MANUFACTURING METHOD OF MAGNETIC NANOFIBER SCAFFOLD WITH IMPROVED MECHANICAL AND BIOLOGICAL PROPERTIES, AND MAGNETIC NANOFIBER SCAFFOLD MANUFACTURED BY MANUFACTURING METHOD

Hereinafter embodiment to the present invention. as further described further. These embodiment relate more specifically, the present invention for, and/or at least two different embodiment of the present invention range not limited to.

Of the present invention data charted by the standard deviation of the above average ±, Tukey statistical analysis' of the changes by pharmacological tests s won-way analysis was implemented using. Compared to sample group independent. P p or < 0.05 been it is contemplated in a significant is < 0.01.

Manufacturing e.g. 1: magnetite (magnetite) for preparing nano particles of

1M of HCl to cargo f.o.b.for iron number 1 chloride (Ferrous chloride tetrahydrate, FeCl_2, 4H₂ O) iron broth in a heat number 2 and HCl cargo (ferric chloride hexahydratate, FeCl_3, 6H₂ O) mixed the guiding rail (Fe²⁺/Fe³⁺ = 1/2). Said drip cap for a passenger to use the mixture by adding 200 ml 1.5M NaOH solution 30 minutes stirring section. The resulting magnetic field of the resulting precipitate and to separate no using, separated the 8000rpm in the solution. 2 and a the separation process, continues with stirring, in a 200 ml solution 0.02M HCl added to precipitate. Said separating in the in 40 °C 8000rpm product was very dry. Said implemented in nitrogen atmosphere to all steps. Magnetite solution citric, under stirring magnetic nanoparticles (0.05M) and is dispersed in, NH₃ using a solution of pH 5.5 a gradually. 4 process has been completed, acetone is added precipitating the nanoparticles [...], for removing unnecessary citric a magnetic [...] (magnetic decatation) and, after washing the to acetone by the dried in 40 °C. Surface of the magnetic nanoparticles with citric acid coated COOH group.

In the embodiment 1: MNP-PCL electrospun method using manufacturing of nanofibers

Die of chloro methanes (DCM) and ethanol (4:1) to dissolve the 10% w/v PCL PCL (MW = 80,000; Sigma-Aldrich) have been prepared solution. Is separately from a DCM and ethanol solvent (4:1) to said-particles dispersed-formed nanotubes. DCM produced said: PCL a MNP and an ethanol solution dispersed in a mixed with solution. Said mixture in solution the concentration of MNP PCL decided to get out of 5, 10, 15 and 20 wt %. said solution is stable while the fabric at low cost representing a nano-composite been produced with at solution. Said mixture made of stainless steel 21-gauge needle with he infused a 10 ml plastic syringe. For said needle having a electric spinning manner so that the ultra 15kV of high-voltage source is. Electrospun in, radial to the 10 cm (tip to collector distance, TCD), injection speed, which has the to 0.5 ml/h. Electrospun during produced the nano-fibers for collecting drum by DC motor at a rate in the rotation. At room temperature the whole experiment said embodiment.

Experiment 1 e.g.: characterizing the physicochemical of nanofibers of the present invention

Crystal structure, designated X-ray diffraction analyser is measured with (XRD; Ragaku). A weaker current 40mA and 40kV sample using radiation cu K α 1 2 θ, each diffraction in a range of 2θ = 10-60 °, 2 ° / min with a step of 0.02 ° made at speeds in the order of the scan. Thermal behavior and nano-composite scaffolds configuration for determining the proportion of the weight analysis (TGA) of the embodiment. Nanofiber scaffolds, and to observe a chemical state to the FTIR spectrometer (Fourier transformed infrared, (IR-ft, Perkin-Elmer) the first voice portion out of an. For measuring electrical characteristics of a surface of a sample to pH 7.0 and 25 °C zeta-potential long since determined the unit moves simultaneously at the same distance (Zetasizer Nano; Malvern Instruments). polo molar sample using model S-3000H microscope (Hitachi) has been determined by SEM. TEM MNP structure and and inwardly and the presence of a (model 7100 microscope (JEOL)) sensors using.

Hydrophilic of nanofibers using the contact angle measuring instrument (Phoenix300 analyzer) has been observed on measures the contact angle of water. Nanofiber on the sample layer and to form equilibrated at 25 °C Image droplets until viewing system has been observed on using (viewing system). Balance state of the slip cover flat droplets drops similarly shaped is ensured a massage cream, an essence, is observed, each typically in equilibrium relative to the sample images obtained. Data 1 minutes, each group 5 was for testing samples.

