KR20200110495A - Hydrogel for bone regeneration and preparation method thereof - Google Patents

Hydrogel for bone regeneration and preparation method thereof Download PDF

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KR20200110495A
KR20200110495A KR1020190028576A KR20190028576A KR20200110495A KR 20200110495 A KR20200110495 A KR 20200110495A KR 1020190028576 A KR1020190028576 A KR 1020190028576A KR 20190028576 A KR20190028576 A KR 20190028576A KR 20200110495 A KR20200110495 A KR 20200110495A
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bone
gel
nac
gnps
gold nanoparticles
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권일근
이동현
허동녕
이재서
나하람
문호진
윤연희
이상진
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경희대학교 산학협력단
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Abstract

The present invention relates to a hydrogel for promoting bone regeneration or bone formation comprising gold nanoparticles, antioxidants, or a combination of gold nanoparticles and antioxidants, a method for preparing the same, and a use thereof. A composition for promoting bone regeneration or bone formation of the present invention is not toxic and thus has excellent biocompatibility.

Description

골 재생용 수화젤 및 이의 제조방법{HYDROGEL FOR BONE REGENERATION AND PREPARATION METHOD THEREOF}Hydration gel for bone regeneration and its manufacturing method {HYDROGEL FOR BONE REGENERATION AND PREPARATION METHOD THEREOF}

본 발명은 금 나노입자, 항산화 물질, 또는 금 나노입자 및 항산화 물질의 결합체를 포함하는 골 재생 또는 골 형성 촉진용 수화젤, 이의 제조방법 및 이의 용도에 관한 것이다.The present invention relates to a hydration gel for promoting bone regeneration or bone formation comprising gold nanoparticles, antioxidants, or a combination of gold nanoparticles and antioxidants, a method for preparing the same, and a use thereof.

골 결손은 사고, 골수염, 골종양 등의 원인으로 인해 뼈의 일부분이 소실되어 발생된다. 최근에 이를 치료하기 위해 자가골, 이종골 합성골 등을 이식하여 골 재생을 유도하는 방법이 사용되고 있다. 과거에는 대부분 자가 뼈 이식(autogenous bone grafting)이 시술되었으나 최근 인공 뼈 이식(artificial bone grafting)의 시술 비중이 늘어나고 있는 추세이다. 그러나, 상기와 같은 방법들은 자가골 채취를 위한 추가 수술, 면역반응 우려, 조기 흡수 등이 단점이 존재한다. 이러한 문제 개선을 위해 효과적인 골 재생을 유도하는 성장인자(growth factor), 약물, 단백질 등을 적용한 연구가 진행되고 있다. 하지만 이것들은 대부분 가격이 비싸고 보관 및 사용이 어렵다는 단점이 있어 개선이 필요하다.Bone defects are caused by the loss of part of the bone due to causes such as accidents, osteomyelitis, and bone tumors. Recently, in order to treat this, a method of inducing bone regeneration by transplanting autogenous bone or synthetic bone of a heterogeneous bone has been used. In the past, most autogenous bone grafting has been performed, but recently, the proportion of artificial bone grafting is increasing. However, such methods have disadvantages such as additional surgery for autogenous bone collection, fear of immune response, and early absorption. In order to improve these problems, studies are underway that apply growth factors, drugs, and proteins that induce effective bone regeneration. However, most of these are expensive and have the disadvantages of being difficult to store and use, so improvement is needed.

또한, 골이식재나 차단막 같은 생체재료 자체를 이식재로서 활용하는 경우, 자체가 골전도성을 가지는 어떠한 매개체로서의 역할은 할 수 있어도 치료기간의 단축을 위해 필수적인 초기 골형성을 위한 골유도력은 지니고 있지 않아 수술 후 상당기간 후에 골형성이 이루어지는 한계가 있다.In addition, when a biomaterial such as a bone graft material or a barrier membrane itself is used as a graft material, surgery itself does not have bone induction power for initial bone formation, which is essential for shortening the treatment period, although it can serve as any medium having bone conductivity. There is a limit to bone formation after a considerable period of time.

이에, 생체적합성이 우수하여 부작용을 최소화하면서도 골 재생 또는 골 형성 효과가 우수한 제재의 개발이 지속적으로 요구되고 있다.Accordingly, there is a continuous demand for the development of a material excellent in bone regeneration or bone formation effect while minimizing side effects due to excellent biocompatibility.

