감마선 조사에 의한 헤파린이 도입된 박테리아 셀룰로오스의 골조 직공학용 지지체로서의 개발 및 특성분석

정성린 ( Sung In Jeong ),정진오 ( Jin Oh Jeong ),최종배 ( Jong Bae Choi ),신영민 ( Young Min Shin ),박종석 ( Jong Seok Park ),권희정 ( Hui Jeong Gwon ),노영창 ( Young Chang Nho ),안성준 ( Sung Jun An ),박만용 ( Man Young Park ) 한국조직공학과 재생의학회 2014 조직공학과 재생의학 Vol.11 No.2s

Bacterial cellulose(BC) scaffolds are generated from gel by gluconacetobacter hansenii TL-2C. BC has good properties such as high-burst pressure and the ultrafine highly nanofibrous structure of mimic natural extra celluar matrix(ECM) for tissue engineering. In this study, 2-aminoethyl methacrylate(AEMA) was grafted onto BC surface using gamma-irradiation and then heparin immobilized it using EDC/NHS reaction. The surface morphology of scaffolds were confirmed BC membranes by SEM for heparin and AEMA grafted it, so that it was not changed surface morphology. The result of TBO, fluorescamine staining, ATR-FTIR confirmed that heparin and AEMA immobilized it. Also result of in vitro test, heparin immobilized AEMA-BC proliferation of hMSC to higher than unmodified BC by CCK-8 and ALP activity then heparin immobilized AEMA-BC cumulative of BMP-2 was confirmed decrease by release test. Therefore, heparin and AEMA grafted BC scaffolds have been possible to tissue scaffold in bone tissue engineering.

방사선 가교된 유착방지용 Carboxymethyl Cellulose/Porcine Cartilage Acellular Matrix 수화젤 필름의 물리적 특성 및 부착 방지 평가

정성린 ( Sung In Jeong ),박종석 ( Jong-seok Park ),권희정 ( Hui-jeong Gwon ),안성준 ( Sung-jun An ),송보람 ( Bo Ram Song ),김영직 ( Young Jick Kim ),민병현 ( Byoung Hyun Min ),김문석 ( Moon Suk Kim ),임윤묵 ( Youn-mook Lim ) 한국화학공학회 2017 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.55 No.1

본 실험은 방사선을 이용하여 유착방지용 Carboxymethyl cellulose sodium salt (CMC)/Porcine Cartilage Acellular Matrix (PCAM) 수화젤 필름에 대해 연구하였다. 방사선 조사시 CMC/PCAM의 농도 및 혼합비율에 따른 필름의 형태학적 구조, 젤화율, 젤강도, 팽윤도 등을 분석하였다. 방사선에 의해 가교된 CMC/PCAM 필름은 CMC 필름보다 물리적인 특성인 젤화율이 낮게 측정되었다. 또한, 가교된 CMC 필름보다는 CMC/PCAM 필름에서 인간 혈관내피세포의 부착 및 증식율이 감소하였다. 우리는 PCAM에 함유한 세포의 항부착 성분이 도입된 CMC/PCAM 필름과 CMC 필름은 세포의 접착 및 증식율을 낮추는 것을 확인하였다. 결론적으로, 본 연구에서는 방사선 가교된 CMC/PCAM 수화젤 필름은 유착방지 향상을 위한 유착방지제로서 응용이 기대된다. In this study, intermolecular crosslinked carboxymethyl cellulose sodium salt (CMC) and porcine Cartilage Acellular Matrix (PCAM) blended hydrogel films for anti-adhesive barriers were prepared by gamma-ray radiation. The effects of the CMC/PCAM concentration and blending ratio on the morphology, gel fraction, gel strength, and degree of swelling were determined. The results indicated that crosslinked CMC/PCAM films show significantly lower the gel-fraction than CMC films. The degree of attachment and proliferation of human vascular endothelial cells on CMC/PCAM films was lower than the CMC films. We show the capacity of the CMC and PCAM to be hydrogel films, and the ability to reduce cell adhesion and proliferation on these films by modification with cell anti-adhesion molecules of PCAM. In conclusion, this study suggests that radiation cross-linked CMC/PCAM hydrogel films endowed with anti-adhesion ligands may allow for improved regulation of cell anti-adhesion behavior for prevent peritoneal adhesions.

