected mice from erosive joint changes compared with LacZ controls (P < 0.05), although treatment appeared less effective on the local inflammatory progress. MicroCT data suggested significant protection against subchondral bone mineral density changes in OPG treated CIA mice. Interestingly, mRNA expressions of IFN-g and MMP3 were noticeably diminished following OPG gene transfer. Overall, gene transfer of OPG effectively inhibited the arthritis-associated periarticular bone erosion and preserved the architecture of arthritic joints, and the study provides evidence that the cartilage protection of the OPG gene therapy may be associated with the down-regulation of MMP3 expression.

Naringin promotes osteoblast differentiation and effectively reverses ovariectomy-associated osteoporosis.

BACKGROUND: Osteoporosis is a common pathological condition that influences 20 % of women over 50 years of age. This condition decreases bone strength and increases the risk of bone fracture. Naringin is a major flavonoid found in grapefruit and an active compound extracted from a Chinese herbal medicine (Rhizoma Drynariae). Studies have shown that naringin possesses many pharmacological effects. The current study evaluated the influence of naringin on osteoblastic cell differentiation and proliferation, and assessed its therapeutic effects on a rat osteoporosis model. METHOD: The proliferation, differentiation, and function of rat bone marrow stromal cells (BMSCs) were determined following treatment with various concentrations of naringin. Ovariectomy (OVX)-induced osteoporotic rats were orally administered naringin daily at low, medium, and high dosages, while a control group received PBS for 2 months. Femoral X-ray images and microCT scans were used for bone mineral density (BMD) and BV/TV (bone volume/total volume) analyses, and histological assessments of left tibiae were employed to check for changes in trabecular thickness (Tb.Th) and trabecular space (Tb.Sp) in the groups. RESULTS: Naringin was effective at enhancing the proliferation and osteogenic differentiation of BMSCs, and a concentration of 10 µg/ml prompted the highest levels of osteocalcin expression among the in vitro study groups. There appeared to be a delayed response pattern of BMSCs to the naringin treatment. Naringin also effectively reversed OVX-induced bone loss via increasing BMD, bone volume, and trabecular thickness. The medium dose (300 mg/kg) appeared to be the optimal dosage for delivering satisfactory therapeutic effects. CONCLUSION: Naringin promotes the proliferation and differentiation of BMSCs, and increases osteocalcin expression. Naringin also effectively reverses ovariectomy-induced osteoporosis in rats. The study suggests that naringin administration may represent an effective treatment for osteoporosis.

Current research in the pathogenesis of aseptic implant loosening associated with particulate wear debris.

Periprosthetic osteolysis is the most common long-term complication of a total joint arthroplasty, often resulting in aseptic loosening of the implant, which occurs in up to 34% of younger implant recipients and usually requires surgical revision. Particulate wear debris, continuously generated by articulating motion at the bearing surfaces, has been implicated as one of the primary causes of periprosthetic bone loss and implant loosening. With developing implants and bearing surfaces designs, various types of wear particles with specific chemical nature, dimension and shape are formed, which may initiate different immune or inflammatory responses. Wear debris induces down-regulation or up-regulation of various pro-inflammatory cytokines and chemokines in a range of cell types at the interface between implants and the surrounding bone, such as macrophages, osteoclast precursor cells, osteoblasts, lymphocytes, fibroblasts etc. Concomitantly, these mediators further affect functions of cells through distinct signaling mechanisms in either an autocrine or a paracrine manner. This review summarizes current concepts of how wear debris causes osteolysis, and describes the interaction and effects of wear debris on functions of primary cell types involved in osteolysis.

Movement Artifact Suppression in Wearable Low-Density and Dry EEG Recordings Using Active Electrodes and Artifact Subspace Reconstruction.

