Treating critical-sized bone defects is an important issue in the field of tissue engineering and bone regeneration. From the various biomaterials for bone regeneration, collagen is an important and widely used biomaterial in biomedical applications, hence, it has numerous attractive properties including biocompatibility, hyper elastic behavior, prominent mechanical properties, support cell adhesion, proliferation, and biodegradability. In the present study, collagen was extracted from duck's feet (DC) as a new collagen source and combined with quercetin (Qtn), a type of flavonoids found in apple and onions and has been reported to affect the bone metabolism, for increasing osteogenic differentiation. Further, improving osteoconductive properties of the scaffold hydroxyapatite (HAp) a biodegradable material was used.We prepared 0, 25, 50, and 100 μM Qtn/DC/HAp sponges using Qtn, DC, and HAp. Their physiochemical characteristics were evaluated using scanning electron microscopy, compressive strength, porosity, and Fourier transform infrared spectroscopy. To assess the effect of Qtn on osteogenic differentiation, we cultured bone marrow mesenchymal stem cells on the sponges and evaluated by alkaline phosphatase, 3-4-2, 5-diphenyl tetrazolium bromide assay, and real-time polymerase chain reaction. Additionally, they were studied implanting in rat, analyzed through Micro-CT and histological staining. From our in vitro and in vivo results, we found that Qtn has an effect on bone regeneration. Among the different experimental groups, 25 μM Qtn/DC/HAp sponge was found to be highly increased in cell proliferation and osteogenic differentiation compared with other groups. Therefore, 25 μM Qtn/DC/HAp sponge can be used as an alternative biomaterial for bone regeneration in critical situations. K E Y W O R D S bone marrow mesenchymal stem cells, bone regeneration, duck's feet collagen, hydroxyapatite, quercetin
Scaffolds mimicking structural and chemical characteristics of the native bone tissues are critical for bone tissue engineering. Herein, we have developed and characterized epigallocatechin gallate/duck's feet collagen/hydroxyapatite (EGCG/DC/HAp) composite sponges that enhanced the bone tissue regeneration. The three-dimensional composite sponges were synthesized by loading various amounts (i.e. 1, 5 and 10 μM) of EGCG to duck feet derived collagen followed by freeze-drying and then coating with hydroxyapatite. Several measuremental techniques were employed to examine the properties of the as-fabricated composite sponges including morphology and structure, porosity, compressive strength, etc. and as well compared with pristine duck feet derived collagen. SEM observations of EGCG/DC/HAp sponges showed the formation of a highly porous collagen matrix with EGCG embodiment. The porosity and pore size of sponges were found to increase by high EGCG content. The compressive strength was calculated as 3.54 ± 0.04, 3.63 ± 0.03, 3.89 ± 0.05, 4.047 ± 0.05 MPa for 1, 5 and 10 μM EGCG/DC/HAp sponges, respectively. Osteoblast-like cell (BMSCs isolated from rabbit) culture and in vivo experiments with EGCG/DC/HAp sponges implanted in nude mouse followed by histological staining showed enhanced cell internalization and attachment, cell proliferation, alkaline phosphatase expressions, indicating that EGCG/DC/HAp sponges have ahigh biocompatibility. Moreover, highEGCG content in the EGCG/DC/HAp sponges have led to increased cellular behavior. Collectively, the 5 μM of EGCG/DC/HAp sponges were suggested as the potential candidates for bone tissue regeneration.
BackgroundThe aim of this study was to investigate the osteogenesis effects of DC and DC/HAp sponge immersed in without and with dexamethasone.MethodsThe experimental groups in this study were DC and DC/HAp sponge immersed in without dexamethasone (Dex(−)DC and Dex(−)-DC/HAp group) and with dexamethasone (Dex(+)-DC and Dex(+)-DC/HAp group). We characterized DC and DC/HAp sponge using compressive strength, scanning electron microscopy (SEM). Also, osteogenic differentiation of BMSCs on sponge (Dex(−)DC, Dex(−)-DC/HAp, Dex(+)-DC and Dex(+)-DC/HAp group) was assessed by SEM, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay, alkaline phosphatase (ALP) activity assay and reverse transcription-PCR (RT-PCR).ResultsIn this study, we assessed osteogenic differentiation of BMSCs on Duck’s feet-derived collagen (DC)/HAp sponge immersed with dexamethasone Dex(+)-DC/HAp. These results showed that Dex(+)-DC/HAp group increased cell proliferation and osteogenic differentiation of BMSCs during 28 days.ConclusionFrom these results, Dex(+)-DC/HAp can be envisioned as a potential biomaterial for bone regeneration applications.
