Real-time assessment of guided bone regeneration in critical size mandibular bone defects in rats using collagen membranes with adjunct fibroblast growth factor-2
Background/purpose/UNASSIGNED:Fibroblast growth factor-2 (FGF-2) regulates bone formation. The concept of guided bone regeneration using a resorbable collagen membrane (RCM) is generally accepted in implant dentistry. This study aimed to investigate the bone healing pattern in rat mandibular bone defects in real-time with and without RCM containing FGF-2 (RCM/FGF-2). Materials and methods/UNASSIGNED:Critical-size circular bone defects (4.0â€¯mm diameter) were created on both sides of the rat mandibular bone. The defects were randomly divided into the following groups: control, RCM alone, RCM containing low (0.5â€¯Î¼g) or high (2.0â€¯Î¼g) concentration of FGF-2. We performed real-time inÂ vivo micro-computerized tomography scans at the baseline and at 2, 4, and 6 weeks, and measured the volume of newly formed bone (NFB), bone mineral density (BMD) of NFB, and the closure percentage of the NFB area. At 6 weeks, the mandibular specimens were assessed histologically and histomorphometrically to evaluate the area of new bone regeneration. Results/UNASSIGNED:Real-time assessment revealed a significant increase in the volume, BMD, and closure percentage of the NFB area in the RCM/FGF-2-treated groups than that in the control and RCM groups. In the H-FGF-2 group, the volume and BMD of NFB exhibited a significant increase at 6 weeks than that at the baseline. Histological evaluation revealed the presence of osteoblasts, osteocytes, and blood vessels within the NFB. Conclusion/UNASSIGNED:The real-time inÂ vivo experiment demonstrated that RCM/FGF-2 effectively promoted bone regeneration within the critical-size mandibular defects in rats and verified new bone formation starting in the early postoperative phase.
Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway
BACKGROUND:We developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, Î²-actin expression was measured after siRNA Î²-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells. RESULTS:mTat/PEI/INT/siRNA produced significant improvement in transfection efficiency with little cytotoxicity compared to other vectors and achieved â‰ˆâ€‰100% knockdown of Î²-actin expression compared to non-treated cells. The electric charge of mTat/PEI/INT/siRNA was significantly higher than INT/siRNA. The particle size of mTat/PEI/INT/siRNA was significantly smaller than INT/siRNA. Filipin III and Î²-cyclodextrin, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI/INT/siRNA transfection, while chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not inhibit mTat/PEI/INT/siRNA transfection. Furthermore, the transfection efficiency of mTat/PEI/INT at 4Â Â°C was significantly lower than 37Â Â°C. CONCLUSIONS:These findings demonstrated the feasibility of using mTat/PEI/INT as a potentially attractive non-viral vector for siRNA delivery.
Released fibroblast growth factor18 from a collagen membrane induces osteoblastic activity involved with downregulation of miR-133a and miR-135a
We have developed a unique delivery system of growth factors using collagen membranes (CMs) to induce bone regeneration. We hypothesized that fibroblast growth factor18 (FGF-18), a pleiotropic protein that stimulates proliferation in several tissues, can be a good candidate to use our delivery system for bone regeneration. Cell viability, cell proliferation, alkaline phosphatase activity, mineralization, and marker gene expression of osteoblastic differentiation were evaluated after mouse preosteoblasts were cultured with a CM containing FGF-18, a CM containing platelet-derived growth factor, or a CM alone. Furthermore, expression of microRNA, especially miR-133a and miR-135a involving inhibition of osteogenic factors, was measured in preosteoblasts with CM/FGF-18 or CM alone. A sustained release of FGF-18 from the CM was observed over 21 days. CM/FGF-18 significantly promoted cell proliferation, alkaline phosphatase activity, and mineralization compared to CM alone. Gene expression of type I collagen, runt-related transcription factor 2, osteocalcin, Smad5, and osteopontin was significantly upregulated in CM/FGF-18 compared to CM alone, and similar to CM/platelet-derived growth factor. Additionally, CM/FGF-18 downregulated expression of miR-133a and miR-135a. These results suggested that released FGF-18 from a CM promotes osteoblastic activity involved with downregulation of miR-133a and miR-135a.