Wearable low-density dry electroencephalogram (EEG) headsets facilitate multidisciplinary applications of brain-activity decoding and brain-triggered interaction for healthy people in real-world scenarios. However, movement artifacts pose a great challenge to their validity in users with naturalistic behaviors (i.e., without highly controlled settings in a laboratory). High-precision, high-density EEG instruments commonly embed an active electrode infrastructure and/or incorporate an auxiliary artifact subspace reconstruction (ASR) pipeline to handle movement artifact interferences. Existing endeavors motivate this study to explore the efficacy of both hardware and software solutions in low-density and dry EEG recordings against non-tethered settings, which are rarely found in the literature. Therefore, this study employed a LEGO-like electrode-holder assembly grid to coordinate three 3-channel system designs (with passive/active dry vs. passive wet electrodes). It also conducted a simultaneous EEG recording while performing an oddball task during treadmill walking, with speeds of 1 and 2 KPH. The quantitative metrics of pre-stimulus noise, signal-to-noise ratio, and inter-subject correlation from the collected event-related potentials of 18 subjects were assessed. Results indicate that while treating a passive-wet system as benchmark, only the active-electrode design more or less rectified movement artifacts for dry electrodes, whereas the ASR pipeline was substantially compromised by limited electrodes. These findings suggest that a lightweight, minimally obtrusive dry EEG headset should at least equip an active-electrode infrastructure to withstand realistic movement artifacts for potentially sustaining its validity and applicability in real-world scenarios.

Fabrication, Characterization, and In Vitro Cytotoxicity Assessment of Tri-Layered Multifunctional Scaffold for Effective Chronic Wound Healing.

Chronic wounds have been a global health risk that demands intensive exploration. A tri-layered biomaterial scaffold has been developed for skin wounds. The top layer of the scaffold is superhydrophobic, and the bottom layer is hydrophilic, both of which were electrospun using recycled expanded polystyrene (EPS) and monofilament fishing line (MFL), respectively. The intermediate layer of the scaffold comprised hydrogel by cross-linking chitosan (CS) with polyethylene glycol. The surface morphology, surface chemistry, thermal degradation, and wettability characteristics of each layer of the scaffold were examined. Also, the antibacterial activity and in vitro cytotoxicity study on the combined tri-layered scaffold were assessed against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Data revealed exceptional water repellency of the heat-treated electrospun top superhydrophobic layer (TSL) with a high-water contact angle (WCA) of 172.44°. A TSL with 15 wt% of micro-/nano-inclusions had the best thermal stability above 400 °C. The bottom hydrophilic layer (BHL) displayed a WCA of 9.91°. Therapeutically, the synergistic effect of the combined tri-layered scaffold significantly inhibited bacteria growth by 70.5% for E. coli and 68.6% for S. aureus. Furthermore, cell viability is enhanced when PEG is included as part of the intermediate CS hydrogel layer (ICHL) composition.

Coronoid Fracture Reconstruction with Ipsilateral Olecranon Osteoarticular Autograft in the Acute Setting: A Case Report.

CASE: An 18-year-old male polytrauma patient sustained a high-energy posterior fracture dislocation of his left elbow associated with a comminuted and irreparable O'Driscoll type 2 subtype 3 anteromedial facet coronoid fracture. He underwent early coronoid reconstruction using ipsilateral olecranon osteoarticular autograft with incorporation of the sublime tubercle attachment of the medial collateral ligament and repair of the lateral ulnar collateral ligament. A 3-year follow-up revealed a functional, painless, congruent, and stable elbow. CONCLUSION: Early reconstruction of a highly comminuted coronoid fracture may be a useful salvage option for the polytrauma patient, thereby avoiding complications associated with late reconstruction of posttraumatic elbow instability.

DEPDC1 as a crucial factor in the progression of human osteosarcoma.

