[보고서]생분해성 졸-젤 전이 고분자와 이의 의학적 응용

보고서 정보

주관연구기관

이화여자대학교 산학협력단
Ewha Womans University

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2014-04

과제시작연도

2013

주관부처

미래창조과학부
Ministry of Science, ICT and Future Planning

등록번호

TRKO201500002460

과제고유번호

1345198114

사업명

중견연구자지원

DB 구축일자

2015-05-16

키워드

졸-젤 전이.생분해성 고분자.자기조립.폴리펩타이드 생체재료.2 차구조.기능성 수화젤.단백질/펩타이드 의약.의약전달 시스템.주사형 조직공학.sol-gel transition.biodegradable polymer.self-assembly.polypeptide biomaterial.secondary structure.functional hydrogel.protein/peptide drug.drug delivery system.injectable tissue engineering.

DOI

https://doi.org/10.23000/TRKO201500002460

표 졸-젤 전이 고분자 수용액. 상온 (20 oC)에서는 저점도의 수용액이나 체온 (37 oC)이 되면 순간적으로 수화젤로 전이가 일어남.
표 단백질 의약품의 세계시장.
표 Epogen과 Aranesp의 매출액 변화. Amgen의 EPO (erythropoeitin) (Epogen)는 1 주일에 3번 투여하나, EPO에 의약전달 개념이 도입된 glycosylated EPO (Aranesp)는 1 또는 3 주에 한번 주사제로 투여함.
표 세계 세포치료를 이용한 조직공학 산업의 성장 추이.
표 Synthesis of the PAF-PLX-PAF.
표 List of polymers studied.
표 a) Phase diagram of the PAF-PLX-PAF (PIII) aqueous solutions determined by the test-tube inverting method. Photos are PAF-PLX-PAF (PIII) aqueous solutions (8.0 wt. %) at 20 oC (sol state) and 37 oC (gel state). b) Increases in storage modulus (G’) and loss modulus (G”) of the PAF-PLX-PAF (PIII) aqueous solutions (8.0 wt. %) during the sol-to-gel transition. The plot on a linear scale is inserted. A PLX aqueous solution (8.0 wt. %) was compared as a control.
표 a) CD spectra of PAF-PLX-PAF (PIII) aqueous solution at 10 oC as a function of concentration (wt. %) in water. b) Absorbance of hydrophobic dye (1,6-diphenyl-1,3,5-hexatriene) at 10 oC as a function of PAF-PLX-PAF (PIII) concentration in water. The legends in a and b are the concentration (wt. %) of PAF-PLX-PAF in water. c) Determin ation of CMC of the PAF-PLX-PAF (PIII) in water at 10 oC by the CD and the dye solubilization method (DSM).
표 a) CD spectra of PAF-PLX-PAF (PIII) aqueous solution (0.05 wt. %) as a function of temperature by an increment of 10 oC from 10 oC to 50 oC. The subtracted spectra at a temperature from the spectra at 10 oC (ΔΘ) show the increase in the magnitude of positive band at 200 nm and negative band at 218 nm. b) FTIR spectra of PAF-PLX-PAF (PIII) aqueous solution (8.0 wt. %) as a function of temperature by an increment of 10 oC from 10 oC to 50 oC. The subtracted spectra at a temperature from the spectra at 10 oC(DAbsorbance) show the decrease in absorbance at 1640 cm-1 and increase in absorbance at 1620cm-1.
표 a)Structure-property relationship of phase diagram of the PAF-PLX-PAF aqueous solutions determined by the test-tube inverting method (n=3). b) FTIR spectra of PAF-PLX-PAF aqueous solution (8.0 wt. %) in D2O.
표 Synthetic scheme of PVP-PA.
표 List of polymer studied.
표 a)Phase diagram of the PVP-PA (PIII) aqueous solutions determined by the test-tube inverting method (n=3). b) Changes in storage modulus (G’) and loss modulus (G”) of the PVP-PA (PIII) aqueous solutions (12.0 wt. %) as a function of temperature. G” of PVP aqueous solution (12.0 wt. %) is shown as a reference. G’ of the PVP is less than a tenth of G” over the temperature range studied (data not shown). The data were collected with a heating rate of 0.5 oCmin-1 and a frequency of 1.0 rad./s.
표 a) FTIR spectra of PVP-PA (PIII) aqueous solution (12.0 wt. % in D2O) as a function of temperature by an in crement of 5 oC from 10 oC to 50 oC. FTIR spectra of PVP-II aqueous solution (12.0 wt. % in D2O)at 20 oC was comp ared as a reference. b) Deconvolution of the carbonyl peak (50 oC)by the XPSPEAK41 program. The orange line is the original carbonyl band at 50 oC and the thick black line is the simulated curve from the two deconvoluted peaks (black thin lines). c) Replot of a) by the ratio (fβ) of a peak area centered at 1624 cm-1 to the whole carbonyl peak.