감마선 조사에 의한 조직공학용 알긴산 나노섬유의 분해 영향

정성린 ( Sung In Jeong ),최종배 ( Jong Bae Choi ),신영민 ( Young Min Shin ),박종석 ( Jong Seok Park ),권희정 ( Hui Jeong Gwon ),노영창 ( Young Chang Nho ),강성수 ( Seong Soo Kang ),임윤묵 ( Youn Mook Lim ) 한국조직공학과 재생의학회 2014 조직공학과 재생의학 Vol.11 No.2s

Alginate, a linear un-branched polysaccharide derived from seaweed has shown great potential as a cell scaffold for the regeneration of many tissues. However, alginate is not naturally enzymatically degraded in ionically crossliked alginate htdrogels exhibit a remarkably slow degradation rate, which is typically months to years for their complete removal from injection site. The ionizing irradiation degrades polysaccharides through the free radicalinduced scission of glycosidic bonds of alginate. In this work, alginate/PEO nanofiber was irradiated 60Co γ-rays in the dose range of 50-300 kGy. This approach offers control over the degradation rate by varying the gamma-irradiation dose degree, as increasing the gamma-irradiation dose degree can increase the vulnerability of alginate nanofibers to hydrolysis under in vitro condition. And, the ability of electrospun a cell adhesive peptide Gly-Arg-Gly-Asp- Ser-Pro (GRGDSP) modified alginate and unmodified alginate nanofibrous scaffolds to support human mesenchymal stem cell (hMSCs) attachment and spreading was greatly enhanced on the adhesion ligand-modified compared to unmodified nanofibers, demonstrating the initial promise of this electrospun polysaccharide material with defined nanoscale architecture and cell adhesive properties for tissue regeneration applications.

백서 두개골 결손모델에서 골형성단백질이 함유된 헤파린 결합 박테리아 셀룰로오스 지지체가 골재생에 미치는 영향

정성린(Sung In Jeong),안성준(Sung-Jun An),김세은(Se Eun Kim),이소현(So-Hyoun Lee),박종석(Jong-Seok Park),권희정(Hui-Jeong Gwon),강성수(Seong Soo Kang),허중보(Jung-Bo Huh),임윤묵(Youn-Mook Lim) 한국고분자학회 2017 폴리머 Vol.41 No.2

조직공학용 나노섬유 지지체의 3차원 고공극 구조는 세포의 부착과 성장에 적합하고, 지지체 내에 영양분과 불순물이 통과되어야 한다. 최근에 우리 연구실에서는 감마선과 EDC/NHS 화학반응을 이용하여 조직공학용 bacterial cellulose(BC) 지지체에 heparin과 2-aminoethyl methacrylate(AEMA)을 표면 개질하였다. 본 연구에서는 백서 두개골 결손 모델을 이용하여 recombinant human bone morphogenic protein-2(rhBMP-2) 함유 유무에 따른 헤파린 결합 박테리아 셀룰로오스 지지체가 신생골 형성에 미치는 영향을 평가하려고 한다. 지지체의 골형성 효과를 평가하기 위해서 지지체를 백서 두개골에 식립 8주 후에 방사선학적 및 조직학적 평가를 통해 지지체에 대한 임상학적인 측면에서 골결손부 크기, 신생골 재생속도, 염증반응 등을 평가하였다. 골형성 단백질이 함유된 헤파린이 결합 박테리아 셀룰로오스는 두개골 결손 모델에서 골형성을 촉진하였다. 이는 조직공학적으로나 임상학적으로 우수한 특성을 가지고 있다고 사료된다. Nanofibrous scaffolds for tissue regeneration need to be three-dimensional and highly porous to support uniform cell attachment and proliferation, and need to have an interconnected and permeable pore network to promote nutrient and waste exchange. Our laboratory recently developed heparin and 2-aminoethyl methacrylate (AEMA) immobilized bacterial cellulose (BC) scaffolds for tissue engineering using gamma-irradiation and EDC/NHS chemistry. Here, we examined in vivo efficiency of a heparin immobilized AEMA-BC nanofibrous scaffolds under the condition of with or without recombinant human bone morphogenic protein-2 (rhBMP-2) for a therapeutic substrate in bone healing. To evaluate the bone healing effect of the scaffolds, they were evaluated at 8 weeks post-operatively according to the following criteria: size of residual defect area, speed of new bone formation, inflammation, and clinical applicability of the material. This heparin immobilized AEMA-BC scaffolds and rhBMP-2 independently facilitated a bone healing of the calvarial defect for its good biocompatibility. It holds a great potential for a clinical application and tissue engineering.

