Does Stocking Density Affect Growth Performance and Hematological Parameters of Juvenile Olive Flounder Paralichthys olivaceus in a Recirculating Aquaculture System?

Olive flounder Paralichthys olivaceus is a representative culture species in South Korea. Recirculating aquaculture systems (RASs) have received increased attention because they can provide sustainable and environmentally friendly productivity. However, to maintain economic sustainability, the system generally requires high productivity, achieved through a high stocking density, which compromises animal welfare. The reduction in growth based on the stocking density may be due to the social hierarchy resulting from the growth suppression of subordinate individuals. Species, size, culture systems, and other management regimes can affect the social hierarchy. Therefore, a more practical approach must be taken to adjust the stocking density for a particular fish species and fish size in a specific culture system. This study investigated the effect of stocking density on juvenile olive flounder in an RAS. Juvenile olive flounder (61.0 ± 0.3 g) were initially stocked at 3.29, 4.84, 7.14, and 8.56 kg/m2 (T1, T2, T3, and T4, respectively). After 8 weeks, growth performance, in terms of feed conversion, specific growth rate, and daily feed intake rate, was measured. In addition, the blood levels of insulin-like growth factor (IGF-1), growth hormone, glucose, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase and the whole-body composition were evaluated as stress indicators. Growth performance increased as stocking density increased, but fish at the highest stocking density showed signs of growth reduction toward the end of the experiment. In addition, as stocking density increased, IGF-1 decreased, and cortisol increased. The whole-body protein level was significantly lower in T4 compared with the other treatments. Olive flounder seemed to tolerate a stocking density up to 20.16 kg/m2. Based on the growth performance, hematological response, and whole-body composition results in the present study, a final stocking density of 20 kg/m2 (from the initial stocking density of 4.84-7.14 kg/m2) may be desirable for juvenile olive flounder in an RAS for fish weighing ~60-180 g.

A rapamycin derivative, biolimus, preferentially activates autophagy in vascular smooth muscle cells.

Although rapamycin is a well-known conformational inhibitor of mTORC1, it is now widely used for treating arterial restenosis. Various rapamycin analogues (rapalogue) have been made for applying to drug-eluting stents. Here we show that two major rapalogues, everolimus and biolimus, exert a differential effect on the mTORC1-mediated signaling pathways in vascular smooth muscle cells. In balloon-injured carotid arteries, both rapalogues strongly inhibit neointimal hyperplasia. Signaling pathway analyses reveal that everolimus exert cytotoxicity by increasing cellular reactive oxygen species and consequently reduce energy metabolism. By contrast, biolimus confers a preferential induction of autophagy by more strongly activating major autophagy regulator, ULK1, in vascular smooth muscle cells than everolimus does. As a consequence, the implantation of biolimus-eluting stent reduces endothelial loss, which in turn reduces inflammation, in porcine coronary arteries. Thus, this study reveals that a chemical derivatization can cause a change among mTORC1-dependent signaling pathways in vascular smooth muscle cells, thereby enabling to elicit a differential efficacy on arterial restenosis.

Generation of an rhBMP-2-loaded beta-tricalcium phosphate/hydrogel composite and evaluation of its efficacy on peri-implant bone formation.

Dental implant insertion on a site with low bone quality or bone defect should be preceded by a bone graft or artificial bone graft insertion to heal the defect. We generated a beta-tricalcium phosphate (ß-TCP) and poloxamer 407-based hydrogel composite and penetration of the ß-TCP/hydrogel composite into the peri-implant area of bone was evaluated by porous bone block experiments. The maximum penetration depth for porous bone blocks and dense bone blocks were 524 µm and 464 µm, respectively. We report the in-vivo performance of a composite of ß-TCP/hydrogel composite as a carrier of recombinant human bone morphogenetic protein (rhBMP-2), implanted into a rabbit tibial defect model. Three holes drilled into each tibia of eight male rabbits were (1) grafted with dental implant fixtures; (2) filled with ß-TCP/hydrogel composite (containing 5 µg of rhBMP-2), followed by grafting of the dental implant fixtures. Four weeks later, bone-implant contact ratio and peri-implant bone formation were analyzed by radiography, micro-CT and histology of undecalcified specimens. The micro-CT results showed a significantly higher level of trabecular thickness and new bone and peri-implant new bone formation in the experimental treatment compared to the control treatment. Histomorphometry revealed a significantly higher bone-implant contact ratio and peri-implant bone formation with the experimental treatment. The use of ß-TCP/poloxamer 407 hydrogel composite as a carrier of rhBMP-2 significantly promoted new bone formation around the dental implant fixture and it also improved the quality of the new bone formed in the tibial marrow space.