표 (좌) Effect of the PA molecular weight on the phase diagram of the PVP-PA aqueous solution determined by the test-tube inverting method (n=3). (우) Effect of the PVP molecular weight on the phase diagram of the PVP-PA aqueous solutions determined by the test-tube inverting method (n=3).
표 Effect of the L-alanine/DL-alanine ratio of PA on the phase diagram of the PVP-PA aqueous solutions determined by the test-tube inverting method (n=3).
표 List of polymers
표 Mean residue ellipticity of PEG-L-PA aqueous solution(0.01wt.%)at 15oC as a function of PEG and L-PA length. Unconjugated L-PA(L0-7), PEG-DL-PA(DL10-7), and PEG are shown for comparison. Legends are the molecular weight of each block in hundred Daltons.
표 a) FTIR spectra of PEG-L-PA aqueous solution (6.0wt.%) at 15 oC as a function of PEG and L-PA length. Unconjugated L-PA(L0-7), PEG-DL-PA(DL10-7), and PEG are shown for comparison. α.h., β.s., and r.c. indicate α-helix, β-sheet, and random coil, respectively. Legends are the molecular weight of each block in hundred Daltons. b) Curve fitting of the amide I band of the PEG-L-PA. Original spectra (thick curve) and simulated spectra (dashed curve) consisting of various secondary structures were compared.
표 Relative content of secondary structure of L-PApolypeptide analyzed by FTIR spectra.
표 cryo-TEM images of PEG-L-PA aqueous solution (0.1wt.%) at 15 oC as a function of PEG and L-PA length. The scale bar is 100 nm.
표 a)Phase diagram of the PAL-PLX-PAL aqueous solutions determined by the test-tube inverting method (n=3). b) Storage modulus (G’) and loss modulus (G”) of the PAL-PLX-PAL aqueous solutions as a function of temperature and concentration. The legends are the concentrations of the polymers in water.
표 Apparent size distribution of PAL-PLX-PAL in water as a function of temperature at 0.05 wt %.
표 a) Circular dichroism spectra of PAL-PLX-PAL aqueous solution (0.01 wt. %) as a function of temperature. b) FTIR spectra of PAL-PLX-PAL aqueous solution (10.0 wt. % in D2O)as a function of temperature. c) 13C-NMR spectra of the PAL-PLX-PAL (10.0 wt. % in D2O) as a function of temperature.
표 Deconvolution of amide I band in FTIR spectra of A7.0L1.8-PLX-A7.0L1.8 (a) and A5.5L2.1-PLX-A5.5L2.1 (b) aqueous solutions (10.0 wt. % in D2O) at 20 oC. c) Structure-property relationship of phase diagram of the PAL-PLX-PLXPAL aqueous solutions determined by the test-tube inverting method (n=3).
표 Enzymatic degradation of the PAL-PLX-PAL. a) Mass loss of the gel in the presence of enzymes (n=3). b) Gel permeation chromatogram of the remaining gel 6 days after the incubation. Control is the PAL-PLX-PAL incubated in phosphate buffered saline (pH=7.4).
표 In vivo gel formation by subcutaneous injection of a PAL-PLX-PAL aqueous solution (0.5 mL) containing FITC-BSA into rats. The photos were taken one day, 31 days, and 47 days after the subcutaneous injection.
표 Tissue biocompatibility around the implant 31 days after subcutaneous injection of a PAL-PLX-PAL aqueous solution (0.5 mL) containing FITC-BSA into rats. Optical microscopic images (a), H&E stained images with different magnifications (b: x40, d: x100, e: x200, and f: x400), and fluorescence microscopic images (c) around the implant. g) Gel permeation chromatogram of the remaining gel 31 days after subcutaneous injection of the PAL-PLX-PAL aqueous solut ion into rats. Control is the raw PAL-PLX-PAL.