초록: 합성고분자인 poly(lactic-co-glycolic acid)(PLGA)는 우수한 생분해능을 가진 생체재료로서 널리 이용되고 있 으며, 천연유래 고분자인 실크와 소장 점막하 조직, 탈 미네랄화 골분 그리고 오리발 유래의 콜라겐은 생체 적합한 특성을 가지고 있다. 본 논문에서는 여러 가지 천연유래 고분자를 함유한 PLGA를 제작한 후, 지지체의 물성 및 표 면을 확인하였으며 토끼 골수유래줄기세포를 이용하여 지지체에 파종 후, 세포의 생존율과 세포분화 정도를 측정하 였고 in vivo 실험에서 면역조직학적 염색인 H&E와 Von Kossa을 실시하여 골분화 정도를 확인하였다. 결과적으로 오리발 유래 콜라겐과 소장 점막하 조직을 함유한 지지체에서 빠른 골 분화의 모습을 확인할 수 있었다.Abstract: Poly(lactic-co-glycolic acid) (PLGA) has been most widely used owing to its advantages such as good biodegradability and biocompatibility. Scaffolds made of natural source bio-materials like small intestinal submucosa (SIS), demineralized bone particles (DBP), silk fibroin (SF), and collagen-derived duck's feet collagen (DC) have been proved to be biocompatible. We manufactured composite scaffolds of all these natural biomaterials/PLGA. After making hybrid scaffolds, we measured mechanical strength of scaffolds and observed a porosity and surface morphology of the scaffolds by scanning electronic microscopy. Rabbit bone marrow mesenchymal stem cells (rBMSCs) were cultured, expanded and seeded on scaffolds. Inflammatory responses were checked at 1, 4 and 8 weeks using H&E & Von Kossa staining. DC and SIS group were found to be faster in bone regeneration than other groups. Finally, we have observed that scaffolds using biomaterials are effective in cell attachment, stem cell differentiation, and mineralized tissue formation.전하얀 · 김수민 · 전유신 · 국연지 · 송정은 · 강길선 폴리머, 제41권 제5호, 2017년 심근세포로 분화되어 조직 재생을 위한 시도가 진행되고 있 다. 4,6 이러한 골수유래줄기세포를 이식하기 위해 다공성 지지 체를 제작하는데, 이는 다공에 세포가 부착되고 증식하며 분 화되어 결국 특정 조직이 되도록 유도하는 역할을 한다. 또 한 이식 후 분해되고, 일정 수준의 기계적 강도를 가져야 한 다. 따라서 다공성 지지체는 조직공학의 필수 요소이기 때문 에 재생의학 산업기술로의 개발에 상당한 잠재력을 가지고 있다. 8,9 생체재료는 크게 분류하면 생분해성 합성고분자와 천연 분 자로 나뉜다. 10 조직공학에서 생분해성 합성고분자는 특정 세 포의 부착, 증식 및 분화를 위해 다공성 지지체로써 널리 이 용되고 있다. 합성고분자 중 poly(lactic-co-glycolic acid) (PLGA)는 poly(lactic acid)(PLA)와 poly(glycolic acid)(PGA) 의 공중합체로, 물리적, 화학적, 생분해성 특징이 많은 생물 학적 부분에서 이미 증명되었다. 8,11 특히, 공중합체의 비율을 다양하게 함으로써 생체적합성과 분해 속도, 기계적 특징이 쉽게 조절 가능하므로 가장 널리 사용되는 생분해성 고분자 중 하나이다. 12 반면, 천연 생체재료들은 합성물질보다 생체 조직 환경과 유사하여, 줄기 세포가 부착되고 증식하고 분화 하는데 더 많은 기능성 리간드를 제공할 수 있다. 6,13 그러나 다양한 응용에 필수적인 물리적, 화학적 성질은 합성물질보 다 조절하기 어렵고 분해 능력이 떨어진다. 13-15 따라서 본 연구에서는 합성고분자인 PLGA에 천연고분자 인 실크(silk), 동물의 소장점막하 조직(small intestinal submucosa, SIS), 탈 미네랄화 골분(dimineralized bone particle, DBP) 및 오리발 유래 콜라겐(collagen-derived duck's feet collagen, DC)을 첨가하여 지지체를 제작하고자 하였다. 실크는 피브로인과 세리신으로 이루어진 천연 고분자로, 글 리신, 알라닌, 세린과 같은 작은 분자량을 가진 아미노산이 전체 아미노산의 약 80~85%의 높은 농도로 피브로인을 구 성하고 있다. 16-21 이는 높은 인장 강도, 다양한 공정, 조절 가능한 생분해성, 쉬운 화학적 변형, 낮은 항원성과 비염증성을 가지고 있기 때 문에 생체재료로서 다양하게 적용되고 있다. 16,22-24 SIS는 세포가 존재하지 않는 조직으로 면역반응이 거의 일 어나지 않으며, 구성 성분의 90% 이상이 피부에 있는 제 I형 및 제 III형 콜라겐으로 구성되어 있고, 그 외에는 소량의 제 IV, V 및 VI형 콜라겐으로 구성된다. 25-28 이는 천연소재로서 높은 생체적합성을 가지며 다양한 사이토카인을 다량 함유하 고 있어 세포의 점착이나 성장, 이동, 분화 등의 세포의 기능 적인 면에 도움을 줌으로써 조직 재생 이외에도 많은 분야에 응용될 수 있다. 17-19 DBP는 골 형성 단백질과 성장인자를 포함하는 골 유도성 인자들로 구성하고 있어, 신생골 형성을 촉진시키기 위한 골 유도성 생체재료로 조직공학 분야에서 널리 사용되고 있다. 29 콜라겐은 동물 내에 다양한 결합 조직의 주요 구조 단백질
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