Fabrication of an rhBMP-2 loaded porous ß-TCP microsphere-hyaluronic acid-based powder gel composite and evaluation of implant osseointegration.

Methods to improve osseointegration that include implantation of rhBMP-2 with various kinds of carriers are currently of considerable interest. The present study was conducted to evaluate if the rhBMP-2 loaded ß-TCP microsphere-hyaluronic acid-based powder-like hydrogel composite (powder gel) can act as an effective rhBMP-2 carrier for implantation in host bone with a bone defect or poor bone quality. The release pattern for rhBMP-2 was then evaluated against an rhBMP-2-loaded collagen sponge as a control group. Dental implants were also inserted into the tibias of three groups of rabbits: an rhBMP-2 (200 µg) loaded powder gel composite implanted group, an implant only group, and a powder gel implanted group. Micro-CT and histology of the implanted areas were carried out four weeks later. The rhBMP-2 powder gel released less rhBMP-2 than the collagen sponge, but it continued a slow release for more than 7 days. The rhBMP-2 powder gel composite improved osseointegration of the dental implant by increasing the amount of new bone formation in the implant pitch and it improved the bone quality and bone quantity of new bone. The histology results indicated that the rhBMP-2 powder gel composite improved the osseointegration in the cortical bone as well as the marrow space along the fixture. The bone-to-implant contact ratio of the rhBMP-2 (200 µg) loaded powder gel composite implanted group was significantly higher than those of the implant only group and the powder gel implanted group. The powder gel appeared to be a good carrier and could release rhBMP-2 slowly to promote the formation of new bone following implantation in a bone defect, thereby improving implant osseointegration.

Negative effect of rapidly resorbing properties of bioactive glass-ceramics as bone graft substitute in a rabbit lumbar fusion model.

BACKGROUND: Bioactive glass-ceramics have the ability to directly bind to bones and have been widely used as bone graft substitutes due to their high osteoconductivity and biocompatibility. CaO-SiO2-P2O5-B2O3 glass-ceramics are known to have good osteoconductivity and are used as bone graft extenders. METHODS: This study aimed to evaluate the effects of the resorbing properties of glass-ceramics in bone fusion after producing and analyzing three types of CaO-SiO2-P2O5-B2O3 glass-ceramics with high osteoconductivity that had enhanced resorption by having an increased B2O3 content. The three types of CaO-SiO2-P2O5-B2O3 glass-ceramics with B2O3 contents of 8.0, 9.0, and 9.5 weight % were designated and grouped as P20B80, P10B90, and P5B95, respectively. Glass-ceramic types were tested for fusion rates and bone formation by employing the lumbar 5-6 intertransverse process fusion model in 51 New Zealand male rabbits. Bioactivity was assessed by soaking in simulated body fluid (SBF). RESULTS: In vitro study results showed sufficient hydroxycarbonate apatite layer formation occurred for P20B80 in1 day, for P10B90 in 3 days, and for P5B95 in 5 days after soaking in SBF. For the rabbit lumbar spine posterolateral fusion model, the autograft group recorded a 100% fusion rate with levels significantly higher than those of P20B80 (29.4%), P10B90 (0%), and P5B95 (14.3%), with high resorbing properties. Resorbing property differences among the three glass-ceramic groups were not significant. Histological results showed new bone formation confirming osteoconductivity in all three types of glass-ceramics. Radiomorphometric results also confirmed the resorbing properties of the three glass-ceramic types. CONCLUSIONS: The high resorbing properties and osteoconductivity of porous glass-ceramics can be advantageous as no glass-ceramics remain in the body. However, their relatively fast rate of resorption in the body negatively affects their role as an osteoconductive scaffold as glass-ceramics are resorbed before bony fusion.