표 Synthesis of CS-g-(PA-PEG). x+y=m, p=11, and q=45.
표 a)Changes in viscosity of chitosan(CS), PEG-PA, and CS-g-(PA-PEG) aqueous solutions(6.0wt.%) as a function of temperature. b) Changes in modulus of the CS-g-(PA-PEG) aqueous solutions (6.0 wt. %) as a function of temperature.
표 Change in pH of chitosan and CS-g-(PA-PEG) aqueous solution (1.2 wt. %) as function of temperature.
표 IR spectra (a), and 13C-NMR spectra (b) of the CS-g-(PA-PEG) aqueous solution (6.0wt.%)as a function of temperature.
표 pH dependence of degree of ionization of CS (a), FTIR (b), circular dichroism (CD) (c) and 13C-NMR spectra (d) of the CS-g-(PA-PEG) aqueous solution at 30oC. The polymer concentration was 6.0 wt. % except for CD spectra (0.05 wt %).
표 Modulus of polymer aqueous solutions as a function of temperature. The modulus at 3 oC (sol state) is less than 10 Pa, which is significantly smaller that the modulus at 37 oC (gel state).
표 Nanostructure of the polymer. (a) IR spectra of polymer aqueous solutions (10.0 wt. %) at room temperature.
표 Comparison of the cell morphology in 28th day for 2D (culture dish) and 3D (PI, PII, PIII, and MatrigelTM in vitro cultured systems. The scale bar is 200 µm.
표 In vitro 3D culture. (좌) Amount of sGAG in 3D-cultured samples assayed by the DMB method. (우) Gene expression of collagens in 3D-cultured samples after 28th day analyzed by RT-PCR. n=3 for all data.
표 In vivo assay 14th and 28th days after the injection of PII (10.0 wt. %)/chondrocytes aqueous system. (a) In situ gel formation after the subcutaneous injection of the PII (10.0 wt. %)/chondrocytes aqueous system (left). The gel recovered in 14th day (right). Amount of sGAG per recovered gel (μg/mg) assayed by the DMB method (n=3) (b), and by alcian blue staining method (c). PA-PLX-PA (PII) hydrogel itself (control) is not stained by the alcian blue method. (d) Gene expressions of collagens analyzed by RT-PCR.
표 Changes in the modulus of the PA-PLX-PA aqueous solutions as a function of concentration at 37 oC. The modulus of 10.0 wt. % in a sol state at 3 oC is shown for comparison.
표 a) FTIR spectra of polymer aqueous solutions as a function of concentration (7.0, 10.0, 15.0 wt. %) at 37 oC. b) Deconvolution of the amide I band after baseline correction of the PA-PLX-PA aqueous solutions (7.0 wt. % in D2O) FTIR spectrum.
표 Cryo-TEM images of polymer developed from polymer aqueous solutions as a function of concentration at 37 oC. The scale bar is 100 nm.
표 A) Proliferation of chondrocytes in PA―PLX―PA hydrogels analyzed by CCK-8, and B) confocal microscopic images of hydrogel encapsulating chondrocytes analyzed using a Live/Dead kit. Green and red images indicate live and dead cells, respectively. The scale bar is 200 μm.
표 A) Amounts of sGAG per well in hydrogel encapsulating chondrocytes by DMB assay, and B) alcian blue staining of hydrogel encapsulating chondrocytes at 28 days. The scale bar is 200 mm.
표 A) Relative mRNA expression of type I and type II collagen at 14 and 28 days to 0 days for 3D-cultured chon drocytes. GAPDH was used as an internal control. The numbers on the X-axis are the initial concentrations of polymer. M and 2D indicate the MatrigelTM and two dimensional cultured system, respectively. B) Type II collagen immunostaining of hydrogel encapsulating chondrocytes at 28 days. The scale bar is 200 mm.
표 A set of divergent Rabs is associated with phagocytic uptake or digestion. (Bright, L. J., Kambesis, N., Nelson, S. B., Jeong, B., Turkewitz, A. P. Plos Genetics, 2010, 6, e1001155, 1-18).
