Decellularized Human Adipose Tissue as an Alternative Graft Material for Bone Regeneration.

BACKGROUND: Tissue engineering approaches to treat damaged bone include various tissue transplants such as autologous, allogeneic, and xenografts. Artificial materials have been widely introduced to meet the demand for graft materials, but insufficiency in supply is still not resolved. In this study, human adipose tissue, easily obtained from the human body, was harvested, and the tissue was decellularized to fabricate a decellularized human adipose tissue matrix (DM) as an alternative graft material. METHODS: Human adipose tissue was obtained via liposuction. The obtained fresh adipose tissue sample was cut into pieces then put into decellularization solution (1% antibiotic-antimycotic solution and 1% phenylmethanesulphonyl fluoride). Lipids were further removed via treatment in isopropanol. The sample was then subjected to another enzymatic digestion and lipid removal processes. The obtained decellularized adipose tissue matrix was lyophilized to form a graft material in disc shape. RESULTS: Decellularization was confirmed by nuclear staining methods and detection of RNA and DNA via PCR. Bone morphogenetic protein 2 (BMP2)-loaded DM showed the ability to form new bone tissue when implanted in subcutaneous tissue. In recovery of a mouse calvarial defect model, BMP2-loaded DM exhibited similar levels of bone tissue regeneration efficiency compared with a well-defined commercial product, BMP2-loaded CollaCote®. CONCLUSION: The DM developed in this study is expected to address the problem of insufficient supply of graft materials and contribute to the treatment of bone defects of critical size as an alternative bone graft material with preserved extracellular matrix components.

Semi-Rigid Fixation Using a Sliding Plate for Treating Fractures of the Mandibular Condylar Process.

Occlusal displacement often occurs after surgery for condylar process fractures because it is difficult to reduce these fractures precisely. However, performing semi-rigid fixation using a sliding plate may overcome this limitation. A retrospective clinical comparison between semi-rigid and rigid fixations was performed. Among 34 patients who had unilateral condylar process fractures, 17 were treated with rigid fixation and the remaining with semi-rigid fixation using a sliding plate. For all patients, panoramic radiographs were collected 1 day and 6 months after surgery. In these radiographs, ramus height and condylar process inclination were measured, and the differences between the fractured and normal sides were assessed. Additionally, the radiographic density of the fracture area was measured. Differences in surgical outcomes and operative times between the two groups and changes in postoperative deviations within each group were analyzed. There was no statistically significant difference in ramus height and condylar process inclination between the two groups at postoperative day 1 and 6 months. Radio-density was observed to be higher in the rigid fixation group, and it increased with time in both groups. The semi-rigid fixation group had a significantly shorter operative time than the other group did. Semi-rigid and rigid fixations showed no differences in terms of effectiveness and outcomes of surgery. In terms of operative time, semi-rigid fixation was superior to rigid fixation.

Micro-CT and Histomorphometric Study of Bone Regeneration Effect with Autogenous Tooth Biomaterial Enriched with Platelet-Rich Fibrin in an Animal Model.

The aim of this study was to evaluate the potential of tooth biomaterials as bone graft biomaterials for bone healing in rabbits. We prepared tooth biomaterial and platelet-rich fibrin (PRF) to fill the round-shaped defect in the skull of New Zealand white rabbits. These cranial defects were treated with different conditions as follows: group 1, a mixture of tooth biomaterials and platelet-rich fibrin (PRF); group 2, only tooth biomaterials; group 3, only PRF; and group 4, the unfilled control group. Specimens of the filled sites were harvested for analysis with microscopic computerized tomography (micro-CT) and histomorphology at 4 and 8 weeks. As a result of micro-CT, at 4 weeks, the bone volume percentages in groups 1 and 2 were 50.33 ± 6.35 and 57.74 ± 3.13, respectively, and that in the unfilled control group was 42.20 ± 10.53 (p = 0.001). At 8 weeks, the bone volume percentages in groups 1 and 2 were 53.73 ± 9.60 and 54.56 ± 8.44, respectively, and that in the unfilled control group was 37.86 ± 7.66 (p = 0.002). The difference between the experimental group 3 and the unfilled control group was not statistically significant. Histomorphologically, the total new bone was statistically different.