O. J. Sul, J. C. Kim, T. W. Kyung, H. J. Kim, Y. Y. Kim, S. H. Kim, J. S. Kim and H. S. Choi, Biosci., Biotechnol., Biochem., 2010, 74, 2209-2213 O. J. Sul, J. C. Kim, T. W. Kyung, H. J. Kim, Y. Y. Kim, S. H. Kim, J. S. Kim and H. S. Choi, Biosci., Biotechnol., Biochem., 2010, 74, 2209-2213

본 발명의 목적은 금 나노입자, 항산화 물질, 또는 금 나노입자 및 항산화 물질의 결합체를 포함하는, 골 재생 또는 골 형성 촉진용 수화젤을 제공하는 것이다. An object of the present invention is to provide a hydrogel for promoting bone regeneration or bone formation, comprising gold nanoparticles, antioxidants, or a combination of gold nanoparticles and antioxidants.

본 발명의 다른 목적은 상기 골 재생 또는 골 형성 촉진용 수화젤을 포함하는 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition comprising a hydrogel for promoting bone regeneration or bone formation.

본 발명의 또 다른 목적은 상기 약학적 조성물을 투여하는 단계를 포함하는 골질환의 치료방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating bone diseases comprising administering the pharmaceutical composition.

본 발명의 또 다른 목적은 상기 골 재생 또는 골 형성 촉진용 수화젤의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method of preparing a hydrogel for promoting bone regeneration or bone formation.

상기와 같은 목적을 달성하기 위한 본 발명의 일 측면은 i) 금 나노입자 및 항산화 물질의 결합체 또는 ii) 금 나노입자 또는 금 나노입자 및 항산화 물질의 결합체를 포함하는 수화젤을 포함하는, 골 재생 또는 골 형성 촉진용 조성물에 관한 것이다.One aspect of the present invention for achieving the above object is i) a combination of gold nanoparticles and an antioxidant, or ii) a hydration gel comprising gold nanoparticles or a combination of gold nanoparticles and antioxidants, bone regeneration Or it relates to a composition for promoting bone formation.

본 발명에서, “골 재생”은 골 세포의 증식 또는 골 세포로의 분화를 통해 골이 재생산되는 모든 과정 및 형태를 말하며, 세포의 증식 등을 통해 조직이 재생산되어 골 조직 또는 골 결손부가 수복되는 것 역시 포함된다.In the present invention, "bone regeneration" refers to all processes and forms in which bone is regenerated through proliferation of bone cells or differentiation into bone cells, and bone tissue or bone defects are repaired by regenerating tissue through cell proliferation or the like. It also includes.

본 발명에서, “골 형성 촉진”은 골 세포의 증식 또는 골 형성 세포로의 분화를 촉진하거나, 골 형성과 관련된 유전자의 발현이나 단백질의 활성을 촉진하는 등 골의 형성이 유도되거나 빠르게 형성될 수 있는 모든 과정 및 형태를 말한다.In the present invention, "promoting bone formation" may induce or rapidly form bone formation, such as promoting the proliferation of bone cells or differentiation into bone forming cells, or promoting the expression of genes or proteins related to bone formation. All processes and forms that are present.

본 발명에서, “수화젤”은 친수성을 가진 고분자가 결합을 통해 3차원적으로 가교된 구조를 가져 수분을 많이 포함할 수 있는 생체 내의 세포 외 기질(extracellular matrix)을 모방한 물질이다. 체내 환경과 유사하게 많은 수분을 포함하고 있어서 친수성인 단백질 약물의 구조와 생리활성을 보존할 수 있는 최적의 환경을 제공할 수 있으며, 내부에 약물을 담지하고 조절함으로써 약물의 서방출이 가능하다. In the present invention, the “hydration gel” is a material that mimics an extracellular matrix in a living body that can contain a large amount of moisture by having a structure in which a polymer having hydrophilicity is crosslinked in three dimensions through bonding. Similar to the body environment, it contains a lot of water, so it can provide an optimal environment for preserving the structure and physiological activity of a hydrophilic protein drug, and sustained release of the drug is possible by carrying and controlling the drug inside.

구체적으로, 상기 수화젤은 젤라틴 및 티라민의 결합체를 포함하는 것일 수 있다. 상기 젤라틴은 생체 조직의 상당부분을 구성하는 섬유상 단백질인 콜라젠으로부터 얻어진 물질로 콜라젠에 비해 분자량이 낮고 생분해성과 생체 적합성이 뛰어난 바, 본 발명의 젤라틴 및 티라민의 결합체를 포함하는 수화젤은 독성 없이 특히 우수한 생체 적합성을 나타낸다.Specifically, the hydrating gel may include a combination of gelatin and tyramine. The gelatin is a material obtained from collagen, a fibrous protein constituting a significant part of living tissue, and has a lower molecular weight compared to collagen, and has excellent biodegradability and biocompatibility.The hydrogel containing the combination of gelatin and tyramine of the present invention is not particularly toxic. It shows excellent biocompatibility.