표 a) Synthetic scheme of the polymer. b) Gel permeation chromatogram of PEG, PEG-PA, and PEG-PA-Z-PA-PEG. N,N-dimethyl formamide was used as an eluting solvent.
표 (좌) UV-VIS spectra of PEG-PA-Z-PA-PEG aqueous solutions (0.01 wt.%) as a function of time. After exposure of the polymer aqueous solution to UV at 365 nm for 10 minutes, it was exposed to atmospheric visible light. Inset is the change in absorbance at 325 nm of the polymer aqueous solution after exposure to UV or VIS light as a function of time. (우) Storage modulus (G’: filled) and loss modulus (G": unfilled) of the PEG-PA-Z-PA-PEG aqueous solutions (20.0 wt.%) as a function of temperature. After exposure of the polymer aqueous solution to UV at 365 nm for 10minutes (UV), itwas exposed to atmospheric visible light for 2 hours (VIS).
표 Molecular weight of triblock copolymers studied.
표 a) Phase diagram of PEG-L-PA-DL-PA and PEG-DL-PA-L-PA aqueous solutions determined by the test-tube inverting method. Each data point is an average of three measurements. * is the temperature at which squeezing of the gel begins to be observed. b) Change in absorbance at 550 nm of PEG-L-PA-DL-PA (20.0wt.%) and PEG-DL-PA-L-PA (5.0wt.%) aqueous solutions as a function of temperature. PEG (M.W.=2000 Daltons) aqueous solution (20.0wt.%) was compared as a control. c) Change in storage modulus (G’) and loss modulus (G“) of PEG-L-PA-DL-PA (20.0wt.%) and PEG-DL-PA-L-PA (5.0wt.%) aqueous solutions as a function of temperature. PEG (M.W.=2000 Daltons) aqueous solution (20.0wt.%) was compared as a control.
표 a) Photos of sol (5 oC)/gel (37 oC or 60 oC) states of PEG-L-PA-DL-PA (20.0 wt.% in water) (A), and sol (5oC)/gel (37 oC)/squeezed gel (60 oC) states of PEG-L-PA-DL-PA (5.0 wt.% in water) (B). b) Scanning electron micros copy images of gels. Images of the gel of PEG-L-PA-DL-PA at 37 oC (A), the gel of PEG-DL-PA-L-PA at 37 oC (B-1), and the squeezed gel of PEG-DL-PA-L-PA at 60 oC (B-2) are compared. The scale bar is 10 mm.
표 CD spectra of PEG-L-PA-DL-PA (a) and PEG-DL-PA-L-PA (b) as a function of temperature at 0.025 wt. % in water.
표 Cryo-transmission electron microscopyimages of PEG-L-PA-DL-PA (a) and PEG-DL-PA-L-PA (b) aqueous solutions (1.0 wt. %) at 30 oC. The scale bar is 100 nm. c) Apparent size of PEG-L-PA-DL-PA and PEG-DL-PA-L-PA (1.0 wt. % in water) measured by dynamic light scattering at 30 oC.
표 a) Phase diagram of PEG-PAF aqueous solutions determined by the test-tube inverting method (n=3). b)Modulus of the PEG-PAF aqueous solutions (5.0 wt.%) as a function of temperature. G” of α-amino-w-methoxy-poly(ethylene glycol) (PEG) aqueous solution (5.0 wt.%) is compared as a control. G’ of the PEG aqueous solution was < 0.001 Pa(data not shown).
표 Apparent size of PEG-L-PAF(a) and PEG-D-PAF (b) as a function of temperature studied by dynamic light scattering of their aqueous solutions(1.0 wt.%). c) TEM images of PEG-L-PAF and PEG-D-PAF developed from their aqueous solutions (0.01 wt.%).
표 CD spectra of PEG-L-PAF and PEG-D-PAF aqueous solution (0.01 wt.%) as a function of temperature. The legends of LN and DN indicate PEG-L-PAF at N oC and PEG-D-PAF at N oC, respectively.
표 GPC profile of PEG-L-PAF (a) and PEG-D-PAF (b) after in vitro enzymatic degradation.
표 Gel duration of PEG-L-PAF (a) and PEG-D-PAF (b) after subcutaneous injection of the PEG-PAF aqueous solution (5.0 wt.% 0.5 mL/rat) into rats. The photos were taken 1 day, 5 days , and 15 days after the subcutaneous injection.