Microcomputed Tomography and Histological Study of Bone Regeneration Using Tooth Biomaterial with BMP-2 in Rabbit Calvarial Defects.

Our study was aimed to analyze the osteoinductive effect of powdered and block type autogenous bone graft along with bone morphogenetic protein (BMP-2) as compared to synthetic bone graft. Three circular bicortical defects were made in the calvaria of each rabbit and randomly divided into three groups as follows: powdered tooth biomaterial+BMP-2, block tooth biomaterial+BMP-2, and control group: synthetic bone+BMP-2. The samples taken from these defects after 4 and 8 weeks were analyzed histologically along with micro CT analysis. In our study, both powered and block type tooth autogenous bone graft successfully stimulated mesenchymal cells leading to endochondral ossification and bone regeneration. We observed that the powered bone graft material which is acid insoluble especially is preferable as a carrier for BMP-2.

Histomorphometric Evaluation of Socket Preservation Using Autogenous Tooth Biomaterial and BM-MSC in Dogs.

This study is aimed at assessing the dimensional alterations occurring in the alveolar bone after premolar extraction in dogs with histomorphometric and histological analysis. After atraumatic premolar extraction, tooth-derived bone graft material was grafted in the extraction socket of the premolar region in the lower jaws of six dogs in two experimental groups. In the second experimental group, BM-MSCs were added together with the graft. The control was left untreated on the opposite side. After twelve weeks, all six animals were sacrificed. Differences in alveolar bone height crests lingually and buccally, and alveolar bone width at 1, 3, and 5 mm infracrestally, were examined. Histologic study revealed osteoconductive properties of tooth biomaterial. A statistically significant difference was detected between the test and control groups. In the test groups, a reduced loss of vertical and horizontal alveolar bone dimensions compared with the control group was observed. Tooth bone graft material may be considered useful for alveolar ridge preservation after tooth extraction, as it could limit the natural bone resorption process.

Biosilicated collagen/ß-tricalcium phosphate composites as a BMP-2-delivering bone-graft substitute for accelerated craniofacial bone regeneration.

BACKGROUND: Bioceramic ß-tricalcium phosphate (ß-TCP) is used as a bone-grafting material and a therapeutic drug carrier for treatment of bone defects in the oral and maxillofacial regions due to the osteoconductivity and biocompatibility. However, the low mechanical strength and limited osteoinductivity of ß-TCP agglomerate restrict bone regenerating performance in clinical settings. METHODS: Herein, a biomimetic composite is proposed as a bone morphogenetic protein-2 (BMP-2)-delivering bone graft substitute to achieve a robust bone grafting and augmented bone regeneration. RESULTS: The sequential processes of brown algae-inspired biosilicification and collagen coating on the surface of ß-TCP enable the effective incorporation of BMP-2 into the coating layer without losing its bioactivity. The sustained delivery of BMP-2 from the biosilicated collagen and ß-TCP composites promoted in vitro osteogenic behaviors of pre-osteoblasts and remarkedly accelerated in vivo bone regeneration within a rat calvarial bone defect. CONCLUSIONS: Our multicomposite bone substitutes can be practically applied to improve bone tissue growth in bone grafting applications with further expansion to general bone tissue engineering.

Effect of Decompression on Jaw Cystic Lesions Based on Three-Dimensional Volumetric Analysis.

Background and objectives: This study aimed to evaluate the effectiveness of decompression on jaw cysts according to various parameters by volumetric analysis using three-dimensional computed tomography. Materials and methods: Fifty patients who underwent surgical decompression of the jaw cystic lesion were selected, and their preoperative and postoperative computed tomography results between 3 and 27 months were collected. Volumetric analysis was performed to evaluate any differences in the rate of volumetric change according to the sex, age, initial volume of the lesion, duration, location of the lesion, tooth extraction, expansion of the cortical layer, and pathological diagnosis. Multiple linear regression and generalised linear mixed models were used for statistical analyses. Results: The mean reduction rate among all patients was 54.68%. Multiple linear regression analysis revealed that higher reduction rates were associated with a long decompression period, young patient age, and location of the cyst in the posterior maxilla. Generalised linear mixed models revealed that higher reduction rates were associated with a long decompression period and young patient age. Conclusions: Decompression was an effective treatment for reducing the cyst size in all patients. Its effectiveness increased with a long treatment duration, young patient age, and cyst location in the posterior maxilla three-dimensionally.