또한 구체적으로, 상기 항산화 물질은 N-아세틸 시스테인(N-acetyl cysteine)인 것일 수 있으나, 이에 제한되는 것은 아니다. 더욱 구체적으로 금 나노입자와 결합하기 위하여 상기 항산화 물질은 -SH 또는 -NH2 그룹이 존재하는 것일 수 있다. 상기 항산화 물질은 라디칼의 생성이나 혹은 이미 생성된 라디칼의 기능 전해, 라디칼에 의한 세포 손상 복구를 통해 산화스트레스를 줄이는데 사용되는 것으로서, N-아세틸 시스테인 외에도 알부민, 세룰로플라스민(ceruloplasmin), 페리틴(ferritin), 아스코르브산, 글루타치온, 우린산(uric acid), 토코페롤, 카로티노이드 (폴리)페놀류 등 역시 제한없이 적용될 수 있다. In addition, specifically, the antioxidant may be N-acetyl cysteine, but is not limited thereto. More specifically, in order to bind to gold nanoparticles, the antioxidant material may have -SH or -NH 2 groups. The antioxidant is used to reduce oxidative stress through the generation of radicals, electrolysis of the function of already generated radicals, and repair of cell damage by radicals.In addition to N-acetyl cysteine, albumin, ceruloplasmin, and ferritin ( ferritin), ascorbic acid, glutathione, uric acid, tocopherol, carotenoid (poly)phenols, etc. can also be applied without limitation.

본 발명 일 실시예에서는 i) 금 나노입자 및 항산화 물질의 결합체(G-NAC), ii) 금 나노입자 또는 금 나노입자 및 항산화 물질의 결합체를 포함하는 수화젤(Gel-Ty/GNPs, Gel-Ty/G-NAC)을 제조하였으며, 상기 물질들이 독성이 없으며 높은 알칼리 포스파타아제(Alkaline phosphatase, ALP) 활성을 나타내는 것을 확인하였다. 알칼리 포스파타아제는 세포막 효소로서 초기 골 재생시 풍부하게 존재하는 것으로 알려져 있으며, 알칼리 포스파타제 발현은 골 재생 증가와 연관되는 바, 본 발명의 수화젤은 골 재생 및 골 형성 용도로 활용될 수 있다.In one embodiment of the present invention, i) a combination of gold nanoparticles and an antioxidant (G-NAC), ii) a hydration gel (Gel-Ty/GNPs, Gel-) including gold nanoparticles or a combination of gold nanoparticles and antioxidants. Ty/G-NAC) was prepared, and it was confirmed that the materials were non-toxic and exhibited high alkaline phosphatase (ALP) activity. Alkaline phosphatase is known to be abundantly present during initial bone regeneration as a cell membrane enzyme, and alkaline phosphatase expression is associated with increased bone regeneration, and the hydration gel of the present invention can be used for bone regeneration and bone formation.

구체적으로, 상기 금 나노입자는 10 μM 내지 400 μM 의 농도로 포함되는 것일 수 있다.Specifically, the gold nanoparticles may be contained in a concentration of 10 μM to 400 μM.

본 발명 일 실시예에서는 10 μM 내지 400 μM 농도의 금 나노입자가 포함된 금 나노입자를 포함하는 수화젤을 처리한 경우, 세포 독성없이 생존능이 유지되거나 시간에 따라 증가하면서도(도 20), 금 나노입자가 포함되어 있지 않은 대조군 대비 우수한 알칼리 포스파타아제 활성을 나타냄을 확인하였다(도 21).In an embodiment of the present invention, when a hydrogel containing gold nanoparticles containing gold nanoparticles of 10 μM to 400 μM is treated, the viability is maintained without cytotoxicity or increased with time (FIG. 20), It was confirmed that the alkali phosphatase activity was superior to that of the control group containing no nanoparticles (FIG. 21).

또한 구체적으로, 상기 금 나노입자 및 항산화 물질의 결합체는 10 μM 내지 400 μM 의 농도로 포함되는 것일 수 있다. In addition, specifically, the combination of the gold nanoparticles and the antioxidant may be contained in a concentration of 10 μM to 400 μM.

본 발명 일 실시예에서는 금 나노입자 및 항산화 물질의 결합체를 처리한 경우, 세포 생존능이 높으면서도(도 10), 대조군 대비 우수한 알칼리 포스파타아제 활성을 나타냄을 확인하였다(도 11). In one embodiment of the present invention, when the gold nanoparticles and the combination of antioxidants were treated, it was confirmed that the cell viability was high (FIG. 10) and showed superior alkaline phosphatase activity compared to the control (FIG. 11).