표 a) Phase diagram of the PEG-PAF aqueous solutionsdetermined by the test-tube inverting method (n=3). b) Storage modulus (G’) and loss modulus (G") of PEG-PAF aqueous solution (10.0 wt.%) as a function of temperature.
표 Storage stability of PEG-PAF. GPC traces of PEG-PAF (a) and change in pH of PEG-PAF aqueous solutions (b) during the storage of the polymer as an aqueous solution (10.0 wt.%) at room temperature of 15 oC. The polymer was sampled in 1 (1 D), 7 (7 D), 15 (15 D), 21 (21 D), and 28 (28 D) days of storage. 0 D and Raw indicate GPC traces of the PEG-PAF aqueous solution sampled in 0th day and the original PEG-PAF before preparingthe polymer aqueous solution, respectively.
표 (좌)In vivo gel formation by subcutaneous injection of a PEG-PAF aqueous solution (10.0 wt.%, 0.5 mL) into rats. The photos around the implanted site were taken 1 day(1D), 7 days (7D), and 15 days (15D) after the subcutaneous injection. (우) Tissue biocompatibility around the implant 7days after subcutaneous injection of a PEG-PAF aqueous solution (10.0 wt.%, 0.5 mL) into rats. H&E stained images with different magnifications (a: x4, b: x10, c: x20) around the implant.
표 a) In vitro hGH release profile from in-situ formed gel prepared from of a PEG-PAF aqueous formulation (0.5mL). The initialpolymer concentration was 10.0 wt.%, and loaded drug was 1.0 mg. n=3. b) Plasma drug concentration (Cp) of hGH after subcutaneous injection of PEG-PAF aqueous formulation (0.5 mL) containing hGH into rats. The initialpolymer concentration was 10.0 wt.% and the dose was 5.0 mg/kg rat. n=3. Control is the Cpof rats without injection of the hGH. ELISA assay was used for in vivo measurements of hGH.
표 Cryo-TEM images (a) and SEM images (b) of PA-PLX-PA gels at 37 oC prepared from polymer aqueous solutions (8.0 wt.%) as a function of HA content.
표 (right) 1H-NMR spectra of chitosan (D2O),PEG-PAF (CF3COOD)and CS-g-(PAF-PEG) (CF3COOD).
표 a)Phase diagram of CS-g-(PAF-PEG) aqueous solutions. The sol-to-gel transition temperatures were determined by the test-tube inverting method (n=3). b) Changes in modulus of the CS-g-(PAF-PEG) aqueous solutions (6.0wt.%) as a function of temperature. An aqueous solution (6.0 wt.%) of PEG (M.W=2000 Daltons) was compared. c) Frequency sweep test in the sol (10 oC) and gel(37 oC)phases of the polymer aqueous solution (6.0 wt.%).
표 Changes in pH of CS-g-(PAF-PEG) aqueous solution (1.0 wt.%) as a function of temperature. During the heating and cooling cycle, pH was measured with an increment of 1 oC per step. Phosphate buffer (10 mM) (PB) was used for comparison.
표 a) In situ gel formation and gel duration after subcutaneous injection of the CS-g-(PAF-PEG) aqueous solution (6.0 wt.%; 0.5 mL/rat) into rats. Photos around the implants (marked as white-dotted curves) were taken 6 hours (0 day), 1 day, 7 days, and 14 days after the injection. b) H & E stained image around the implanted gel 7 days after the injection. Arrows indicates vascular capillaries formed inside of the gel.
표 List of polymer studied.
표 Circular dichroism spectra of PI, PIII, and PV aqueous solutions (0.01 wt.%) as a function of temperature.
표 FTIR spectra of (10.0 wt.%) aqueous solutions in D2O as a function of temperature.
표 Schematic presentation of the PEG-PAF depending on the D-Alanine. Thin blue curves and thick black curves indicate PEG and PAF, respectively.
표 a)Phase diagram of PEG-PAF aqueous solutions determined by the test-tube inverting method. b) Changes in the storage modulus (G’) and the loss modulus (G”) of the PI (18.0 wt.%), PIII (18.0 wt.%), and PV (10.0 wt.%) aqueous solutions as a function of temperature.