감마선을 이용한 돼지 무세포 연골세포외기질이 함유된 CMC 수화젤의 제조 및 특성 연구

정성린(Sung In Jeong),박종석(Jong-Seok Park),권희정(Hui-Jeong Gwon),안성준(Sung-Jun An),임윤묵(Youn-Mook Lim),송보람(Bo Ram Song),김영직(Young Jick Kim),민병현(Byoung-Hyun Min),김문석(Moon Suk Kim) 한국고분자학회 2017 폴리머 Vol.41 No.1

본 연구는 감마선을 이용하여 가교된 유착방지용 carboxymethyl cellulose sodium salt(CMC-Na)와 porcine cartilage acellular matrix(PCAM) 수화젤을 제조하였다. CMC는 높은 농도의 수용액에서 감마선에 의해 가교가 되는 천연고분자 중에 하나이다. 방사선 가교된 CMC/PCAM 수화젤의 물리/화학적 특성을 알기 위해 SEM, 젤화율, 젤강도, 팽윤도, FTIR-ATR 등 분석을 통해 특성을 확인하였다. 감마선으로 가교된 CMC/PCAM 수화젤에서 PCAM 비율이 증가할수록 젤화율과 젤강도는 감소하였고 팽윤도는 증가하였다. 또한 CMC/PCAM 수화젤에서 세포의 부착도와 증식도가 CMC 수화젤에 비해 낮음을 확인하였다. 결론적으로, 본 연구에서는 방사선으로 가교된 CMC/PCAM 수화젤은 유착방지를 위한 유착방지제로서 응용이 기대된다. In this study, a novel crosslinked carboxymethyl cellulose sodium salt (CMC) and porcine cartilage acellular matrix (PCAM) hydrogel for adhesion prevention were prepared by gamma-irradiation. CMC is known as a kind of cross-linked natural polymer at highly concentrated aqueous solution under action of gamma-irradiation. The radiationcrosslinked CMC/PCAM hydrogels were characterized by scanning electron microscope (SEM), gel-fraction, gel strength, swelling behavior, and FTIR-ATR spectroscopy. The results showed that the PCAM addition increased the swelling behavior and decreased the gel-fraction and gel strength of CMC/PCAM hydrogels. The degree of attachment and proliferation of human fibroblast on CMC/PCAM hydrogel was lower than the CMC hydrogels. In conclusion, this study suggests that the radiation cross-linked CMC/PCAM hydrogels may be useful as an anti-adhesion barrier.

Preparation and Characterization of Radiation Fabricated β-Glucan Hydrogels for Wound Dressing

Hui-Jeong Gwon(권희정),Sung-Jun Ahn(안성준),Jin-Oh Jeong(정진오),Su-Min Kim(김수민),Jong-Seok Park(박종석),Sung In Jeong(정성린),Young-Chang Nho(노영창),Youn-Mook Lim(임윤묵) 한국고분자학회 2017 폴리머 Vol.41 No.4

화학가교제의 사용없이 감마선을 이용하여 β-glucan, poly(vinyl alcohol)(PVA), poly(vinyl pyrrolidone)(PVP), κ-carrageenan(κC), glycerin으로 구성된 고분자 하이드로젤을 제조하였다. 25, 50, 75 kGy로 감마선 조사선량을 달리하여 하이드로젤을 제조한 후, 젤화율, 팽윤도, 압축강도의 측정을 통해 제조된 하이드로젤의 물리적 특성을 확인하였다. 조사선량의 증가에 따라 하이드로젤의 가교밀도가 증가되어 젤화율과 압축강도는 증가하였고, 팽윤도는 감소되었다. 제조된 베타글루칸 하이드로젤로 손상된 조직의 재생 특성을 평가해 보기 위해 동물실험을 수행하였고, 그 결과 베타글루칸이 함유된 하이드로젤이 빠른 상처 치유능을 나타내었고, 상용제품과 비교 시에도 동등한 치료효과를 관찰할 수 있었다. 따라서 감마선으로 제조된 β-glucan/PVA/PVP/κC/glycerin 하이드로젤은 화학가교제 독성의 우려 없이 β-glucan에 의한 향상된 치료효과와 더불어 향후 조직재생용 소재로서 유용하게 사용될 수 있을 것으로 사료된다. Hydrogels consisted of β-glucan, poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP), κ-carrageenan (κC) and glycerin were prepared by gamma-ray irradiation for damaged tissue regeneration. Irradiation doses of 25, 50, and 75 kGy were exposed, respectively, to the β-glucan hydrogel to evaluate the effect of irradiation dose on physical properties. The physical properties were examined such as gel fraction, absorption ratio, and compressive strength. It was found that the gel fraction and the compressive strength increased with increasing the irradiation dose. This is due to the fact that the crosslinking density increases with increasing the irradiation dose, whereas the absorption ratio decreased with increasing the irradiation dose. On observing the wound healing of rat skin, the resulting hydrogels accelerated the wound repair, which can be attributed to the release of β-glucan from the hydrogel. Therefore, radiation fabricated β-glucan/PVA/PVP/κC/glycerin blended hydrogel was suitable for wound healing and could be considered as good tissue regeneration biomaterials without chemical toxicity.