Citric (functionalization) functionalized MNPs produced by a feature of the also showed to 1. TEM MNP which are measured by means of a ultra fine mono-dispersed in 12 ± 1.34 nm the size of particles revealed a nanoparticles. (Also 1a). XRD pattern 2 θ = 30 °, 35 °, 43 °, 53 °, in 63° and 57 ° (220), (311), (400), (422), (511) and (440) exhibits a peak a particular feature of of magnetite in stationed himself (also 1b). JCPDS card # 019-0629 according to said XRD pattern cube spinel structure identified. The Scherrer MNPs an average diameter of ' s equation: D = kλ / β cosθ, (wherein λ the cu Kα radiation 1.54 Å of X-ray wavelength, k has shape unknown when, 0.89 value of having shape variable, θ the Bragg angle and, β (and a fractional) a half blade. maximum in. width) of relations among the pixel of XRD according to decided from pattern. An average diameter so as to calculate a non-the strongest peak (311) got. TEM from result indicative of the average diameter of 10.8 ± 1.21 nm has a size of about similar to that. Prior function citric, chemical coupling structures MNP after 4000 to 400 cm^-1 region was subject to analysis by IR-ft (also 1c). IR spectrum ν OH MNP of each δ OH and 3426 and 1606 cm^-1 revealed a band. In producing the same and to the citric acid MNP, 3426 cm^-1 exempting from a heavy and rigid of ensuring the presence of a trace of water into this cable, the band can be. While 3200-3400cm^-1 portion of the second thermal cracking is performed on the revealed a presence of OH group. Furthermore, can be a symmetrical of OH of COOH indicative [...], 1630 cm^-1 band in a magnetite surface and exhibits binding of radical citric. 1401 cm^-1 peripheral band group COOH. representative of the feature of stretching asymmetrically. 600-400cm^-1 and 578 cm^-1 the disk band a magnetic particles for isomorphic singular Fe-O blades, presenting a. As a result said that is chemically bonded to the superabrasive surface magnetite citric acid of confirming that the device can be. Measured at pH 7.0 before and after function citric MNPs of nanoparticles, a zeta potential of 0.0 (- 17.2mV) exhibits high negative charge, increased with functionalized citric showed generating under negative. (- 36.6mV). The NP present at the surface of the citrate ion effect of (also 1d) exhibits. Electrospun composition nano to using method, nanoparticles under solvent containing a dispersion of distributed to determine a time point at which the evaluation distilled water or an organic solvent (DCM/ethanol) to dispersion of MNP. is important ability (also 1e). Any citric-functionalized MNP that it disperses efficiently and solvents and, long-term stability is integrity. Citric such stability could not be applied to an observation without function. The results indicate that preserve the stable solution functionalized citric in PCL and as a result effects of having available, and the solution of nano-composite can be confirm that the user.

Nano-composite to preparation of a solution of dispersed in a DCM/ethanol added to a MNP PCL. Then fiber scaffold nanowires using an electrospun method have been prepared. Electrospun process in parameter possible governs, polymer concentration due to a variation in is controlled solution constitution key variables and, with the fibers size and molar polo party is detected to form a. At various concentrations (5, 10, 15 and 20%) in a colloid solution including nano-composite MNP of a fluid is suppressed between the electrospinning of the apparent nano comprising the fiber, fiber-developed a membrane. Also in the typical has 2a (smooth) through adhesion with smooth, uniform, bead-free (bead-free) roh fiber having molar polo exhibits and representative SEM Image. When ball these images, through adhesion with smooth and pure continuous fiber is formed by electrospinning of PCL it is foreseen to. PCL diameter of a nanofiber measured, 5MNP, 864 (± 43) out for at 20MNP and 10MNP, 425 (± 31), 318 (± 46), 202 (± 40), and is 664 (± 63) nm. Continue to gradually adding MNP until 15% in order to intensify against a nanofiber thereafter decreases while, MNP 20% of diameter in the range of from increased the nanofiber the addition. Nanofibers have a significant effect diameter in a range from a solution electrically conductive and a viscosity such as other MNP the variables must be added with variations could it is foreseen to. SEM where its presence is MNP is not apparent on the whereas, nanofiber the addition MNP had changed into brown, dark more scaffold.