OBJECTIVE: Novel therapeutic strategies are emerging with the increased understanding of the underlying mechanisms of human osteosarcoma. This current study tends to decipher the potentially critical role of DEP domain-containing 1 (DEPDC1), a tumor-related gene, during the progression of osteosarcoma. METHODS: Bioinformatics analysis of 25,035 genes from the National Center for Biotechnology Information (NCBI) databases was performed to screen differentially expressed genes between osteosarcoma and normal control groups, complemented by the examination of 85 clinical osteosarcoma specimens. Furthermore, the manipulation of DEPDC1 expression levels by using silencing RNA (siRNA) or lentiviral vector intervention on human osteosarcoma cells was performed to reveal its role and interactions in in vitro and in vivo settings. RESULTS: Gene expression profile analysis and immunohistochemical (IHC) examination suggested that DEPDC1 is highly expressed in human osteosarcoma cells and tumor tissue. The silencing of DEPDC1 arrested osteosarcoma cell proliferation, promoted apoptosis, and ceased tumor metastasis. Studies involving clinical human osteosarcoma cases exhibited a strong correlation of DEPDC1 over-expressed osteosarcoma specimens with a reduced patient survival rate. CONCLUSIONS: Collectively, this study demonstrated that DEPDC1 is a critical driver in the promotion of osteosarcoma progression and results in poor patient prognosis. Genetically targeting or pharmacologically inhibiting DEPDC1 may serve as a promising strategy for treating human osteosarcoma.

Higenamine Promotes Osteogenesis Via IQGAP1/SMAD4 Signaling Pathway and Prevents Age- and Estrogen-Dependent Bone Loss in Mice.

Osteoporosis is a common bone disease caused by an imbalance of bone resorption and formation that results in a loss of total bone density. SMAD2/3 signal transduction is known to play a crucial role in osteogenic differentiation through transforming growth factor-beta (TGF-ß). By screening a library of small-molecule compounds, the current study identifies higenamine (HG) as an active osteogenic agent that could be a therapeutic candidate for osteoporosis. In vitro data demonstrated that HG effectively induced expressions of osteogenic markers in mouse bone marrow stromal cell (BMSCs) and preosteoblastic cell cultures. Further, HG treatment resulted in enhanced bone formation and prevented accelerated bone loss on two animal models that mimic spontaneous senile osteoporosis and postmenopausal osteoporosis. IQ motif-containing GTPase-activating protein 1 (IQGAP1) was confirmed as a novel target of HG, where HG appears to bind to the Glu-1019 site of IQGAP1 to exert its osteogenic effects. Data subsequently suggested that HG promoted phosphorylation of SMAD2/3 and regulated the SMAD2/3 pathway by inhibiting SMAD4 ubiquitination. Overall, the findings highlight HG as a new small-molecule drug to promote bone formation through SMAD2/3 pathway in osteoporosis. © 2023 American Society for Bone and Mineral Research (ASBMR).

The Role of IL-6 and TMEM100 in Lumbar Discogenic Pain and the Mechanism of the Glycine-Serine-Threonine Metabolic Axis: A Metabolomic and Molecular Biology Study.

Background: It is well established that discogenic low back pain (DLBP) is often caused by the inflammatory response during intervertebral disc degeneration (IDD). However, it remains unclear how inflammatory mediators such as Interleukin-6 (IL-6) are involved in discogenic pain caused by degeneration and intervertebral disc (IVD) metabolism. The purpose of this study is to study the relationship between IL-6 and Transmembrane protein 100 (TMEM100), and to analyze the different metabolites and metabolic pathways in various rat intervertebral discs by metabonomics. Methods: We established a rat model of IDD-DLBP by disc punctures and PBS infusion to examine the rat pain behaviors. Intervertebral disc tissues were harvested for molecular biology experiments to explore the relationship between IL-6 and TMEM100. High-resolution mass spectrometry (HRMS) was performed for untargeted metabolomics, and nuclear magnetic resonance spectroscopy metabolomics (MRS) for differential metabolites and metabolic pathways. Results: The results showed a significant decrease in vonFrey test, hot plate test and acetone test (P < 0.05). The expression of IL-6 and TMEM100 in DLBP model was significantly higher than that in sham control group and IDD discs without PBS infusion group (P < 0.05). There were 15 major contributing metabolites identified in the DLBP intervertebral discs through metabolomic studies, involving 6 major metabolic pathways. The main differential metabolites included nitric oxide (NO), ammonia, and lactic acid as the metabolic endpoints; and the differential metabolic pathways included the glycine-serine-threonine (Gly-Ser-Thr), which is gradually weakened with the progression of inflammation. Conclusion: The change of TMEM100 expression mediated by il-6 is related to the Gly-Ser-Thr metabolic axis and plays an important role in the relief of discogenic pain.