방사선 이용 미생물 발효 셀룰로오스 다공성 폼 제조 및 특성

권희정 ( Hui Jeong Gwon ),신영민 ( Young Min Shin ),최종배 ( Jong Bae Choi ),임종영 ( Jong Young Lim ),정진오 ( Jin Oh Jeong ),정성린 ( Sung In Jeong ),박종석 ( Jong Seok Park ),김진규 ( Jin Kyu Kim ),임윤묵 ( Youn Mook Lim ),최 한국환경생물학회 2013 환경생물 : 환경생물학회지 Vol.31 No.4

Microbial fermented cellulose gel, citrus gel (CG), was successfully fabricated to porous foam by radiation treatment and freeze drying. The chemically induced radiation was used to create highly porous foam and further freeze drying of the CG produced tough foams with interconnected open pores for use in tissue engineering. The microstructure of the CG foam was controlled by varying the irradiation dose and quenching temperature with pore size ranging from several microns to a few hundred microns. Tensile strength and Gurley value of the CG foam were influenced by irradiation dose. These radiation induced CG foams are promising scaffolds for tissue engineering.

방사선을 이용한 느릅나무 추출 다당류가 함유된 하이드로젤의 제조 및 항염증성 평가

권희정(Hui Jeong Gwon),박은지(Eun Ji Park),최종배(Jong Bae Choi),임종영(Jong Young Lim),정진오(Jin Oh Jeong),신영민(Young Min Shin),정성린(Sung In Jeong),박종석(Jong Seok Park),임윤묵(Youn Mook Lim),최영훈(Young Hun Choi),김상숙(Sang 한국고분자학회 2014 폴리머 Vol.38 No.1

본 연구에서는 방사선 기술을 이용하여 항염증 특성을 가지는 천연 추출물을 함유하는 고분자 하이드로젤을 제조하여 항염증 패치로서의 유용성을 확인하였다. 천연 추출물로는 오래 전부터 전통 민간요법으로 항염제나 궤양 및 종양 치료 등에 사용되고 있는 느릅나무를 사용하였고, 생체 적합성 고분자인 poly(vinyl alcohol)(PVA)와 보습률 향상을 위해 글리세린을 첨가하여 감마선 가교에 의해 다당류 함유 고분자 하이드로젤을 제조하였다. 제조된 하이드로젤은 항염증 패치로 사용하기에 우수한 기계적 물성을 나타내었고, 사이토카인 분비를 효과적으로 억제함은 물론 세포독성도 나타나지 않는 것을 확인하였다. 결론적으로 느릅나무 추출 다당류가 함유된 하이드로젤은 항염증패치로써 유용하게 사용될 수 있음을 확인하였다. In this study, polysaccharide-poly(vinyl alcohol) (PVA) hydrogels were prepared by using γ-ray and evaluatedfor potential application as an anti-inflammation patch. Ulmus davidiana var. japonica (UD), one of polysaccharides hasbeen particularly used as an oriental remedy for the treatment of inflammation and ulcers. PVA as a biocompatible polymerand glycerin as a moisturizer were blended with the UD, and its hydrogels were prepared by radiation crosslinking. Characterizations for UD hydrogels were performed by using cytotoxicity assay, antioxidant activity test, and physicochemicaltest such as gel fraction ratio, and swelling behavior. The results showed that these UD hydrogels had excellentphysical properties, anti-inflammation activity, and non-cytotoxicity on the cells. Therefore, these polysaccharidebased-UD hydrogels can be effectively used as an inflammation patch.

감마선을 이용한 아크릴산이 도입된 골조직공학용 PCL/BCP 나노섬유 지지체의 개발

정진오(Jin-Oh Jeong),정성린(Sung In Jeong),신영민(Young Min Shin),박종석(Jong-Seok Park),권희정(Hui-Jeong Gwon),안성준(Sung-Jun An),허중보(Jung-Bo Huh),신흥수(Heungsoo Shin),임윤묵(Youn-Mook Lim) 한국고분자학회 2015 폴리머 Vol.39 No.3