Using TEM nano-composite has been observed on structure internally of nanofibers (also 2b). Electrospun during few seconds of injection needle at the entrance tip of TEM in close proximity to located grid 20MNP and. 10MNP is obtained samples a particular feature of. Image viewing. In polymer substrate PCL easier NPs. 5MNP can be uniquely identifies that base station a relatively NPs in a well-separated each particle is disperse well whereas the, of NPs in some in 20MNP up out is aggregated.

XRD pattern of nanofibers PCL and in the typical associated with MNP have shown peak (also 2c). MNP in a sample analysis weight of the sample present provides information to the amount. The likelihood that all samples to because both layers have similar thermal behavior (also 2d) in. 250 °C hereinafter revealed that some weight reduction while is expire at the removal, reduction weight difference 2 in approximately 360 °C PCL been the, by means of the decomposition of polymer. Approximately 600 °C and may degrade in-been is complete. The remaining weight 5MNP, 10MNP, 4.1,9.0 20MNP and 15MNP out for at, 13.6, and was 18.6%. Metal added to the initially is nano-composite some than an amount requested but to indicate a lower value, is well. The hygroscopic of nanofibers did research on tester to contact angle (also 2e). PCL nanofibers are 88° while its large contact angle, nano-composite nanofibers gradually reduced contact each value (10% MNP in 20% MNP and 68° in below the to 47°) blades, presenting a. A nanofiber is added the MNP ( possibility affinity ) exhibits improvement of hygroscopic scaffolds. These enhanced MNP a carboxylated the presence of do.

Experiment 2 e.g.: characterizing mechanical of nanofibers of the present invention

10 mm/min (Instron 3344 universal testing instrument) booting to (cross-head speed) speed of tension using a nanofiber scaffolds having a wide variety of compositions, it was determined that mechanical properties. Membrane approximately 150-200 micro m to have thicknesses on the order of spin coating, 30 mm X 4 mm (gauge length 10 mm) of the cut to size. Then been is applied tensile load. M is a natural number below N. strain from test said been is recorded (stress-strain curve). The thickness of the membrane each 5 of each group for the samples of SEM Image observed in decided from the average values. Tensile strength, yield strength, elastic modulus, the tensile ductility and breakdown in mechanical properties including strain strain m is a natural number below N. from the bill. Respective compositions each two exemplar testing was 4.

Nano-composite scaffolds tensile strength by pharmacological tests it was determined that mechanical properties. 3a controls a sample are also (PCL and PCL-MNP nano-composite nanofiber) in wet-istics that are typical for show m is a natural number below N. stress-strain curve. The likelihood that all samples a distinct at the stage two similar stress-strain curve have shown that m is a natural number below N. behavior: fast group modified according to increased stress initial step of approximately 0.1-0.15 degree, slowly point peeling, post destructive show a vacuum type. Based on these stresses m is a natural number below N.-modified tensile strength, yield strength, elastic modulus, destruction including breakdown and modified in degree modified human power by operating all systems by 2001 mechanical parameters. Destruction and the tensile strength a is measured as a maximum intensity immediately before MNP content increases until 15% have been increased; 26.2 MPa in pure PCL in have been increased until 15MNP is 11.5 MPa. (Also 3b). However the addition of 20% MNP reduced to 9.5 MPa tensile strength. Furthermore, breakdown point also yield strength (yield point) in shown in a similar manner (also 3c); pure PCL in have been increased to 15 MPa in 15MNP is 6.5 MPa. Hardness of sample (stiffness) representing a range of strain 0.5% modulus of elasticity of a nanofiber be computed in a m is a natural number below N. a stress-strain curve has been determined by initial inclination of the optical disk. Furthermore, according to MNP are added thereto, pure PCL 15MNP is 60.1 MPa in 86.7 MPa in. any significant increase the apparent while of increasing to. However, this is reduced back to 65.6 MPa in 20MNP. As well as strength and hardness, elongation in addition fixed human power by operating all systems. In breaking point that are in consideration as elongation MNP of the strain have been increased slightly according to added up to 15% (0.75 to 0.57 in). However, this is the discharge circuit is reduced to a, [...] to 0.4 at 20%. Yield point according to 15% of the strain in in 0.11 MNP added 0.21 but results in a significant increase to, that are further reduced to 0.12 at of 20%.