또한, 금 나노입자 및 항산화 물질의 결합체를 포함하는 수화젤을 처리한 경우, 대조군에 비해 현저히 높은 알칼리 포스파타아제 활성을 나타냄을 확인하였는 바, 우수한 골 분화 효과를 나타냄을 확인하였다(도 22).In addition, when the hydrogel containing a combination of gold nanoparticles and antioxidants was treated, it was confirmed that the alkali phosphatase activity was significantly higher than that of the control group, and excellent bone differentiation effect was observed (FIG. 22). .

이에 따라, 본 발명의 금 나노입자 및 항산화 물질의 결합체, 금 나노입자 또는 금 나노입자 및 항산화 물질의 결합체를 포함하는 수화젤을 포함하는 조성물은 골 재생 또는 골 형성 촉진에 우수한 효과를 나타내며, 이에 따라 골 재생, 골 형성 등 골 관련 조직의 재생 및 수복에 활용될 수 있으며, 골 재생 또는 골 형성이 필요로 되는 골 질환에 활용될 수 있다.Accordingly, the composition comprising a hydration gel comprising a combination of gold nanoparticles and an antioxidant material of the present invention, gold nanoparticles, or a combination of gold nanoparticles and an antioxidant material exhibits excellent effects in promoting bone regeneration or bone formation. Accordingly, it can be used for regeneration and repair of bone-related tissues such as bone regeneration and bone formation, and can be used for bone diseases that require bone regeneration or bone formation.

본 발명의 다른 측면은 상기 골 재생 또는 골 형성 촉진용 조성물을 포함하는, 골 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다. 구체적으로, 상기 약학적 조성물은 골다공증, 암세포의 골전이에 의해 초래되는 뼈의 손상, 골연화증, 구루병, 섬유성 골염, 무형성 골질환, 대사성 골질환, 골용해, 백혈구 감소증, 뼈의 기형, 고칼슘혈증, 류마티스성 관절염, 골관절염, 골수염, 변성 관절증 및 골종양으로 구성되는 군으로부터 선택되는 하나 이상의 질환의 골 재생 또는 골 형성 촉진용에 관한 것일 수 있다.Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating bone diseases, including the composition for promoting bone regeneration or bone formation. Specifically, the pharmaceutical composition is osteoporosis, bone damage caused by bone metastasis of cancer cells, osteomalacia, rickets, fibrotic osteopathy, amorphous bone disease, metabolic bone disease, osteolysis, leukopenia, bone malformation, hypercalcemia , Rheumatoid arthritis, osteoarthritis, osteomyelitis, degenerative arthritis and bone tumors may be related to the promotion of bone regeneration or bone formation of one or more diseases selected from the group consisting of.

본 발명에서, “골 질환”은 골 밀도가 감소되어 발생하는 모든 질환을 말한다. 상기 골다공증, 암세포의 골전이에 의해 초래되는 뼈의 손상, 골연화증, 구루병, 섬유성 골염, 무형성 골질환, 대사성 골질환, 골용해, 백혈구 감소증, 뼈의 기형, 고칼슘혈증, 류마티스성 관절염, 골관절염, 골수염, 변성 관절증 및 골종양으로 구성되는 군으로부터 선택되는 하나 이상의 질환은 직접적 또는 타 질환에 의해 부수적으로 발생되는 골 감소에 기인한 것으로서, 예방 또는 치료를 위해서는 골 재생 또는 골 형성이 요구된다.In the present invention, "bone disease" refers to all diseases caused by a decrease in bone density. Osteoporosis, bone damage caused by bone metastasis of cancer cells, osteomalacia, rickets, fibrotic osteopathy, amorphous bone disease, metabolic bone disease, osteolysis, leukopenia, bone deformity, hypercalcemia, rheumatoid arthritis, osteoarthritis, One or more diseases selected from the group consisting of osteomyelitis, degenerative arthrosis, and bone tumors are caused by bone loss that is directly or incidentally caused by other diseases, and bone regeneration or bone formation is required for prevention or treatment.

더욱 구체적으로, 본 발명의 약학적 조성물은 골다공증의 예방 또는 치료에 적용될 수 있다. 상기 골다공증은 원발성(일차성) 골다공증과 속원성(이차성)골다공증으로 나눌 수 있다. 원발성 골다공증은 칼슘과 비타민 D를 적절히 섭취하지 않거나 육체적 운동을 적절히 하지 않거나 흡연하거나 폐경기 이후 여성들이나 노년기의 남성들에게 주로 생길 수 있는 골다공증을 의미한다. More specifically, the pharmaceutical composition of the present invention can be applied to the prevention or treatment of osteoporosis. The osteoporosis can be divided into primary (primary) osteoporosis and somatogenous (secondary) osteoporosis. Primary osteoporosis refers to osteoporosis, which can occur mainly in postmenopausal women or older men, such as not taking adequate calcium and vitamin D, not exercising properly, smoking, or smoking.