Combined Delivery of Two Different Bioactive Factors Incorporated in Hydroxyapatite Microcarrier for Bone Regeneration.

BACKGROUND: The delivery of growth factors using a carrier system presents a promising and innovative tool in tissue engineering and dentistry today. Two of the foremost bioactive factors, bone morphogenetic protein-2 and vascular endothelial growth factor (VEGF), are widely applied using a ceramic scaffold. The aim of this study was to determine the use of hydroxyapatite microcarrier (MC) for dual delivery of osteogenic and angiogenic factors to accelerate hard tissue regeneration during the regenerative process. METHODS: Two MCs of different sizes were fabricated by emulsification of gelatin and alpha-tricalcium phosphate (α-TCP). The experimental group was divided based on the combination of MC size and growth factors. For investigating the in vitro properties, rat mesenchymal stem cells (rMSCs) were harvested from bone marrow of the femur and tibia. For in vivo experiments, MC with/without growth factors was applied into the standardized, 5-mm diameter defects, which were made bilaterally on the parietal bone of the rat. The animals were allowed to heal for 8 weeks, and samples were harvested and analyzed by micro-computed tomography and histology. RESULTS: Improved proliferation of rat mesenchymal stem cells was observed with VEGF loaded MC. For osteogenic differentiation, dual growth factors delivered by MC showed higher osteogenic gene expression, alkaline phosphatse production and calcium deposition. The in vivo results revealed statistically significant increase in new bone formation when dual growth factors were delivered by MC. Dual growth factors administered on a calcium phosphate matrix showed significantly enhanced osteogenic potential. CONCLUSION: We propose this system has potential clinical utility in providing solutions for craniofacial bone defects, with the added benefit of early availability.

The influence of bone graft procedures on primary stability and bone change of implants placed in fresh extraction sockets.

BACKGROUND: This study was to evaluate the effect of bone graft procedure on the primary stability of implants installed in fresh sockets and assess the vertical alteration of peri-implant bone radiographically. METHODS: Twenty-three implants were inserted in 18 patients immediately after tooth extraction. The horizontal gap between the implant and bony walls of the extraction socket was grafted with xenografts. The implant stability before and after graft procedure was measured by Osstell Mentor as implant stability quotient before bone graft (ISQ bbg) and implant stability quotient after bone graft (ISQ abg). Peri-apical radiographs were taken to measure peri-implant bone change immediately after implant surgery and 12 months after implant placement. Data were analyzed by independent t test; the relationships between stability parameters (insertion torque value (ITV), ISQ abg, and ISQ bbg) and peri-implant bone changes were analyzed according to Pearson correlation coefficients. RESULTS: The increase of ISQ in low primary stability group (LPSG) was 6.87 ± 3.62, which was significantly higher than the increase in high primary stability group (HPSG). A significant correlation between ITV and ISQ bbg (R = 0.606, P = 0.002) was found; however, age and peri-implant bone change were not found significantly related to implant stability parameters. It was presented that there were no significant peri-implant bone changes at 1 year after bone graft surgery. CONCLUSIONS: Bone graft procedure is beneficial for increasing the primary stability of immediately placed implants, especially when the ISQ of implants is below 65 and that bone grafts have some effects on peri-implant bone maintenance.

Effect of BMP-2 Delivery Mode on Osteogenic Differentiation of Stem Cells.