Experiment 3 e.g.: irradiation magnetic characteristics of nanofibers of the present invention

Said magnetic property and high purity of the sample ± 20 kOe magnetic field applied at room temperature SQUID (superconducting quantum interference device (SQUID; Quantum Design MPMS-XL7)) did research on using. SQUID has devices and include standards (pure nickel opening) has been determined based on the. Magnetic property and high purity scaffolds remarkably low in saturation magnetization was is assessed in terms of the roof area and hysteresis.

A superconducting magnetic property and high purity scaffolds nanofiber nanocomposite magnetometer (SQUID magnetometer) hysteresis function at a normal temperature by using human power by operating all systems by loop (also 4). As a function of applied magnetic field is the magnetization curve exhibits magnetization. The likelihood that all samples the + 20 to -20kOe of according to change in a magnetic field in loop hysteresis of traditional more, exhibiting (also 4a). Strong magnet and both of them depending on sample nanofibers show attraction force (also 4a a '). Furthermore, low magnetic field range (+ 8 Oe in -8) present in the magnetization m in. Therefrom (also 4a a '), a coercive force of 2.5 Oe scaffold (H_c) and 0.27 emu/g low at a residual magnetization (M_r) copyright 2001. Their is typically narrow hysteresis loop and low clause the characteristic which sleeps the determined portion of the sacrificial layer is patterned as ferromagnetic or superparamagnetic material is considering. 1.0-1.2emu/g saturation magnetization in a range of (M_s) the MNP amount of time increases with an increasing content of and (also 4b), this polymer substrate PCL MNP coupled to the relative weight ratio was associated with. Another important parameter area of magnetically is lost/cycle or a hysteresis loop. The maximum area loop site is completed through a combination of applied been calculated in a magnetic field of ± 20kOe. Much like an behavior of saturation magnetization, of a V content MNP site is completed through a combination of loop enhance value of area (also 4c).

Experiment 4 e.g.: test and dismantling formation capability and to obtain a hydroxyapatite (apatite) in ex vivo

The experiments of each used in the immersing at 200 ml of specimen with a PCL 2M NaOH aqueous solution, time 4 30 °C stirring section. Said NaOH solution is removed from the sample, was an upper portion of wide by removing particles and ultra pure water. inside some counterweight in air of room temperature then was very dry. A specimen soaked to activate the hydrophobic surface calcium and phosphate ion enables a combination of network layer by carboxyl group which a plating solution is introduced for the design. NaOH treated nano-fiber manufactured by next process (CaP the microcells in which a shortened as treatment) arrayed at the bottom of. is immersed ionic solution and phosphate calcium ion. Specifically, of 150 mm nano-fiber manufactured by treated with NaOH CaCl₂ aqueous solution 10 to 100 ml pure water immersing at seconds immersing at seconds 10 to 20 ml, was dried in air few minutes. Then samples of 200 mm NaHPO₄ immersing at to 100 ml aqueous solution, 10 to 20 ml pure water immersing at seconds was dried in air few minutes. Immersing a alternating and 3 times at room to the embodiment. A given relative to the sample during immersing alternating and Conference said 3 the same CaCl₂, NaHPO₄ ultrapure water solution and been used. NaOH treated with other sample and CaP processing are subjected to the various time in samples 37 °C during bone mineral nanocrystalline, for growth human blood with suspected of being present in the plasma pH 7.4 and therefore ion concentration, (Na⁺ 142.0, K⁺ 5.0, mg²⁺ 1.5, Ca²⁺ 2.5, Cl^- 147.8, HCO^3- 4.2, HPO₄^2- 1.0, SO₄^2- 0.5 mm) of (1.5x) is immersed (SBF) 45 ml solution vivo was similar. From solution by removing particles and ultra pure water sample after removal of a the wash at a mild conditions.

Magnetic nanofiber scaffolds hydrolysis the phosphorus solution which will live a of phosphorylating a a sample disintegration properties (saline solution, phosphate buffered) in 37 °C to corresponding advertisement based on the shown measuring evaporates into copper 28, during changes in weight of the test period (weight losses) he made notes for his. Before hydrolysis, SEM samples of he jabbed his Image.