A collagen membrane containing osteogenic protein-1 facilitates bone regeneration in a rat mandibular bone defect
OBJECTIVES: Osteogenic protein-1 (OP-1) has shown osteoinductive activities and is useful for clinical treatments, including bone regeneration. Regenerative procedures using a bioabsorbable collagen membrane (BCM) are well established in periodontal and implant dentistry. We evaluated the subsequent effects of the BCM in combination with OP-1 on bone regeneration in a rat mandibular circular critical-sized bone defect in vivo. DESIGN: We used 8 rats that received surgery in both sides of the mandible, and created the total 16 defects which were divided into 4 groups: Group 1; no treatment, as a control, Group 2; BCM alone, Group 3; BCM containing low dose 0.5mug of OP-1 (L-OP-1), and Group 4; BCM containing high dose 2.0mug of OP-1 (H-OP-1). Newly formed bone was evaluated by micro computed tomography (micro-CT) and histological analyses at 8 weeks postoperatively. In quantitative and qualitative micro-CT analyses of the volume of new bone formation, bone density, and percentage of new bone area was evaluated. RESULTS: BCM with rhOP-1 significantly increased and accelerated bone volume, bone mineral density, and percentage of new bone area compared to control and BCM alone at 8 weeks after surgery; these enhancements in bone regeneration in the OP-1-treated groups were dose-dependent. CONCLUSIONS: OP-1 delivered with a BCM may have effective osteoinductive potency and be a good combination for bone regeneration. The use of such a combination device for osteogenesis may result in safer and more predictable bone regenerative outcomes in the future.
The potential of stromal cell-derived factor-1 delivery using a collagen membrane for bone regeneration
Stromal cell-derived factor-1 (SDF-1) is a cytokine that is important in stem and progenitor cell recruitment in tissue repair after injury. Regenerative procedures using collagen membranes (CMs) are presently well established in periodontal and implant dentistry. The objective of this study is to test the subsequent effects of the released SDF-1 from a CM on bone regeneration compared to platelet-derived growth factor (PDGF) in vitro and in vivo. For in vitro studies, cell proliferation, alkaline phosphatase activity, and osteoblastic differentiation marker genes were assessed after MC3T3-E1 mouse preosteoblasts were cultured with CMs containing factors. In vivo effects were investigated by placement of CMs containing SDF-1 or PDGF using a rat mandibular bone defect model. At 4 weeks after the surgery, the new bone formation was measured using micro-computed tomography (microCT) and histological analysis. The results of in vitro studies revealed that CM delivery of SDF-1 significantly induced cell proliferation, ALP activity, and gene expression of all osteogenic markers compared to the CM alone or control, similar to PDGF. Quantitative and qualitative microCT analysis for volume of new bone formation and the percentage of new bone area showed that SDF-1-treated groups significantly increased and accelerated bone regeneration compared to control and CM alone. The enhancement of bone formation in SDF-1-treated animals was dose-dependent and with levels similar to those measured with PDGF. These results suggest that a CM with SDF-1 may be a great candidate for growth factor delivery that could be a substitute for PDGF in clinical procedures where bone regeneration is necessary.
Ex vivo nonviral gene delivery of mu-opioid receptor to attenuate cancer-induced pain
Virus-mediated gene delivery shows promise for the treatment of chronic pain. However, viral vectors have cytotoxicity. To avoid toxicities and limitations of virus-mediated gene delivery, we developed a novel nonviral hybrid vector: HIV-1 Tat peptide sequence modified with histidine and cysteine residues combined with a cationic lipid. The vector has high transfection efficiency with little cytotoxicity in cancer cell lines including HSC-3 (human tongue squamous cell carcinoma) and exhibits differential expression in HSC-3 ( approximately 45-fold) relative to HGF-1 (human gingival fibroblasts) cells. We used the nonviral vector to transfect cancer with OPRM1, the mu-opioid receptor gene, as a novel method for treating cancer-induced pain. After HSC-3 cells were transfected with OPRM1, a cancer mouse model was created by inoculating the transfected HSC-3 cells into the hind paw or tongue of athymic mice to determine the analgesic potential of OPRM1 transfection. Mice with HSC-3 tumors expressing OPRM1 demonstrated significant antinociception compared with control mice. The effect was reversible with local naloxone administration. We quantified beta-endorphin secretion from HSC-3 cells and showed that HSC-3 cells transfected with OPRM1 secreted significantly more beta-endorphin than control HSC-3 cells. These findings indicate that nonviral delivery of the OPRM1 gene targeted to the cancer microenvironment has an analgesic effect in a preclinical cancer model, and nonviral gene delivery is a potential treatment for cancer pain.