Polycaprolactone(PCL)과 biphasic calcium phosphate(BCP)는 생체적합성 및 골 형성 촉진 등으로 인해 정형외과 소재로 사용되고 있다. 하지만, PCL은 표면이 소수성으로 인해 세포의 부착 및 증식에 제한적이기 때문에 이를 극복하기 위해 본 연구에서는 감마선을 이용하여 골 재생을 위한 친수성이 향상된 PCL/BCP 나노섬유를 제조하였다. 나노섬유는 전기방사를 통해 제작했으며, 감마선을 이용하여 acrylic acid(AAc)를 도입하였다. SEM을 통해 나노섬유 표면을 확인하였고, AAc가 도입된 나노섬유 위에서 MG63의 초기 생존율이 현저히 증가한 것을 확인하였다. 알칼리성 포스파테이즈 활성은 1.239±0.226 nmole/μg/min으로 개질되지 않은 나노섬유(0.590±0.286 nmole/μg/min) 보다 증가하였다. 따라서, AAc가 도입된 PCL/BCP 나노섬유는 골조직 재생에 활용될 수 있을 것으로 기대된다. Polycaprolactone (PCL) and biphasic calcium phosphate (BCP) have been considered as useful materials for orthopedic devices and osseous implants because of their biocompatibility and bone-forming activity. However, PCLbased scaffolds have hydrophobic surfaces reducing initial cell adhesion or proliferation. To overcome the limitation, we fabricated surface-modified PCL/BCP nanofibers using gamma-irradiation for bone tissue engineering. PCL/BCP nanofibers were prepared by electrospinning and then we supplemented hydrophilicity by introducing acrylic acid (AAc) through gamma-irradiation. We confirmed the surface of nanofibers by SEM, and then the initial viability of MG63 was significantly increased on the AAc grafted nanofibers, and alkaline phosphatase activity(1.239±0.226 nmole/μg/min) improved on the modified nanofibers than that on the non-modified nanofibers(0.590±0.286 nmole/μg/min). Therefore, AAc-grafted nanofibers may be a good tool for bone tissue engineering applications.

감마선 이용 AEMA 도입 PCL 나노섬유의 제조 및 특성분석

서다은(Da-Eun Seo),정성린(Sung In Jeong),박종석(Jong-Seok Park),권희정(Hui-Jeong Gwon),안성준(Sung-Jun An),이병헌(Byoung Hun Lee),임돈순(Don-Sun Im),신흥수(Heungsoo Shin),임윤묵(Youn-Mook Lim) 한국고분자학회 2017 폴리머 Vol.41 No.1

폴리카프로락톤(polycaprolactone, PCL)은 생체적합성이 우수하고 시간이 지나면 체내에서 생분해되어 조직공학용 생체재료로 많이 사용되고 있다. 하지만, 소수성 표면과 낮은 세포 부착성을 나타내는 단점이 있다. 따라서 본 연구에서는 PCL 필름 및 전기방사를 통한 나노섬유 지지체를 제조하고 감마선을 이용하여 친수성과 양전하의 아민기를 갖는 2-아미노에틸메타크릴레이트(2-aminoethylmethacrylate, AEMA)로 표면을 개질하였다. 감마선을 이용하여 표면 개질된 PCL 필름과 나노섬유의 분석 결과, ATR-FTIR을 통해 감마선 선량과 반응 표면적이 증가할수록 표면의 아민기가 증가함을 확인하였다. 아민기의 증가로 인해 친수성이 증가했음을 확인하였고 단백질 흡착량이 정전기적 인력으로 인해 증가함을 확인하였다. 이를 통해 AEMA가 도입된 PCL 필름보다 나노섬유 지지체의 경우 세포적합성이 향상된 조직공학용 지지체로서의 응용 가능성이 매우 높을 것으로 기대된다. Polycaprolactone (PCL) has been applied to tissue engineering because it has excellent biocompatibility and biodegradable properties. However, there is a limit to hydrophobic surface and low cell adhesion, therefore the PCL requires surface modification for increasing hydrophilicity and cell adhesion capacity. In this study, PCL films and nanofibers were manufactured by solvent casting and electrospinning, respectively. They were modified by 2-aminoethylmethacrylate (AEMA) that had hydrophilicity and amine groups of positive charge using gamma-irradiation. The surface properties of AEMA-grafted PCL films and nanofibers were confirmed by ATR-FTIR, and amine groups were increased by surface area and gamma-irradiation dose. A significantly higher amount of negative-charged proteins adhered to hydrophilic and cationic surface of AEMA-grafted PCL nanofibers than that of AEMA-grafted PCL films, because of increased amine groups and electrostatic interaction. The results show that AEMA-grafted PCL nanofibers are possible to apply tissue engineering scaffolds.