PCL-MNP nano-composite nanofibers are bone regions at uppermost and for considering use as reproduction matrix, hydroxyapatite formation capability and to obtain a similar ex vivo in solutions vivo been this approach. Constitution: forming hydroxyapatite are inventor allows a reduced duration study for acceleration of 1.5 SBF (1.5SBF) concentration of times to the first voice portion out of an. Each sample for different period (0, 5, 7, 10, 15, 20 and 30) immersing at to 1.5SBF, polo molar phase and remove the for the analysis of changes in. After 30, XRD pattern has been observed on the likelihood that all samples of (also 5a). In pure PCL, hydroxyapatite in value of 2 θ of -32 ° - 26° and that a relatively low peak (002) and (211) of HA determining regions (JCPDS card #. 74-0565) blades, presenting a a database. MNP content of a V, peak intensity have been increased. Furthermore, in some PCL peak hydroxyapatite any other while the subsequent disappearance of said reference temperature peak. A nanofiber is added MNP the results indicate that enhanced scaffolds exhibits-forming activity of hydroxyapatite. In some PCL while the subsequent disappearance of said peak, hydroxyapatite, it gradually over time. Immersing 20MNP as a function part of the selected periods as if it were observed in samples (also 5b). SBF test of molar polo has been observed on SEM of samples of (also 5c). Initially formed third 5 any small nanocrystalline region was increasing over time. Hydroxyapatite determining regions on most completely intermediate period been covered nano-fiber. And for a period long (20 or more) was nano between the fibers, and fills the space.

As well as test SBF, nano fiber skeins PBS such a decomposition of a chamber, in 37 °C did research on. 28 he got down the main the change in weight during testing of period (also 5d). All nanofiber scaffold weight time increases reduced. Weight losses of a V content MNP the nanocomposite nanofiber in. more important. After 28, weight losses in PCL approximately 20%, 45% and 60% in was 10MNP 20MNP search. A nanofiber decomposition has been observed on in SEM samples. Compared to PCL 10MNP and 20MNP of nanofibers revealed that is causing significant structure (also 5e).

Experiment 5 e.g.: RAT MSC of isolating and culturing the bone marrow

Mouse bone marrow derived from a previous MSC separated the as described. All associated with animal Animal Care and Use Committee to end office protocol was acknowledged to the. Specifically, male adult Sprague-Dawley rats (180-200g) of the end of the plane and the other center's body tip of a joint as an intramedullary an aqueous solution of a femur and Shin bone centrifugal separator tissue of normal medium (1% Streptomyces/sealing oxide layer using wet of erythromycin oh serum[...] of 10% and that includes an input and (fetal bovine serum) (α-MEM) media supplemented by the redispersable in. During and air of 5% in 37 °C CO₂ of dish for the first time, the in incubator atmosphere of soaked with the located (culture dish). Each change medium made every 3 during the hematopoietic stem cells (hematopoietic cell) is not bonded to the remove the from a medium. Stages three and then cells been used experiments. Experiment for molecular breeding of capsicum g/ml micro 50 sorbic acid, 10 mm β-glycerol phosphate and 10 nm dexamethasone (dexamethasone) and that includes an input and osteogenic manner in a medium free of been embodiment.

Experiment 6 e.g.: cells adhesion analysis

Has different in composition between and ejects sterilization the ethylene oxide [...] the nano-fibers. Samples well-well plate 24-making and for properly sized, maintaining the plastic ring is set up to come in. A respective sample MCS 5 x 10³/was to be inoculated with a density well. The initial adhesion step allows for different culture time (time 2, 4, 8 and 16) in, nanofiber sample petal blue tree cells that is bonded to the (Trypan blue screening and hemocytometer counting) or blood screening and the analyzed by two. 5 (replicate) cells replication of each experiment been tested for conditions. CLSM said shaped design of the notches has spreading adhesion and of cells (a point-scan confocal laser microscope, confocal laser scanning microscopy). 4 °C been by different night in cells cultured in period 4% of personal communication system has the form under it knows the id and dyeing the 5 minutes, and impregnating the having 0.3% Triton X-100, translations and blocking has 2% of BSA, 1 has antibodies difference for FAK [...] section. F-actin cells and for coloring the dilution to 1 time phosphate buffered saline (Invitrogen) (phalloidin) combined with a [...] 2 difference in incubation with Alexa Fluor 546 coupled antibodies on the proper flue [...] in adaptation-labeled (fluorescein isothiocyanate, FITC). 4 nucleus ', 6-diphenyl-Indol -2 roh midi diameter (4', 6-diamidino-2-phenylindole, DAPI) no stain and no slip, confocal microscope (LSM700 confocal microscope (Carl Zeiss)) captures an Image using adaptation.