속발성 골다공증은 특정 질환이나 약물에 의해 골다골증이 유발되는 골다공증을 의미한다. 이 경우 젊은 사람에서도 골다공증이 발생할 수 있으며, 질환이나 약물의 심각성과 노출된 기간에 비례하여 골강도가 감소하고 골절의 발생률이 증가한다. 상기한 특정 질환으로는 갑상선 기능항진증, 부갑상선 기능항진증, 쿠싱증후군(부신피질호르몬 과다분비질환), 조기 폐경, 인위적 수술(난소 절제)에 의한 폐경, 성기능 저하증, 만성 간질환(간경화증), 류마티스 관절염, 만성 신부전증, 위절제수술 등을 들 수 있으며, 상기한 약물로는 스테로이드(부신피질 호르몬제), 항경련제(간질약), 헤파린 등이 있다.Secondary osteoporosis refers to osteoporosis in which osteoporosis is caused by a specific disease or drug. In this case, osteoporosis can occur even in young people, and bone strength decreases and the incidence of fracture increases in proportion to the severity of the disease or drug and the duration of exposure. The specific diseases described above include hyperthyroidism, hyperparathyroidism, Cushing's syndrome (adrenocorticotropic hormone hypersecretion disease), early menopause, menopause due to artificial surgery (oophorectomy), hypogonadism, chronic liver disease (cirrhosis), rheumatoid arthritis. , Chronic renal failure, gastrectomy, and the like, and the above-described drugs include steroids (corticosteroid drugs), anticonvulsants (epileptic drugs), and heparin.

본 발명의 골 재생 또는 골 형성 촉진용 조성물을 포함하는 약학적 조성물은 파골세포의 활성을 억제하는 방식이 아닌 골 재생 또는 골 형성을 유도하거나 촉진하는 효과를 나타내는 것으로서, 골 재생 또는 골 형성이 필요한 모든 골 질환에 적용될 수 있으며, 상기 질환에 제한되는 것은 아니다.The pharmaceutical composition comprising the composition for promoting bone regeneration or bone formation of the present invention exhibits an effect of inducing or promoting bone regeneration or bone formation rather than inhibiting the activity of osteoclasts, and requires bone regeneration or bone formation. It can be applied to all bone diseases, but is not limited to the disease.

또한 구체적으로, 상기 조성물은 기형 교정, 치과 교정, 골절 치료, 골접합술, 가관절에서의 골 재생, 골 형성 또는 골 이식에 사용되는 것일 수 있으나, 상기 사용 형태에 제한되는 것은 아니다.In addition, the composition may be used for deformity correction, dental orthodontic treatment, fracture treatment, bone splicing, bone regeneration in a false joint, bone formation, or bone transplantation, but is not limited to the use form.

본 발명의 약학적 조성물은 투여를 위하여, 상기 본 발명의 골 재생 또는 골 형성 촉진용 조성물 외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.For administration, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier, excipient, or diluent in addition to the composition for promoting bone regeneration or bone formation of the present invention. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

또한, 본 발명의 약학적 조성물은 어떠한 제형으로도 적용가능하며, 보다 구체적으로 비경구용 제형일 수 있다. 비경구용 제형으로는 주사용, 도포용, 에어로졸 등의 스프레이 형일 수 있다. 더욱 구체적으로는 주사제 형태일 수 있다.In addition, the pharmaceutical composition of the present invention may be applied in any formulation, and more specifically, may be a parenteral formulation. The parenteral formulation may be a spray type such as injection, application, or aerosol. More specifically, it may be in the form of an injection.

비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성 용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.

주사형 제형으로 제제화하기 위해서는 본 발명의 골 재생 또는 골 형성 촉진용 조성물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알의 단위 투여용으로 제제할 수 있다.In order to formulate in an injectable dosage form, the composition for promoting bone regeneration or bone formation of the present invention may be mixed in water with a stabilizer or buffer to prepare a solution or suspension, which may be formulated for unit administration in ampoules or vials.