Differentiation of stem cells is an important strategy for regeneration of defective tissue in stem cell therapy. Bone morphogenetic protein-2 (BMP-2) is a well-known osteogenic differentiation factor that stimulates stem cell signaling pathways by activating transmembrane type I and type II receptors. However, BMPs have a very short half-life and may rapidly lose their bioactivity. Thus, a BMP delivery system is required to take advantage of an osteoinductive effect for osteogenic differentiation. Previously, BMP delivery has been designed and evaluated for osteogenic differentiation, focusing on carriers and sustained release system for delivery of BMPs. The effect of the delivery mode in cell culture plate on osteogenic differentiation potential was not evaluated. Herein, to investigate the effect of delivery mode on osteogenic differentiation of BM-MSCs in this study, we fabricated bottom-up release and top-down release systems for culture plate delivery of BMP-2. And also, we selected Arg-Gly-Asp- (RGD-) conjugated alginate hydrogel for BMP-2 delivery because alginate is able to release BMP-2 in a sustained manner and it is a biocompatible material. After 7 days of culture, the bottom-up release system in culture plate significantly stimulated alkaline phosphate activity of human bone marrow-mesenchymal stem cells. The present study highlights the potential value of the tool in stem cell therapy.

Demineralized dentin matrix combined with recombinant human bone morphogenetic protein-2 in rabbit calvarial defects.

OBJECTIVES: The aim of this study was to compare the osteogenic effects of demineralized dentin matrix (DDM) combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) in rabbit calvarial defects with DDM and anorganic bovine bone (ABB) combined with rhBMP-2. MATERIALS AND METHODS: Four round defects with 8-mm diameters were created in each rabbit calvaria. Each defect was treated with one of the following: 1) DDM, 2) ABB/rhBMP-2, or 3) DDM/rhBMP-2. The rhBMP-2 was combined with DDM and ABB according to a stepwise dry and dip lyophilizing protocol. Histological and microcomputed tomography (µCT) analyses were performed to measure the amount of bone formation and bone volume after 2- and 8-week healing intervals. RESULTS: Upon histological observation at two weeks, the DDM and ABB/rhBMP-2 groups showed osteoconductive bone formation, while the DDM/rhBMP-2 group showed osteoconductive and osteoinductive bone formation. New bone formation was higher in DDM/rhBMP-2, DDM and ABB decreasing order. The amounts of bone formation were very similar at two weeks; however, at eight weeks, the DDM/rhBMP-2 group showed a two-fold greater amount of bone formation compared to the DDM and ABB/rhBMP-2 groups. The µCT analysis showed markedly increased bone volume in the DDM/rhBMP-2 group at eight weeks compared with that of the DDM group. Notably, there was a slight decrease in bone volume in the ABB/rhBMP-2 group at eight weeks. There were no significant differences among the DDM, ABB/rhBMP-2, and DDM/rhBMP-2 groups at two or eight weeks. CONCLUSION: Within the limitations of this study, DDM appears to be a suitable carrier for rhBMP-2 in orthotopic sites.

Histological analysis of in vitro co-culture and in vivo mice co-transplantation of stem cell-derived adipocyte and osteoblast.

Many researchers have focused on the role of adipocytes in increasing efficient bone tissue engineering and osteogenic differentiation of stem cells. Previous reports have not reached a definite consensus on whether adipocytes positively influence in vitro osteogenic differentiation and in vivo bone formation. We investigated the adipocyte influence on osteogenic differentiation from adipose-derived stromal cells (ADSCs) and bone formation through histological analysis in vitro and in vivo. Using the direct co-culture system, we analyzed the influence of adipocytes to promote the differentiation fate of ADSCs. Using co-transplantation of ADSC-derived adipocytes and osteoblasts into the dorsal region of mice, the osteogenesis and bone quality were determined by histological morphology, radiography, and the measurement of the Ca2+ concentration. The adipocyte negatively affected the osteoblast differentiation of ADSCs in the in vitro system and induced osteogenesis of osteoblasts in the in vivo system through co-transplantation. Interestingly, in the co-transplanted adipocytes and osteoblasts, the bone formation areas decreased in the osteoblast only group compared with the mixed adipocytes and osteoblast group 6 weeks after transplantation. Conversely, co-transplantation and osteoblast transplantation had similar degrees of calcification as observed from radiography analysis and the measurement of the Ca2+ concentrations. Our results revealed that adipocytes inhibited osteoblast differentiation in vitro but enhanced the efficacy of osteogenesis in vivo. In addition, the adipocytes controlled the activity of osteoclasts in the newly formed bone tissue. Our approach can be used to reconstruct bone using stem cell-based tissue engineering and to enhance the understanding of the role adipocytes play.