Expression of Vascular Endothelial Growth Factor correlates with the advance of clinical osteosarcoma.

PURPOSE: Osteosarcoma is the most common primary malignancy in orthopaedic surgery. Studies suggest that expression of VEGF and high vascularity within osteosarcoma may correlate with poor prognosis. The purpose of this study was to determine whether there was a correlation of VEGF expression with clinical tumour stage and metastasis. METHODS: This retrospective case series examined 54 cases of osteosarcoma patients who were treated during a ten-year period. Relevant clinical information included age, gender, tumour location, stage, adjuvant therapy, morbidity, mortality, and tumour subtypes. The clinical information was analysed for correlation of VEGF expression and tumour prognosis. Tumour sections were examined by routine H&E and by immunohistochemistry for VEGF, CD31, and the oncogenes c-myc and c-fos. RESULTS: There was a significantly positive correlation between VEGF expression and tumour stages among these cases (p < 0.01). The data also suggested a higher cancer recurrence and more frequent cases of remote metastasis in the high-VEGF group compared to the low-VEGF group. VEGF expression also positively associated with c-fos and c-myc expressions in the primary tumour sections. CONCLUSION: The results of this study highlight the role of VEGF in angiogenesis and tumour burden. Data also suggest the influence of VEGF may associate with the elevations of c-fos and c-myc expression. The development of novel therapies to target the VEGF pathway in osteosarcoma may lead to improved survival.

Synthesis and Properties of Magnetic Fe3O4/PCL Porous Biocomposite Scaffolds with Different Sizes and Quantities of Fe3O4 Particles.

In clinical practice, to treat diseases such as osteosarcoma or chondrosarcoma with broad surgical ostectomy, it would be ideal to have scaffolds that not only fill up the bone void but also possess the ability to regulate the subsequent regimes for targeted chemotherapy and/or bone regeneration. Magnetic targeting of therapeutic agents to specific sites in the body provides certain advantages such as minimal side-effects of anti-cancer drugs. The objective of this study was to characterize novel magnetic scaffolds that can be used as a central station to regulate the drug delivery of a magnetic nanoparticle system. Different sizes and quantities of Fe3O4 particles were mixed with poly-ε-caprolactone (PCL) to construct the magnetic scaffolds, and their mechanical properties, degradation performance, and cell biocompatibility were evaluated. It appeared that the presence of Fe3O4 particles influenced the magnetic, mechanical, and biological performances of the scaffolds. The prepared bio-nanocomposite scaffolds provided predominantly magnetic/superparamagnetic properties. Scaffolds with a micron-sized Fe3O4 to PCL weight (wt) ratio of 0.1:0.9 exhibited higher mechanical performances among samples, with Young's modulus reaching 1 MPa and stiffness, 13 N/mm. Although an increased Fe3O4 particle proportion mildly influenced cell growth during the biocompatibility test, none of the Fe3O4/PCL scaffolds showed a cytotoxic effect.

Identification and coregulation pattern analysis of long noncoding RNAs following subacute spinal cord injury.

Long noncoding RNAs (lncRNAs) have been demonstrated to play critical regulatory roles in posttranscriptional and transcriptional regulation in eukaryotic cells. However, the characteristics of many lncRNAs, particularly their expression patterns in the lesion epicenter of spinal tissues following subacute spinal cord injury (SCI), remain unclear. In this study, we determined the expression profiles of lncRNAs in the lesion epicenter of spinal tissues after traumatic SCI and predicted latent regulatory networks. Standard Allen's drop surgery was conducted on mice, and hematoxylin and eosin staining was used to observe the damaged area. High-throughput sequencing was performed to identify the differential expression profiles of lncRNAs. Quantitative real-time polymerase chain reaction was conducted to evaluate the quality of the sequencing results. Bioinformatics analyses, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, coexpression analysis, and protein-protein interaction analysis, were performed. Targeted binding of lncRNA-miRNA-mRNA was predicted by TargetScan and miRanda. A total of 230 differentially expressed lncRNAs were identified and preliminarily verified, and some potential regulatory networks were constructed. These findings improve our understanding of the mechanisms underlying subacute SCI; differentially expressed lncRNAs are closely involved in pathophysiological processes by regulating multiple pathways. Further studies are essential for revealing the exact mechanism underlying competing endogenous RNA pathways in vivo and in vitro.