본 발명의 또 다른 측면은 상기 수화젤을 포함하는 약학적 조성물을 치료를 필요로 하는 개체에 약학적으로 유효한 양으로 투여하는 단계를 포함하는, 골 질환 치료방법을 제공한다. 구체적으로 상기 골 질환은 골다공증, 암세포의 골전이에 의해 초래되는 뼈의 손상, 골연화증, 구루병, 섬유성 골염, 무형성 골질환, 대사성 골질환, 골용해, 백혈구 감소증, 뼈의 기형, 고칼슘혈증, 류마티스성 관절염, 골관절염, 골수염, 변성 관절증 및 골종양으로 구성되는 군으로부터 선택되는 하나 이상일 수 있다.Another aspect of the present invention provides a method for treating bone diseases, comprising administering a pharmaceutical composition containing the hydrogel to an individual in need thereof in a pharmaceutically effective amount. Specifically, the bone disease is osteoporosis, bone damage caused by bone metastasis of cancer cells, osteomalacia, rickets, fibrotic osteoitis, amorphous bone disease, metabolic bone disease, osteolysis, leukopenia, bone deformity, hypercalcemia, rheumatism It may be one or more selected from the group consisting of arthritis, osteoarthritis, osteomyelitis, degenerative arthritis and bone tumors.

본 발명의 용어 "약학적으로 유효한 양"은 의학적 치료에 적용가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 성병, 연령, 질병의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있다. 또한 본 발명의 약학적 조성물은 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다. The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the patient's sexually transmitted disease, age, type of disease, severity, It can be determined according to the activity of the drug, its sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. In addition, the pharmaceutical composition of the present invention may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.

본 발명의 용어 "개체"는 본 발명에 따른 약학적 조성물의 투여에 의해 증상이 호전될 수 있는 골 질환을 가진 말, 양, 돼지, 염소, 낙타, 영양, 개 등의 동물 또는 인간을 포함한다. 본 발명에 따른 치료용 조성물을 개체에게 투여함으로써, 골 질환을 효과적으로 예방 및 치료할 수 있다. The term "individual" of the present invention includes animals or humans such as horses, sheep, pigs, goats, camels, antelopes, dogs, etc. with bone disease whose symptoms can be improved by administration of the pharmaceutical composition according to the present invention. . By administering the therapeutic composition according to the present invention to an individual, bone diseases can be effectively prevented and treated.

본 발명의 용어 "투여"는 어떠한 적절한 방법으로 인간 또는 동물에게 소정의 물질을 도입하는 것을 의미하며, 본 발명에 따른 치료용 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 경구 또는 비경구 투여될 수 있다. 또한, 본 발명에 따른 치료용 조성물은 유효성분이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다. The term "administration" of the present invention means introducing a predetermined substance to humans or animals by any suitable method, and the route of administration of the therapeutic composition according to the present invention is through any general route as long as it can reach the target tissue. It can be administered orally or parenterally. In addition, the therapeutic composition according to the present invention can be administered by any device capable of moving the active ingredient to target cells.

본 발명에 따른 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the condition and weight of the patient, the degree of the disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art.

본 발명의 또 다른 측면은 a) 금 나노입자, 항산화 물질, 또는 금 나노입자 및 항산화 물질의 결합체를 준비하는 단계; b) 젤라틴 및 티라민을 혼합하여 수화젤을 제조하는 단계; 및 c) 상기 a) 단계의 금 나노입자, 항산화 물질, 또는 금 나노입자 및 항산화 물질의 결합체와 상기 b) 단계에서 제조된 수화젤을 혼합하는 단계를 포함하는, 골 재생 또는 골 형성 촉진용 수화젤의 제조방법에 관한 것이다. 구체적으로, 상기 항산화 물질은 N-아세틸 시스테인(N-acetyl cysteine)일 수 있다.Another aspect of the present invention is a) preparing a gold nanoparticle, an antioxidant, or a combination of gold nanoparticles and an antioxidant; b) preparing a hydrogel by mixing gelatin and tyramine; And c) mixing the gold nanoparticles, antioxidants, or a combination of gold nanoparticles and antioxidants in step a) with the hydration gel prepared in step b), hydration for promoting bone regeneration or bone formation It relates to a method of manufacturing a gel. Specifically, the antioxidant may be N-acetyl cysteine.

본 발명 일 실시예에서는 금 나노입자, 항산화 물질, 또는 금 나노입자 및 항산화 물질의 결합체 및/또는 젤라틴 및 티라민을 혼합하여 제조한 수화젤로부터 골 재생 또는 골 형성 촉진용 조성물을 제조하였으며, 상기 제조방법에 의해 제조된 본 발명 조성물이 우수한 세포 생존능 및 골 분화능을 나타냄을 확인하였다. In one embodiment of the present invention, a composition for promoting bone regeneration or bone formation was prepared from a gold nanoparticle, an antioxidant, or a combination of gold nanoparticles and an antioxidant, and/or a hydration gel prepared by mixing gelatin and tyramine, and the preparation It was confirmed that the composition of the present invention prepared by the method shows excellent cell viability and bone differentiation ability.