Combination of nanoparticles with photothermal effects and phase-change material enhances the non-invasive transdermal delivery of drugs.

We describe a promising non-invasive transdermal delivery system comprising block copolymer composite micelles that contained a phase-change material (PCM), photothermal Au nanoparticles (AuNPs), and hydrophobic drugs in the core. To minimize cell toxicity, we developed block copolymer micelles with a poly(ɛ-caprolactone) (PCL) biodegradable core and a hyperbranched polyglycol (hbPG) shell. The hbPG block formed micelles at a low-molecular-weight fraction of a low-molecular-weight block copolymer. The composite micelles showed excellent biocompatibility with cell viability at high concentrations. Visible light irradiation (λ=520 nm) of the composite micelles induced the photothermal effects of the AuNPs and melting of the PCM (lauric acid); hence, the drugs were released along with the PCM liquid. The release rate was controlled by the light intensity. Based on in vitro and in vivo skin penetration studies, the skin permeability of the drug remarkably improved under mild light irradiation (18 J/cm(2)) that was much lower than the dose that causes skin damage.

Cathepsin B Imaging to Predict Quality of Engineered Cartilage.

Cathepsin B (CB)-specific molecular imaging probe is applied to monitor the changes of CB expression in 3D cultured chondrocytes during the chondrogenesis. The probe is synthesized with a CB-cleavable peptide linked to a near infrared fluorescence (NIRF) dye and a dark quencher, which can recover the NIRF signal in the presence of CB enzyme. The CB activities in two different sets of chondrocytes are comparatively analyzed. The chondrocytes with higher CB activity show more extensive area of chondrogenesis that CB molecular imaging directly reflects the chondrogenic potency of the cells. The CB probe is expected to provide a reliable prediction for the quality of engineered cartilage by visualizing the activity of the relevant enzyme in chondrocytes.

Combination therapy with BMP-2 and BMSCs enhances bone healing efficacy of PCL scaffold fabricated using the 3D plotting system in a large segmental defect model.

The three-dimensional (3D) plotting system is a rapidly-developing scaffold fabrication method for bone tissue engineering. It yields a highly porous and inter-connective structure without the use of cytotoxic solvents. However, the therapeutic effects of a scaffold fabricated using the 3D plotting system in a large segmental defect model have not yet been demonstrated. We have tested two hypotheses: whether the bone healing efficacy of scaffold fabricated using the 3D plotting system would be enhanced by bone marrow-derived mesenchymal stem cell (BMSC) transplantation; and whether the combination of bone morphogenetic protein-2 (BMP-2) administration and BMSC transplantation onto the scaffold would act synergistically to enhance bone regeneration in a large segmental defect model. The use of the combined therapy did increase bone regeneration further as compared to that with monotherapy in large segmental bone defects.

Proteasome inhibition promotes functional recovery after peripheral nerve reperfusion injury.

BACKGROUND: The proteasome degrades NF-kappaB blocking protein (I-kappaB) and activates NF-kappaB that plays as a key transcriptional factor to regulate inflammatory factors that are involved in the tissue reperfusion injury. This study was designed to assess whether the proteasome inhibitor can attenuate peripheral nerve ischemia/reperfusion (I/R) injury and consequently promote motor functional recovery after ischemic insult. METHODS: Rat sciatic nerves were exposed to 2 hour of ischemia followed by various periods of reperfusion. Rats were administered either proteasome inhibitor (bortezomib) or phosphate-buffered saline 30 minutes before reperfusion start. Results were evaluated using a walking track test, and an isolated muscle contraction test, and by muscle weight, and histology. RESULTS: Bortezomib treatment induced an earlier improvement in sciatic functional index and a more rapid restoration of contractile force and wet weight of extensor digitorum longus muscle. Bortezomib reduced early axonal degeneration and promoted regeneration. CONCLUSION: This study indicates that bortezomib; a proteasome inhibitor, is effective at promoting the functional recovery of reperfused peripheral nerve. The proteasome inhibition may play a role as one of the clinical strategy in the peripheral nervous system I/R injury with further understanding its mechanism of action.