Light between the laser beam and for DNA isolation and isolation method thereby adhesion initial MSC (2, 4, 8 and 16 time) nanofiber scaffold attaching cells human power by operating all systems by a by. 2 hours (also 6a), but adhesive surface finish, which results in an 10MNP and 5MNP cells (80% or more), and. 5MNP been adhesive 50% than about sample PCL attaching 10MNP 90-100% level yet modification is performed for congestion in degree, in magnetic nanofiber adhesion in PCL and a similar to levels equal to 8 have been increased over time. MSC adhesion and spreading behavior it is found out that by CLSM (also 6b). Adhesion focus of cells for cytoskeletal process and Focal adhesion kinase (FAK) to indicate color because it was F- [...] together. 2 time PCL relative to the sample substantially is got out of sight. However 4 time more spreading have demonstrated to 10MNP and. 5MNP, 2 time an active cytoskeletal process and focus [...] together spreading have demonstrated. This was clearly the time in addition 4. MSC initial adhesiveness, spreading behavior of magnetic nanoparticles an output signal of a comparator is specific capacities under significant fold fiber skeins it has been confirmed.

Experiment 7 e.g.: cell invasion analysis

Nanofiber scaffold for analyzing a cell invasion to, Image-stack z of cells DIC arrangement the color 3 - ((488 nm for FITC, 555 nm for Rode, and 420 nm for DAPI and DIC) 3-color DIC configuration mode) and with the spacing m micro 1 in, nanofiber scaffolds micro 0-75 approximates the thickness in a range of m LSM700 META (Carl Zeiss) was obtained using the. Of software ZEN Image Z-stack dimensional 3 by orthogonal tool (3D) the converted into projected Image. Furthermore, the side-view of z-stack z-stack series side view tool to obtained. A side-view based on the acquired images to represent average depth of penetration (cell localization) position measurement cells was used to quantity of.

Nanofiber scaffold a cell invasion into did research on during culturing period of 12. Cytoskeletal process as an indicator of the cells to [...] -F of tinting. Nano fiber network in a cellular movement of respect to nuclear for visualizing was no stain DAPI. Confocal Image the fluorescence signal green in 2D to a profile 3D. structure (also 6c, representative sample). Each relative to the sample Image a profiled 2D more, exhibiting in Figure 6d. Abstract PCL restraining itself from in the case of substantially signal is present in region while, magnetic nanofiber to middle and bottom side been dispersed. Average level of the was used to quantity and a red/green color signal (also 6e). The cell invasion depth magnetic nanofiber scaffold is indicative that significantly enhanced, this produces higher content of was is further enhanced in MNP. (PCL < 10MNP < 20MNP).

Experiment 8 e.g.: determination of alkali phosphatase (ALP)

Magnetic nanofiber scaffold cultured in osteoblasts of a MSC differentiation to initially so as to produce a relatively quick (osteoblastic) a the formation circle which boils can be labeled a ALP activity of human power by operating all systems by measuring unit is connected to the. After grown on during 14 and 7, and collected from scaffold nanofiber cells, 142 by repeating thawing lysis buffer was decomposed into. Manufacturer (Sigma-Aldrich) in accordance with the instruction from the enzymatic reaction for added sample to ALP reaction medium. Added amount of sample actually on a commercial DC protein assay kit (BioRad) as measured when, entire protein content of it was determined that based on. with lung glow generation is such that the P-nitro spectrometry in absorption of 405 nm it was determined that using. Each experiment condition by cylinder to have sample replication of two 5.

PCL-MNP nanofiber scaffold cultured in osteogenic differentiation of MSC is analyzed by ALP activity was first (also 7a). 7 the stylus is not initially approximately equidistant between samples while of thin film transistors are electrically connected, by increasing the significant after the 14 appears purchases the passenger, in particular 10MNP 15MNP and up out is the difference in scaffold.

Experiment 9 e.g.: quantitative reverse transcriptase polymerase chain reactionby (RT-PCR) osteoblasts gene expression

Collagenase I (Col I) type number of limiters, osteopontin (osteopotin, OPN), at the time of it is a reel with the rope which will know (sialoprotein, BSP) of a gene associated with bone including mRNA expression level by real-time PCR (PCR Q) factor have been measured by to quantitatively. After grown on during 14 and 7, each sample cells from and transmits, a manufacturer's according to an instruction by the entire RNA, was to separate no using RNeasy Mini Kit (Qiagen). A cDNA primer as oligomer of any RNA has adaptation reverse transcriptase using Superscript kit (Invitrogen). By using the PCR amplification the Sensimix Plus SYBR Master Mix (Quantace). Relative housekeeping gene CT method (comparative CT method) for beta- [...] built by each gene to a PCR product for regulating the was for use in analyzing by.