Classification and Treatment for Cervical Spine Fracture with Ankylosing Spondylitis: A Clinical Nomogram Prediction Study.

Objective: Through the follow-up analysis of cervical spine fracture cases with ankylosing spondylitis (AS), a treatment-oriented fracture classification method is introduced to evaluate the clinical efficacy guided by this classification method. Method: A retrospective analysis was performed on 128 AS patients who underwent comprehensive treatment in the Spine Surgery Department of Qingdao University Hospital from January 2009 to May 2018. Statistics of patient demographic data, distribution of different fractures corresponding to surgical methods, 3-year follow-up outcomes, and summary of objective fracture classification methods were analyzed. A prospective 5-year follow-up study of 90 patients with AS cervical spine fractures from June 2015 to August 2020 was also included. Statistical differences on the distribution of factors such as case information, cervical spine sagittal sequence parameters, and fracture classification were assessed. Correlations between surgical information, American Spinal Injuries Association grade (ASIA), modified Japanese Orthopaedic Association scores (mJOA), and other factors were analyzed to establish a nomogram predictive model for curative effect outcomes. Overall, three major types and the four subtypes of AS cervical spine fractures were evaluated based on the clinical efficacy of the classification and the selection of surgical treatment methods. Result: The most common type of fracture was type II (30 cases, 33.33%), most of the subtypes were A (37 cases), followed by B (36 cases) and C (17 cases). Twenty-four of 28 patients with type I underwent anterior surgery, and 47 of 62 patients with type II and III underwent posterior surgery. The average follow-up time was 25.76 ± 11.80 months. The results of predicting clinical variables are different but include factors such as fracture type and subtype, type of operation, and age. The predictor variables include the above-mentioned similar variables, but survival is more affected by the fracture type of the patient. Conclusion: This predictive model based on follow-up information delineation points out the impact of ankylosing spondylitis cervical spine fracture classification on surgical selection and clinical efficacy.

Polymeric biomaterials for wound healing applications: a comprehensive review.

Chronic wounds have been a global health threat over the past few decades, requiring urgent medical and research attention. The factors delaying the wound-healing process include obesity, stress, microbial infection, aging, edema, inadequate nutrition, poor oxygenation, diabetes, and implant complications. Biomaterials are being developed and fabricated to accelerate the healing of chronic wounds, including hydrogels, nanofibrous, composite, foam, spongy, bilayered, and trilayered scaffolds. Some recent advances in biomaterials development for healing both chronic and acute wounds are extensively compiled here. In addition, various properties of biomaterials for wound-healing applications and how they affect their performance are reviewed. Based on the recent literature, trilayered constructs appear to be a convincing candidate for the healing of chronic wounds and complete skin regeneration because they mimic the full thickness of skin: epidermis, dermis, and the hypodermis. This type of scaffold provides a dense superficial layer, a bioactive middle layer, and a porous lower layer to aid the wound-healing process. The hydrophilicity of scaffolds aids cell attachment, cell proliferation, and protein adhesion. Other scaffold characteristics such as porosity, biodegradability, mechanical properties, and gas permeability help with cell accommodation, proliferation, migration, differentiation, and the release of bioactive factors.

[Retros Flt-1 decelerates the growth of a murine experimental osteosarcoma].