Nanometer-Scale Features on Micrometer-Scale Surface Texturing: A Bone Histological, Gene Expression, and Nanomechanical Study
Micro- and nanoscale surface modifications have been the focus of multiple studies in the pursuit of accelerating bone apposition or osseointegration at the implant surface. Here, we evaluated histological and nanomechanical properties, and gene expression, for a microblasted surface presenting nanometer-scale texture within a micrometer-scale texture (MB) (Ossean Surface, Intra-Lock International, Boca Raton, FL) versus a dual-acid etched surface presenting texture at the micrometer-scale only (AA), in a rodent femur model for 1, 2, 4, and 8weeks in vivo. Following animal sacrifice, samples were evaluated in terms of histomorphometry, biomechanical properties through nanoindentation, and gene expression by real-time quantitative reverse transcription polymerase chain reaction analysis. Although the histomorphometric, and gene expression analysis results were not significantly different between MB and AA at 4 and 8weeks, significant differences were seen at 1 and 2weeks. The expression of the genes encoding collagen type I (COL-1), and osteopontin (OPN) was significantly higher for MB than for AA at 1week, indicating upregulated osteoprogenitor and osteoblast differentiation. At 2weeks, significantly upregulated expression of the genes for COL-1, runt-related transcription factor 2 (RUNX-2), osterix, and osteocalcin (OCN) indicated progressive mineralization in newly formed bone. The nanomechanical properties tested by the nanoindentation presented significantly higher rank hardness and elastic modulus for the MB compared to AA at all time points tested. In conclusion, the nanotopographical featured surfaces presented an overall higher host-to-implant response compared to the microtextured only surfaces. The statistical differences observed in some of the osteogenic gene expression between the two groups may shed some insight into the role of surface texture and its extent in the observed bone healing mechanisms.
Gene delivery from supercharged coiled-coil protein and cationic lipid hybrid complex
A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP.DNA(8:1).FG lipoproteoplex complex demonstrated enhanced transfection of beta-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat.FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP.DNA(8:1).FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery.
Efficient in vivo gene delivery using modified Tat peptide with cationic lipids
A combination of modified HIV-1 Tat (mTat) peptide and cationic lipids, FuGENE HD (FH), dramatically enhanced transfection efficiency across a range of cell lines when compared to mTat or FH alone (Biomaterials 35:1705-1715 2014). The efficiency of this Tat peptide combination was significantly higher than many commercial non-viral vectors. In this present study, we tested the feasibility of this non-viral vector, mTat/FH, in vivo using plasmid DNA encoding a luciferase gene. The results of the in vivo studies showed that animals administered mTat/FH/DNA intramuscularly had significantly higher and longer luciferase expression ( approximately 7 months) than those with mTat/DNA, FH/DNA, or DNA alone. Histological evaluation showed little immune response in the muscles, livers, and kidneys of mice administered with the mTat/FH. The combination of mTat with FH could significantly improve transfection efficiency, expanding the potential use of non-viral gene vectors in vivo.
The effect of a bioactive collagen membrane releasing PDGF or GDF-5 on bone regeneration
Regenerative procedures using barrier membrane technology are presently well established in periodontal/endodontic surgery. The objective of this study was to compare the subsequent effects of the released platelet-derived growth factor (PDGF) and growth/differentiation factor 5 (GDF-5) from collagen membranes (CMs) on bone regeneration in vitro and in vivo. In vitro studies were conducted using MC3T3-E1 mouse preosteoblasts cultured with or without factors. Cell viability, cell proliferation, alkaline phosphatase (ALP) activity and bone marker gene expression were then measured. In vivo studies were conducted by placing CMs with low or high dose PDGF or GDF-5 in rat mandibular defects. At 4 weeks after surgery new bone formation was measured using muCT and histological analysis. The results of in vitro studies showed that CM/GDF-5 significantly increased ALP and cell proliferation activities without cytotoxicity in MC3T3-E1 cells when compared to CM/PDGF or CM alone. Gene expression analysis revealed that Runx2 and Osteocalcin were significantly increased in CM/GDF-5 compared to CM/PDGF or control. Quantitative and qualitative muCT and histological analysis for new bone formation revealed that although CM/PDGF significantly enhanced bone regeneration compared to CM alone or control, CM/GDF-5 significantly accelerated bone regeneration to an even greater extent than CM/PDGF. The results also showed that GDF-5 induced new bone formation in a dose-dependent manner. These results suggest that this strategy, using a CM carrying GDF-5, might lead to an improvement in the current clinical treatment of bone defects for periodontal and implant therapy.