본 발명의 골 재생 또는 골 형성 촉진용 조성물은 독성이 없어 생체 적합성이 우수하며, 골 재생 및 골 형성 촉진 효과가 우수하여 골 조직의 수복 또는 골질환 치료에 널리 활용될 수 있다.The composition for promoting bone regeneration or bone formation of the present invention is not toxic and thus has excellent biocompatibility, and has excellent effects of promoting bone regeneration and bone formation, and thus can be widely used for repairing bone tissue or treating bone diseases.

본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

도 1은 골 재생용 수화젤의 제조 과정의 모식도를 나타낸 것이다.
도 2는 GNPs에 대한 DLS 분석 결과를 나타낸 것이다.
도 3은 GNPs 및 G-NAC의 흡광도를 측정한 결과를 나타낸 것이다.
도 4는 GNPs, G-NAC 및 NAC의 열분해 특성을 확인한 결과를 나타낸 것이다.
도 5는 NAC 처리에 따른 지방유래 줄기세포의 생존능을 확인한 결과를 나타낸 것이다.
도 6은 GNPs 처리에 따른 지방유래 줄기세포의 생존능을 확인한 결과를 나타낸 것이다.
도 7은 G-NAC 처리에 따른 지방유래 줄기세포의 생존능을 확인한 결과를 나타낸 것이다.
도 8은 NAC, GNPs및 G-NAC 각각을 처리한 군들의 생존능을 대비한 결과를 나타낸 것이다.
도 9는 NAC 처리에 따른 지방유래 줄기세포의 ALP 활성을 확인한 결과를 나타낸 것이다.
도 10은 10, 50, 100 및 200 μM 의 GNPs 처리에 따른 지방유래 줄기세포의 ALP 활성을 확인한 결과를 나타낸 것이다.
도 11은 0.1, 1, 10, 20 및 40 μM의 GNPs 처리에 따른 지방유래 줄기세포의 ALP 활성을 확인한 결과를 나타낸 것이다.
도 12는 G-NAC 처리에 따른 지방유래 줄기세포의 ALP 활성을 확인한 결과를 나타낸 것이다.
도 13은 NAC, GNPs및 G-NAC 각각을 처리한 군들의 ALP 활성을 대비한 결과를 나타낸 것이다.
도 14는 DM2500 현미경을 이용하여 GNPs 및 G-NAC의 세포 내 흡수를 확인한 결과를 나타낸 것이다.
도 15는 ImageJ 프로그램을 사용하여 DF 이미지를 정량적으로 비교한 결과를 나타낸 것이다.
도 16은 합성된 Gel-Ty 및 젤라틴의 특성을 확인한 결과를 나타낸 것이다(A: 1H NMR 분석 결과, B: 손실 탄성률(G'), C: 저장 탄성률(G'')).
도 17은 Gel-Ty 수화젤 표면에서 배양된 세포들의 생존능을 형광 이미지를 통해 확인한 결과를 나타낸 것이다.
도 18은 1일 내지 7일의 배양기간 동안 Gel-Ty 수화젤의 농도에 따른 생존능을 확인한 결과를 나타낸 것이다.
도 19는 1일 내지 3일의 배양기간 동안 Gel-Ty 수화젤의 농도에 따른 생존능을 확인한 결과를 나타낸 것이다.
도 20은 Gel-Ty 수화젤에서 배양된 형태를 CLSM을 통해 형광 이미지로 확인한 결과를 나타낸 것이다(5% (A, D, G, J), 7.5% (B, E, H, K), 및 10% Gel-Ty 수화젤(C, F, I, L) / A-C, G-I: 1일 배양, D-F, J-L: 3일 배양).
도 21은 Gel-Ty/GNPs 복합 수화젤에서 GNPs농도에 따른 세포 생존능을 확인한 결과를 나타낸 것이다.
도 22는 포함된 GNPs 농도에 따른 Gel-Ty/GNPs 복합 수화젤의 ALP 활성을 확인한 결과를 나타낸 것이다.
도 23은 Gel-Ty 수화젤, Gel-Ty/GNPs 복합 수화젤 및 Gel-Ty/G-NAC복합 수화젤의 ALP 활성을 대비한 결과를 나타낸 것이다. 1 shows a schematic diagram of a manufacturing process of a hydrogel for bone regeneration.
2 shows the DLS analysis results for GNPs.
3 shows the results of measuring the absorbance of GNPs and G-NAC.
Figure 4 shows the results of confirming the pyrolysis properties of GNPs, G-NAC and NAC.
5 shows the results of confirming the viability of adipose-derived stem cells according to NAC treatment.
6 shows the results of confirming the viability of adipose-derived stem cells according to GNPs treatment.
7 shows the results of confirming the viability of adipose-derived stem cells according to G-NAC treatment.
Figure 8 shows the results comparing the viability of the groups treated with NAC, GNPs and G-NAC, respectively.
9 shows the results of confirming the ALP activity of adipose-derived stem cells according to NAC treatment.
10 shows the results of confirming the ALP activity of adipose-derived stem cells according to the treatment of 10, 50, 100 and 200 μM GNPs.
11 shows the results of confirming the ALP activity of adipose-derived stem cells according to the treatment of 0.1, 1, 10, 20 and 40 μM of GNPs.
12 shows the results of confirming the ALP activity of adipose-derived stem cells according to G-NAC treatment.
13 shows the results of comparing ALP activity of groups treated with NAC, GNPs and G-NAC, respectively.
14 shows the results of confirming the intracellular uptake of GNPs and G-NACs using a DM2500 microscope.
15 shows the results of quantitative comparison of DF images using the ImageJ program.
Figure 16 shows the results of confirming the properties of the synthesized Gel-Ty and gelatin (A: 1 H NMR analysis result, B: loss modulus (G'), C: storage modulus (G')).
17 shows the results of confirming the viability of cells cultured on the surface of Gel-Ty hydration gel through fluorescence images.
18 shows the results of confirming the viability according to the concentration of Gel-Ty hydration gel during the culture period of 1 to 7 days.
19 shows the results of confirming the viability according to the concentration of Gel-Ty hydration gel during the culture period of 1 to 3 days.
20 shows the results of confirming the form cultured in Gel-Ty hydration gel with a fluorescence image through CLSM (5% (A, D, G, J), 7.5% (B, E, H, K), and 10% Gel-Ty hydration gel (C, F, I, L) / AC, GI: 1 day culture, DF, JL: 3 day culture).
21 shows the results of confirming the cell viability according to the GNPs concentration in the Gel-Ty/GNPs complex hydration gel.
22 shows the results of confirming the ALP activity of the Gel-Ty/GNPs complex hydration gel according to the concentration of GNPs included.
23 shows the results comparing the ALP activity of Gel-Ty hydration gel, Gel-Ty/GNPs complex hydration gel, and Gel-Ty/G-NAC complex hydration gel.