OBJECTIVE: To examine the influence of vascular endothelial growth factors (VEGF) in controlling the growth of an experimental osteosarcoma in mice by performing retrovirus-mediated sFlt-1 gene modification. METHODS: From March to October 2010 human osteosarcoma G-292 cells were in vitro infected with retroviral vectors encoding soluble Flt-1 or LacZ gene before transplanted into proximal tibiae of immune deficient SCID mice to establish experimental orthotopic osteosarcoma. Daily observation and biweekly microCT were performed to monitor tumor development and progression till sacrifice at 8 weeks after tumor cell inoculation for histological and molecular analyses. RESULTS: Successful transgene expression was confirmed in the culture media of sFlt-1 transduced G-292 cells using ELISA, and with positive X-gal staining of the LacZ transduced cells. Noteworthy tumors were grown in all mice on the tibiae receiving G-292 cell inoculation, with clear detection on microCT images starting 2 weeks after inoculation. Over the time period, tumors derived from sFlt-1 transduced G-292 cells were distinctively smaller in size compared to the ones from wide-type G-292 and G-292-LacZ cells. Histology showed typical osteosarcoma characteristics including severe cellular pleomorphism, bone erosions, and neo-vascularization. Real-time polymerase chain reaction indicated significantly higher sFlt-1 expression in sFlt-1 transduced groups than the wild-type G-292 or LacZ treated groups. Strong expression of oncogenes c-myc and c-fos were also obvious, along with the expression of VEGF in the primary tumor tissue. CONCLUSION: Retrovirus-mediated sFLT-1 gene modification decelerates the osteosarcoma tumor growth in this murine model.

Effects of Hypoxia on Proliferation and Apoptosis of Osteosarcoma Cells.

BACKGROUND: Hypoxia can happen during solid tumor growth including osteosarcoma. This study investigated the relationship of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) on osteosarcoma cell growth and apoptosis under hypoxic conditions. MATERIALS AND METHODS: Human osteosarcoma cells were cultured under normal or hypoxic conditions. Inhibitors of HIF-1α and VEGF were applied to the cells separately or in combination to block the respective proteins. Cell proliferation and apoptosis were examined by MTT and TUNEL assays, and real-time PCR and ELISA were performed for mRNA and protein expression. RESULTS: There was a dramatic decrease of cell proliferation and an elevation of apoptosis under hypoxia. Blockage of HIF-1α and VEGFR enhanced the cell growth retardation and promoted apoptotic changes. Moreover, blockage of HIF-1α significantly eliminated the expression of VEGF in the cell culture media, and vice versa. CONCLUSION: HIF-1α and VEGF work closely in regulating osteosarcoma cell growth under hypoxic conditions and blockage of either of them may subsequently influence the presence of the other.

Human Peripheral Nerve-Derived Pluripotent Cells Can Be Stimulated by In Vitro Bone Morphogenetic Protein-2.

We have recently identified a population of cells within the peripheral nerves of adult rodent animals (mice and rats) that can respond to Bone Morphogenetic Protein-2 (BMP-2) exposure or physical injury to rapidly proliferate. More importantly, these cells exhibited embryonic differentiation potentials that could be induced into osteoblastic and endothelial cells in vitro. The current study examined human nerve specimens to compare and characterize the cells after BMP-2 stimulation. Fresh pieces of human nerve tissue were minced and treated with either BMP-2 (750 ng/mL) or a PBS vehicle for 12 h at 37 °C, before being digested in 0.2% collagenase and 0.05% trypsin-EDTA. Isolated cells were cultured in a restrictive stem cell medium. Significantly more cells were obtained from the nerve pieces with the BMP-2 treatment in comparison with the PBS vehicle controls. Cell colonies started to form at Day 3. Expressions of the four transcription factors, namely, Klf4, c-Myc, Sox2, and Oct4, were confirmed at both the transcriptional and translational levels. The cells can be maintained in the stem cell culture medium for at least 6 weeks without changing their morphology. When the cells were transferred to a fibroblast growth medium, dispersed spindle-shaped motile cells were noted and became fibroblast activated protein-α (FAP) positive with immunocytochemistry staining. The data suggest that human peripheral nerve tissue also contains a population of cells that can respond to BMP-2 and express Klf4, Sox2, cMyc, and Oct4-the four transcription factors driving cell pluripotency. These cells are able to differentiate into FAP-positive fibroblasts. In summary, in human peripheral nerves also reside a population of quiescent cells with pluripotency potential that may be the same cells as rodent nerve-derived adult stem (NEDAPS) cells. It is proposed that these cells are possibly at the core of a previously unknown natural mechanism for healing an injury.