표 좌) Comparison of 13C-NMR spectra of PI (gel), PII (sol), PIII (sol), PIV (sol), and PV (gel) aqueous systems at 50 oC. Sol-to-gel transition temperatures are 31 oC (PI), 56 oC (PII), 73 oC (PIII), 70 oC (PIV), and 26 oC (PV), respectively. The concentrations were 18.0 wt.% in D2O except for PV (10.0 wt.%). (우) Scanning electron microscopy images of the gels prepared from the polymers aqueous solution (18.0 wt.% in water except for PV (10.0 wt.%)). The scalebar is 10 μm.
표 Plasma drug concentration (Cp) of hGH after thesubcutaneous injection of PEG-PAF aqueous formulation (0.5 mL) containing hGH into rats.
표 (a) Changes in the modulus of the PEG-L-PA aqueous solution (7.6 wt.%) and MatrigelTM(M)(as received) as a function of temperature. The temperature program of the system varied over 4 oC-37 oC. (b) SEM images of PEG-L-LPA thermogel (PI) and MatrigelTM (M) at 37oC developed from PEG-L-PA aqueous solutions (7.6wt.%) and MatrigelTM (as received).
표 (a) Live and dead images of fibroblasts after 21 days incubation at 37 oC in PEG-L-PA thermogel (PI) and MatrigelTM. (b) Proliferation of fibroblasts in the PEG-L-PA thermogel (PI) and MatrigelTManalyzed by CCK-8. * and ** indicate the p value of p<0.01 and p<0.05, respectively. One hundred percent indicates the value on the day incubation began (day 0). Relative mRNA expression of type I collagen (c) and type III collagen (d) at day 7, 14 and 21 for 3D-cultured fibroblasts in the PEG-L-PA thermogel (PI) and MatrigelTM(M). The β-actin (house keeping gene) was used as an internal control. * and *** indicate the p value of p<0.01 and p<0.20, respectively. The data for 2D cultured fibroblasts on the cell culture plate (polystyrene) for 21 days were used for normalization as 100.
표 (a) Photo images of wounds during the healing process. Cell encapsulating PEG-L-PA thermogel (Cell/PI), cell-free PEG-L-PA thermogel (PI), and cell-free phosphate buffered saline (Control) were compared on day 0, 14, and 21 after injury. (b) Healing rate presented by relative wound area measured on day 0, 14, and 21 post-injury. (c) Histology around the wounds compared on day 14, and 21 post-injury. The space between the arrows indicates the center of original wound where the healing process is undergoing. The scale bar is 200 μm. The scale bar in the enlarged image is 100 μm.
표 온도민감성 졸-젤 전이 고분자 수용액의 의학적 응용.
표 (좌) Gel permeation chromatography 분석을 통한 남아있는 PEG-PAF hydrogel의 분석. (우) HPLC 분석을 통한 PEG-PAF의 효소분해 산물의 분석.
표 PEG-PAF와 음성대조군(Hyaluronic acid)과 양성대조군(poly (lysine))을 농도 별로 처리한 후 하루 뒤, L929 세포의 세포 생존률을 평가한 그래프.
표 PEG-PAF를 5000, 2500, 1000, 500, 250mg/mL의 농도로 처리한 CHO-K1 세포의 DNA fragmentation을 전기영동 한 결과. 음성 대조군으로는 신선한 배양액만 첨가한 것을 실험하였음.
표 (a) The photos of sol (4 oC) and gel (37oC) prepared from the PEG-PA aqueous solution (12.0 wt.%). (b) Modulus of the PEG-PA aqueous polymer solution (12.0 wt.%) at 4 oC and their thermogels at 37oC. MatrigelTM (M) was compared as a control. (c) Scanning electron microscope image of gels prepared from polymer aqueous solution of PEG-PA (12.0 wt.%) and MatrigelTM (M)asreceived. The scale bar is 20 μm.
표 Morphology of cells in the PEG-PA thermogel (top images) and MatrigelTM (bottom images) analyzed by the Live/Dead kit. 1 hour (0d), 7th day(7d),14th day(14d), and 21st day(21d) were compared. The scale bar is 40 µm.
표 (a) Gene expression of the BMSCs. The biomarkers for neurogenesis (βTub III), myogenesis (MyoD 1), chondrogenesis (Col II), and osteogensis (OCN) are shown. The data were normalized by the GAPDH and 0 d (1 hour) data. n=3. *, **, and *** indicate P0.1(Studentt-test), respectively, relative to MatrigelTM(M). (b) Immun ofluorescence of βTub III, MyoD 1, Col II, and OCN at 21stdayexpressedbytheBMSCsinthePEG-PA thermogel (top images) and MatrigelTM (bottom images). The scale bar is 40 μm.