The effects of recombinant human bone morphogenetic protein-2-loaded tricalcium phosphate microsphere-hydrogel composite on the osseointegration of dental implants in minipigs.

Bone formation in tooth defect areas and the osseointegration of dental implants are very important for successful dental implant surgery. The aim of the present study was to assess the strengthening effect of a ß-TCP microsphere-hydrogel composite containing recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone healing and implant osseointegration. The molars and premolars on the left and right sides of the maxilla were extracted from six male minipigs, and dental implants were placed using either the ß-TCP microsphere-hydrogel carrier alone or the carrier loaded with rhBMP-2 (500 µg). The animals were kept alive for a further 8 weeks. The molars and premolars from the left and the right sides of the mandibles of another six minipigs were extracted, and the animals were kept alive for 4 weeks. Two 5-mm-diameter bone defects were then made on both sides of the mandible. The defects were filled with saline, ß-TCP microsphere-hydrogel carrier, or the carrier loaded with rhBMP-2 (300 µg), and dental implant fixtures were inserted. The animals were kept alive for a further 4 weeks. Bone formation was examined using plane radiographs, micro-CT, and the histology of undecalcified specimens. The group treated with the rhBMP-2-loaded carrier composite showed a significantly higher percentage bone volume and a greater trabecular thickness for the newly formed bone in the tooth defect areas when compared to the group treated with the carrier alone. The rhBMP-2 group had a significantly higher osseointegration, a larger percentage bone volume, greater trabecular thickness in the newly formed bone in tooth defect areas, a larger newly formed bone fraction in the fixture pitch, and a greater number of newly formed trabecular bones when compared to the other groups. We confirmed that the rhBMP-2-loaded carrier composite promotes new bone formation after tooth extraction and strengthens osseointegration of dental fixtures by improving the degree of osseointegration around the dental implant fixture.

Finite element modeling of stress distribution in intervertebral spacers of different surface geometries.

Intervertebral disc spacers using bioactive ceramics have been used to treat degenerative spinal disease. Tooth-shaped spacers are commonly used to prevent migration, but there is a possibility of fracture when inserted or after insertion. Intervertebral disc spacers with either an isosceles triangle-shaped tooth (T1) or a right triangle-shaped tooth (T2) were used as a control group. The design factors for the experimental group were modified to prevent fractures induced by stress concentration, and the surfaces of the spacers were designed as either an isosceles triangle-shaped valley (V1) or a right triangle-shaped valley (V2). Linear analysis using finite element model (FEM) was performed, and Von Mises stress distribution was calculated by applying 1000 N of uniformly distributed load. Samples of the V2 design were made with bioactive glass-ceramics (BGS-7) and evaluated for compressive strength, fatigue degree, and impact strength. Von Mises stress was highest at the first tooth from the posterior side for the control group and at the center for the experimental group. Compared with the control group, the experimental group showed 18.4% and 82.5% reduction (V1 vs. T1 and V2 vs. T2, respectively) in the maximum stress at the bottom of the valleys. The FEM analysis revealed that the V2 design had the most even load distribution. The V2 samples with bioactive glass-ceramics were evaluated for compressive strength, and all six samples were not fractured up to 24 000 N. However, the average impact strength was 19.42 kN, suggesting that momentary force caused damage at a lower load than compression with a steady speed. The BGS-7 intervertebral disc spacer with V2 design was not fractured during the fatigue test at maximum pressure of 8000 N, R ≥10, 5 Hz, and 5 million cycles. These data confirm that the BGS-7 spacer with the V2 design may be clinically applicable. Collectively, the modified surface geometry of the experimental group significantly lowered Von Mises stress values at the bottom of the valleys, and thus the possibility of fracture by compressive load was greatly reduced. Also, impact during insertion was confirmed to cause fracture more easily, as the impact strength was lower than the compressive strength in the experimental group.