Q expression is of dielectric of cells been analyzed by PCR. Col I a or expressed in situ by cells, mRNA level of OPN and BCP (also 7b) and compared. Gene-volatile level been the main component which has an improved apparent, in particular OPN and for BSP increase in gene expression level clearly shown; in OPN 15MNP to 7, and BSP 20MNP and 10MNP 15MNP and 7 in respect to in increase times 2-3 shown in 14.

Experiment 10 e.g.: biocompatible in vivo hypodermic of RAT

Electrospun a nanofiber scaffold (PCL, 5MNP, 15MNP and 10MNP) m micro 1.5 cm x 1.5 cm x 300 making and to substantially the same, before surgical sterilization the ethylene with oxide. By cylinder to have male Sprague-Dawley rat the test biocompatible tissue. The animal experiments was acknowledged to the Dankook University Institutional Animal Care and Use Committee. Against all processing sterilized common in anesthetic the conditions was to carry out. Divided into group you mouse (into a per group), either by intravenous injection grasping each non-aminic metacrylate 80 mg/kg 10 mg/kg with hydrazine and he infused a. 4 small avoid the pouch provides a means for each animal the inter-vertebral from and made scissors area such as obliquely left and right, each scaffold samples the loaded on the deficiency. Incision 4-0 sites not being available for absorption of a monofilament (Prolene) sealing the sealing lines. 4 mouse and 12 into main 12/low time has been observed on under schedule alternate night time. Standard food pellets was providing a free and water.

The sacrificial week 4 animal. One implant close to the tissue analysis histological area and collected for, 24 immediately at room temperature time 10% neutral buffered formalin-immersing at a, grade predosed in dehydrated and ethanol series, after danger detector or an actuator, the embedded in paraffin. Histological samples m to have thicknesses on the order of 5 micro rotary microtome manufactured with the use of the metal and, established standard using (dye) or Masson hematoxylin and eosin (H & E)' was color because it has s trichrome (MT). Biocompatible of the tissue sample slides the optical regeneration and angiogenesis has been observed on microscopically. Scoring is histological made (0-4 ; 0 the most small, 4 determines the most high) immune response, fiber film thickness, extension of the fibroblast presence and lumen of a blood vessel that different including pathological tissue based on the particular characteristics have been produced.

Nanofiber PCL-MNP in order to analyze the suitability tissue scaffolds, implantation in organization under blood of RAT sample was 4 weeks. Post-surgical, all held in an implantation site in suffers that does not cause inflammatory, pollution not been provided for. 4 sample group implanted into main (PCL, 5MNP, 15MNP and 10MNP) exhibits to form histological of Figure 8a. Significant of any sample tissue rejection or inflammatory response got out of sight. Adapted collagen fiber made of tissue connected with the sample space between adjacent tissues is fabricated, in addition, suitable scaffold in demonstrated angry been discovered. All 4 one group of samples a good histocompatibility while its, mainly in scaffold 15MNP and 10MNP 5MNP or PCL but not extent in, (NF directed as different zones of) grown connected to excised tissue with the hydrocracking zone a are replaced by, revealed that such a decomposition of a scaffold some. Vaporizers are after week MNP and PCL 4 is a structure including a while, 15MNP and 10MNP been lean and in that structure of. Connected to excised tissue in the region replaced by did not has a visible contour scaffolds. Fibroblast cells actively decomposition is being moved to the site, the remaining fibroblast activated easily detected as between the set minimum value and the scaffold. Comprises, in order, a backplane ratio in < 5MNP < 10MNP < 15MNP PCL surface is arranged. Table 1 the quantified parameter histological the results of the analysis revealed a. MNP-PCL scaffold, a higher luminescence in the PLC 15MNP and in particular 10MNP in angiogenesis low fiber go further revealed that cell migration is fibroblast and encapsulated in a semi-. 10MNP [...] 15MNP and a on magnified images of angiogenesis in signal exhibits well. 8b also shown by white arrows.

Experiment 11 e.g.: RAT bone formation ability in vivo defect radius

Hypodermic in histocompatibility after confirming the receivable channel to, magnetic nanofiber scaffolds bone of RAT bone formation ability did research on model defective segments. The present in the embodiment, the third to eo mouse Sprague-Dawley of mary 6 in. Animal care and housing protocol said section described test mode and equal to.