표 In vivo study. (a) Gene expression of the BMSCs in the recovered gels. The biomarkers for βTub III, MyoD1, Col II, and OCN are shown. The data were normalized by the GAPDH and 0 d (1 hour) data. n= 3. (b) Histology of the implanted gel for 21 days in mice stained by alcian blue (A) and alizarin red S (B) staining methods. The scale bar is 100 μm.
표 List of polymers.
표 a) Phase diagram of PEG-L-PA aqueous solution determined by the test-tube inverting method. The data are an average of three measurements. b) The modulus of gel at 37 oC prepared from PEG-L-PA aqueous solutions of P50-06 (17.0 wt.%), P50-11 (4.6 wt.%), P50-25 (2.0 wt.%). MatrigelTM (M) at 37 oC as received as an aqueous solution is also compared. The modulus in sol states was less than 0.1 Pas at 5 oC (not shown).
표 (좌) SEM images of gels at 37 oC prepared from PEG-L-PA aqueous solutions of P50-06 (17.0 wt.%), P50-11 (4.6 wt.%), P50-25 (2.0 wt.%). The SEM image of MatrigelTM at 37 oC was also compared. The in situ formed gel at 37 oC was quenched into liquid nitrogen, followed by freeze-dried. The scale bar is 10 μm.. (우) Characterization of molecular phenotype of the ADSCs.
표 a) Proliferations of the ADSCs in the hydrogels analyzed by CCK-8. 100% is the cell number on the day when experiments started. The cell number is the average of the triplicateexperiments. The cell proliferation in 14th day could not be assayed in P50-06 gel due to the gel was disintegrated and assumed to be the similar with that of 7th day shown as a dotted graph. Other gels kept their physical integrity over the experimental time of 14 days. b) Live and dead images of cells during the culture. The scale bar is 50 am. M indicates MatrigelTM.
표 Gene expression by the ADSCs in 3D-cultured samples analyzed by RT-PCR. n=3 for all data. The biomarker for neurogenesis (β III Tub: a), myogenesis (Myo D 1: b), and chondrogenisis (Coll II: c) are shown. The data were normalized by GAPDH and 0thday. # and ## indicate p<0.05 and p<0.01 (Student t-test), respectively, relative to Matrige lTM The biomarkers for LPL and OCN were undetectable. M indicates MatrigelTM..
표 In vitro cytotoxicity evaluation by MTT assay. a) The relative cell viability of L929 cell after culture in the polymer solution at seven concentrations (10000, 1000, 100, 20, 10, 5, 1 mg/mL) for 5days. b) The relative cell viability of L929 cell after culture in the polymer solution at 100 mg/mL for 1, 3, 5 days. (PEG-L-PA, PEG-D-PA, PEG-L-PAF, L/DL-PA-PLX-PA-L/DL, Hyauronic acid (410K), Poly L-lysine (150K-300K))
표 In vitro genotoxicity evaluation of the polymer. a) The relative cell viability, b) The cell mutation frequency of CHO-K1 cell after culture in the polymer solution for 5hr by determining of the cloning efficiency. (PEG-L-PA w/o, w/S9, PEG-D-PA w/o, w/S9, PEG-L-PAF w/o, w/S9, L/DL-PA-PLX-PA-L/DL w/o, w/S9, Negative (Fresh media) or Positive (EMS) control w/o, w/S9.)
표 Histocompatibility around the implant 1, 3, 7, 14, 21, and 28days after subcutaneous injection of the in situ hydrogel into BALB/c mice. Optical microscopic images H&E stained images, imnunohistochemical stained by anti-CD68(general), CCR7(pro-inflammatory), CD163 (pro-healing). The scale bars are 100 mm.
표 Overview of the in-situ hydrogel formation through biologically benign processes.

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