Effects of porous beta-tricalcium phosphate-based ceramics used as an E. coli-derived rhBMP-2 carrier for bone regeneration.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) requires carriers for clinical effectiveness. In this study, whether porous beta-tricalcium phosphate (ß-TCP)-based ceramics are ideal carriers for rhBMP-2 was investigated. Hydroxyapatite (HA), ß-TCP, TCP/HA (80 %/20 %), HA with rhBMP-2, TCP with rhBMP-2, and TCP/HA (80 %/20 %) with rhBMP-2 were manufactured by a sponge method with a pore size of 300 µm or more and macro-porosity of 83 %. The alkaline phosphatase (ALP) activity and ALP expression of the cells with 100 % ß-TCP granules were more increased than the those of cells with 100 % HA and TCP/HA (80 %/20 %) at the baseline or when treated with 15 ng/ml of rhBMP-2. In an SD rat calvarial defect model, new bone formation was evidently shown in the TCP 100 %-rhBMP-2 and TCP/HA (80 %/20 %)-rhBMP-2 groups, showing that the most affected area was filled with newly-formed bone, that the percent bone volume and trabecular number were larger when compared to the groups without rhBMP-2 treatment at both 4 and 8 weeks after surgery using micro-CT and histology. Porous TCP-based ceramic granules enhanced the osteoblastic differentiation in the hMSC system when treated with 15 ng/ml of rhBMP-2 and accelerated bone-healing by trabecular number in a rat calvarial defect model. Thus, in this study it was proposed that TCP-based ceramics might be useful carriers of rhBMP-2.

In vivo evaluation of CaO-SiO2-P2O5-B2O3 glass-ceramics coating on Steinman pins.

Surface coating using ceramics improves the bone bonding strength of an implant. We questioned whether a new type of glass-ceramics (BGS-7) coating (CaO-SiO2 -P2 O5 -B2 O3 ) would improve the osseointegration of Steinman pins (S-pins) both biomechanically and histomorphometrically. An in vivo study was performed using rabbits by inserting three S-pins into each iliac bone. The pins were 2.2-mm S-pins with a coating of 30-µm-thick BGS-7 and 550-nm-thick hydroxyapatite (HA), as opposed to an S-pin without coating. A tensile strength test and histomorphometrical evaluation was performed. In the 2-week group, the BGS-7 implant showed a significantly higher tensile strength than the S-pin. In the 4- and 8-week groups, the BGS-7 implants had significantly higher tensile strengths than the S-pins and HA implants. The histomorphometrical study revealed that the BGS-7 implant had a significantly higher contact ratio than the S-pin and HA implants in the 4-week group. The biomechanical and histomorphometrical tests showed that the BGS-7 coating had superior bone bonding properties than the groups without the coating from the initial stage of insertion. The BGS-7 coating of an S-pin will enhance the bone bonding strength, and there might also be an advantage in human bone bonding.

Fabrication and evaluation of osteoblastic differentiation of human mesenchymal stem cells on novel CaO-SiO2-P2O5-B2O3 glass-ceramics.

Apatite-wollastonite glass-ceramics have high mechanical strength, and CaO-SiO2 -B2 O3 glass-ceramics showed excellent bioactivity and high biodegradability. A new type of CaO-SiO2 -P2 O5 -B2 O3 system of bioactive glass-ceramics (BGS-7) was fabricated, and the effect and usefulness was evaluated via bioactivity using simulated body fluid and human mesenchymal stem cells (hMSCs). The purpose of this study was to compare BGS-7 and hydroxyapatite (HA) using hMSCs in order to evaluate the bioactivity of BGS-7 and its possibility as a bone graft extender. Alkaline phosphatase (ALP) staining, ALP activity, cell proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, Alizarin Red-S (AR-S) staining, calcium levels, the mRNA expression of ALP, osteocalcin, osteopontin, and runt-related transcription factor 2 (runx-2) using reverse-transcription polymerase chain reaction (RT-PCR) and the protein expression of osteocalcin and runx-2 using Western blot were measured by transplanting hMSC onto a tissue culture plate, HA, and BGS-7. The ALP staining and AR-S staining of BGS-7 was greater than that of HA and control. The ALP value of BGS-7 was significantly higher than that of HA and control. The MTS results showed that BGS-7 had a higher value than the groups transplanted onto HA and control on day 15. The calcium level was higher than the control in both HA and BGS-7, and was especially high in BGS-7. There were more mineral products on BGS-7 than on the HA when analyzed by scanning electron microscopy. The mRNA expression of ALP, osteopontin, osteocalcin, and runx-2 were higher on BGS-7 than on HA and the control when analyzed by RT-PCR. The relative gene expression of osteopontin and runx-2 were found to be higher on BGS-7 than on HA and the control by Western blot. Accordingly, it is predicted that BGS-7 would have high biocompatibility and good osteoconductivity, and presents a possibility as a new bone graft extender.