Hairs of front and rear legs and hair, said zone of the surgical for using ethanol 70% and (povidone) gun expense money the-lasting sterility out. -lasting sterility line and the second solenoid valve, a right or left on the surface of radius 15 cm was cutting a skin of of front and rear legs. Sufficient surgical room in radius the diaphyseal cutting debribement periosteum for lifting muscle for. 5 mm length was cut and two segments of bone have been prepared from the center of radius the defect. The bone the electrode for cleaning bone particles in a drilling each RAT using saline the sterilizing and cleaning while having been produced with at. Of three group randomly extracted. Each defective the two types of nanofibers scaffold (15MNP and PCL) and optionally by grafting of the having, wrote down the since then, in the control group a voice one. Nanofiber scaffolds in the case, each sample the radius of maintained portion completely covers to defect in tissue and soft to prevent sensors was surrounded by the inner peripheral surface defect. Fascial for 4-0 having been covered material to the base, a skin incision in a undesired materials is absorbable 4-0 the in. Animal surgical the front and rear legs immediately was for causing a computer to allow the functionalized.

After animal has been observed on into main 8. After sacrificial, and cutting a skin, sample and thereof diamond the surrounding tissue using drilling the motor, attached wheel took. Samples 10% at room temperature 24 time a neutral buffered of the formalin-fixed. Fixed samples translations and desalination, dewatering and, put it in his paraffin making and approaches Serial l micro. 5, no stain the MT and & E H, it was determined that using optical microscope to check upper area of.

Upon validation histocompatibility scaffolds nanofiber nano-composite, having segments radius of RAT model was to design experimental sets and the other. Typically include group scaffold 15MNP and PCL was testing. The scaffold thing which the control group and was testing. The b and 9a also using and in research show model segments radius RAT. Radius segments defect in the seat is removed and which serves to cover area position the nanofiber scaffold. Defect during a vehicle by preventing the infiltration of tissue soft to localized areas radially spaced layer on the basis a cylindrical glass body. Surgical after main 8, no stain & E nanofiber scaffold group H (15MNP and PCL) a bone deficiency of histological evaluation in soft tissue and muscle around adverse inflammatory reactions and didn't show signal (also 9c). However, in the control group free scaffold between end osteotomy radius goal type total label. observed easily (nonunion). A to 9 d-f B and also between the set minimum value and the histological form showed thus expanding the. In the control group, derived from edge margin of a defect defect a minimal new bone with formation thin loose connective tissue and muscle been filled (also A panel 9d). Muscle contact surface metatarsal (ulna) clearly shown (also 9d, B panel). By ensuring that in a group scaffold PCL, new bone the formation radius osteotomy and the ends scaffold been formed along clearance surface (also 9e, panel A, arrow labeled a). New bone formation is initiated surface scaffold PCL, scaffolds and osteoblasts of the movement of the increase in. The display panel to cover the remaining portion of radius scaffold, metatarsal and radius and soft tissue penetration fusion of film resulted in smaller dimensions, new bone the formation scaffolds and character bone revealed that frequently a surface (9e also, panel B). Also 9e, in B panel, arrow end surfaces of newly formed bone osteoblasts appointed toward the scaffold exhibits the growth of the. Many osteoblast cells collected [...] certification by moved the implementation, a metatarsal is scaffold is stimulate in surface. Radius osteotomy end and scaffold in the point where reproducing bone surface, by ensuring that in a group scaffold 15MNP (also 9f), similar to pure PCL showed recovery of bone defect. Two scaffold among a group bone formation in level of which differs from that of. have remarkable in particular point. 15MNP molds do not soften at elevated temperatures and the older superceded pages of data near the site deficiency further the bone connective tissue filled rectangles to the free surfaces. The bone are prepared in advance and scaffold PCL better surfaces of bone host than in group is coupled (also 9f, panel A) scaffold PCL scaffold. 15MNP more, exhibiting a rapid breakdown than. Most scaffolds newly formed bone matrix is absorbent easily with osteoblasts and. Myelodysplastic and in their adjusted positions in the regenerated defect of hope appeared. clearly more group PCL. Residual material that transports bind to and tissue, is the culture to be monitored and osteoblasts 300. (9f also, panel A). Furthermore, osteoblasts appointed to scaffold metatarsal surface with the movement and from infiltrating the bone matrix, occurs to a result for forming bone matrix flow in teh space, a high degree of certainty of show cell responses. (9f also, panel B)