Flavonoids purified from Rhus verniciflua Stokes actively inhibit cell growth and induce apoptosis in human osteosarcoma cells.

Many studies have suggested that dietary flavonoids are anticancer agents that induce the apoptosis of cancer cells. However, the effects of flavonoids on the induction of apoptosis in osteosarcoma cells are unclear. Previously, a flavonoid fraction, consisting mainly of protocatechuic acid, fustin, fisetin, sulfuretin, and butein, herein named RCMF (the RVS chloroform-methanol fraction), was prepared from a crude acetone extract of Rhus verniciflua Stokes (RVS). This study evaluated the effects of RCMF on the proliferation and apoptosis using human osteosarcoma (HOS) cells. The mechanism of growth inhibition of the HOS cells by the flavonoid fraction, RCMF, was also assessed. The results demonstrated that RCMF exhibited sensitive growth inhibition and induced apoptosis in HOS cells. PARP cleavage was closely associated with the RCMF-induced apoptosis of the HOS cells. Furthermore, the activation of caspase 8 and Bax, the inhibition of Bcl-2 expression, and the release of cytochrome c are believed to be involved in the RCMF-mediated apoptosis. Collectively, these findings suggest that RCMF is an agent which may be capable of inducing sensitive growth inhibition and apoptosis in HOS cells.

Selective antiproliferative and apoptotic effects of flavonoids purified from Rhus verniciflua Stokes on normal versus transformed hepatic cell lines.

Considerable attention is being concentrated on dietary flavonoids in developing novel cancer-preventive approaches due to their potential ability to induce selective apoptosis of cancer cells. In this study, we prepared a flavonoid-containing fraction from a crude acetone extract of Rhus verniciflua Stokes (RVS), traditionally used as a food additive and as an herbal medicine, and named RVS chloroform-methanol fraction (RCMF). We evaluated the effects of RCMF on proliferation and apoptosis using mouse embryonic primary hepatic cells (MPHC), embryonic normal hepatic cell line (BNL CL.2), and its SV40-mediated transformed cell line (BNL SV A.8). We also investigated the effects of RCMF on the antioxidant defense system in those cells. This study demonstrated that RCMF exhibited a selective growth inhibition and apoptosis induction on transformed cells. BNL SV A.8 cells were more sensitive to RCMF-mediated cytotoxicity than were MPHC or BNL CL.2. RCMF-mediated reduction of MnSOD activity and glutathione (GSH) content in BNL SV A.8 cells is thought to be associated with RCMF-induced apoptosis. Our findings suggest that RCMF is an agent which may be capable of inducing growth inhibition and apoptosis of hepatic tumor cells.

Induction of antigen-specific systemic and mucosal immune responses by feeding animals transgenic plants expressing the antigen.

A report from that the presence of lactogenic immunity in pigs protected suckling piglets from porcine epidemic diarrhea virus (PEDV) infection suggested that inducing mucosal immune responses in lactating pigs is an effective way of protecting swine from PEDV infection. In this study, we developed transgenic tobacco plants that express the antigen protein corresponding to the neutralizing epitope of PEDV spike protein, and tested whether feeding the plants to pigs induced an effective immune response against PEDV infection. First, we confirmed the immunogenicity of the plant-derived antigen by using a plaque reduction neutralization assay with serum obtained after injecting mice with protein extracted from the transgenic plants. Feeding the transgenic plants to mice induced both systemic and mucosal immune responses against the antigen. The induced antibodies inhibited virus infection in the plaque reduction neutralization assay. These results suggest that feeding animals transgenic plants carrying antigen genes is an effective strategy to induce protective immune responses against PEDV infection.