Fabrication and evaluation of porous beta-tricalcium phosphate/hydroxyapatite (60/40) composite as a bone graft extender using rat calvarial bone defect model.

Beta-tricalcium phosphate ( ß -TCP) and hydroxyapatite (HA) are widely used as bone graft extenders due to their osteoconductivity and high bioactivity. This study aims to evaluate the possibility of using porous substrate with composite ceramics ( ß -TCP: HA = 60% : 40%, 60TCP40HA) as a bone graft extender and comparing it with Bio-Oss. Interconnectivity and macroporosity of ß -TCP porous substrate were 99.9% and 83%, respectively, and the macro-porosity of packed granule after crushing was 69%. Calvarial defect model with 8 mm diameter was generated with male Sprague-Dawley rats and 60TCP40HA was implanted. Bio-Oss was implanted for a control group and micro-CT and histology were performed at 4 and 8 weeks after implantation. The 60TCP40HA group showed better new bone formation than the Bio-Oss group and the bone formation at central area of bone defect was increased at 8 weeks in micro-CT and histology. The percent bone volume and trabecular number of the 60TCP40HA group were significantly higher than those of Bio-Oss group. This study confirms the usefulness of the porous 60TCP40HA composite as a bone graft extender by showing increased new bone formation in the calvarial defect model and improved bone formation both quantitatively and qualitatively when compared to Bio-Oss.

Bioactive ceramic coating of cancellous screws improves the osseointegration in the cancellous bone.

BACKGROUND: A number of methods for coating implants with bioactive ceramics have been reported to improve osseointegration in bone, but the effects of bioactive ceramic coatings on the osseointegration of cancellous screws are not known. Accordingly, biomechanical and histomorphometric analyses of the bone-screw interface of uncoated cancellous screws and cancellous screws coated with four different bioactive ceramics were performed. METHODS: After coating titanium alloy cancellous screws with calcium pyrophosphate (CPP), CaO-SiO(2)-B(2)O(3) glass-ceramics (CSG), apatite-wollastonite 1:3 glass-ceramics (W3G), and CaO-SiO(2)-P(2)O(5)-B(2)O(3) glass-ceramics (BGS-7) using an enameling method, the coated and the uncoated screws were inserted into the proximal tibia and distal femur metaphysis of seven male mongrel dogs. The torque values of the screws were measured at the time of insertion and at removal after 8 weeks. The bone-screw contact ratio was analyzed by histomorphometry. RESULTS: There was no significant difference in the insertion torque between the uncoated and coated screws. The torque values of the CPP and BGS-7 groups measured at removal after 8 weeks were significantly higher than those of the uncoated group. Moreover, the values of the CPP and BGS-7 groups were significantly higher than the insertion torques. The fraction of bone-screw interface measured from the undecalcified histological slide showed that the CPP, W3G, and BGS-7 groups had significantly higher torque values in the cortical bone area than the uncoated group, and the CPP and BGS-7 groups had significantly higher torque values in the cancellous bone area than the uncoated group. CONCLUSIONS: In conclusion, a cancellous screw coated with CPP and BGS-7 ceramic bonds directly to cancellous bone to improve the bone-implant osseointegration. This may broaden the indications for cancellous screws by clarifying their contribution to improving osseointegration, even in the cancellous bone area.

A 90-day intravenous administration toxicity study of CaO-SiO2-P2O5-B2O3 glass-ceramics (BGS-7) in rat.

CaO-SiO(2)-P(2)O(5)-B(2)O(3) glass ceramics directly bond to bone and have potential use as a bone substitute material. The present study evaluated the toxicity from subchronic intravenous administration of CaO-SiO(2)-P(2)O(5)-B(2)O(3) glass ceramics to male and female Sprague-Dawley rats. The rats were divided into four groups, each consisting of 10 male and 10 female rats, and administered different amounts of CaO-SiO(2)-P(2)O(5)-B(2)O(3) glass ceramics (aqueous extract 5, 2.5, and 1.25 mL/kg body weight/day and saline) 7 days per week for 90 consecutive days. During the experiment, no deaths were observed in any groups, and there were no remarkable changes in clinical signs, body weight, food and water intake, hematological and serum biochemical parameters, organ weight, and histopathological findings between the control and treated groups. The results show no adverse toxic effects of CaO-SiO(2)-P(2)O(5)-B(2)O(3) glass ceramics (aqueous extract 5 mL/kg body weight/day) to rats of either sex.

In vivo study of novel biodegradable and osteoconductive CaO-SiO2-B2O3 glass-ceramics.

To evaluate the possibility of novel CaO-SiO2-B2O3 glass-ceramics (CS10B) as a new bone replacement material, we compared the biodegradation and osteoconduction properties of CS10B, hydroxyapatite (HA), and tricalcium phosphate (TCP). Porous CS10B implants were prepared by the polymer sponge method. L5-6 single-level posterolateral spinal fusions were performed on 30 New Zealand white male rabbits. The animals were divided into three groups by implant material: CS10B, HA, and TCP. Radiographs were performed every 2 weeks. All animals were euthanized 12 weeks after surgery. The ratio of the area occupied by the ceramics by final and initial radiographs was calculated using radiomorphometric analysis. Uniaxial tensile strength was determined from seven cases in each group. The ratio of the area occupied by HA (88.7%+/-16.1%) was significantly higher than the others (p<0.005), and the ratio of the area occupied by CS10B (28.2%+/-9.3%) was significantly lower than those of HA and TCP (37%+/-9.6%, p<0.05). The mean values of the tensile strengths of the CS10B (182.7+/-19.9 N) and HA (191.4+/-33.5 N) were significantly higher (p<0.05) than that of TCP (141.1+/-28.2 N). CS10B had a fusion mass tensile strength similar to that of HA. Histological analysis confirmed that CS10B was well incorporated into the fusion mass. These findings suggest that CS10B is a possible bone replacement material.

Novel bioactive and biodegradable glass ceramics with high mechanical strength in the CaO--SiO2--B2O3 system.

Novel bioactive and biodegradable glass ceramics with high mechanical strength in the (50-x/2)CaO. SiO(2)--xB(2)O(3) (4.2 < or = x < or = 17.2) system were investigated. The systems consisted of three phases: monoclinic wollastonite, calcium metaborate, and amorphous borosilicate matrix. The glass ceramics containing 4.2 mol% and 8.4 mol% B(2)O(3) showed high bulk density and a dense microstructure. Mechanical strengths of the glass ceramics were higher than those of other bioactive ceramics: high compressive strength (2813 MPa), bending strength of 212 MPa, and fracture toughness of 3.12 MPa. m(1/2). The glass-ceramic formed apatite layer on their surface in the simulated body fluid and showed significant biodegradation. The degree of apatite formation in the glass ceramics depended on the calcium metaborate content and borosilicate glassy matrix. Additional calcium metaborate and borosilicate glassy matrix increased the apatite formation rate on the surface. It might be likely that calcium metaborate causes supersaturation of Ca ions, for its high solubility in SBF and the water-reactive borosilicate glassy matrix formed Sibond;OH groups on the surface to provide nucleation sites for apatite formation. Also, through in vitro test for the biocompatibility of the CaO--SiO(2)--B(2)O(3) glass ceramics, no cytotoxicity of the glass ceramics were found. The results on bioactivity and noncytotoxicity indicated that glass ceramics in the (50-x/2)CaO. SiO(2)--xB(2)O(3) (4.2 < or = x < or = 17.2) system could be useful as a biodegradable bone replacement material.