이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited by the following examples.

실시예 1. 재료 및 분석방법Example 1. Materials and Analysis Method

1-1.재료1-1.Material

젤라틴, 2-모르폴리노에탄술폰산(2-morpholinoethanesulfonic acid, MES), 염화나트륨, 염화금(III)수화물(HAuCl4), 시트르산나트륨(sodium citrate), N-아세틸 시스테인(N-acyl cysteine, NAC) 및 티라민(tyramine, Ty)은 Sigma-Aldrich(St Louis, MO, USA)사로부터 구입하였다.Gelatin, 2-morpholinoethanesulfonic acid (MES), sodium chloride, gold (III) chloride (HAuCl 4 ), sodium citrate, N-acetyl cysteine (N-acyl cysteine, NAC) and Tyramine (Ty) was purchased from Sigma-Aldrich (St Louis, MO, USA).

N-히드록시석신이미드(N-Hydroxysuccinimide, NHS) 및 1-에틸-3-(3-디메틸 아미노프로필)-카르보디이미드히드로클로라이드(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochlorid, EDC)는 도쿄 화학 공업(도쿄, 일본)사로부터 구입하였다.N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochlorid, EDC) was purchased from Tokyo Chemical Industries (Tokyo, Japan).

티라민(tyramine, Ty), HRP(Horseradish peroxidase) 및 Alexa Fluor 488 Phalloidin은 Invitrogen(Thermo Fisher Scientific, Waltham, MA, USA)사로부터 구입하였다.Tyramine (Ty), HRP (Horseradish peroxidase) and Alexa Fluor 488 Phalloidin were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA).

Spectra/Por 4 dialysis tubing(3.5 및 10 kDa MWCO)은 Spectrum Laboratories Inc (Rancho Dominguez, CA, USA)사로부터 구입하였다.Spectra/Por 4 dialysis tubing (3.5 and 10 kDa MWCO) was purchased from Spectrum Laboratories Inc (Rancho Dominguez, CA, USA).

DMEM, FBS, 항생제(페니실린/스트렙토마이신, PS), TrypLETM Express, PBS 및 Dubeccox phosphate buffered saline (DPBS)은 Gibco(Thermo Fisher Scientific)사로부터 구입하였다. 탈이온 증류수(Deionized distilled water)는 초순수 물 시스템(Puris-Ro800, Bio Lab Tech, Republic of Korea)을 이용하여 제조하였다.DMEM, FBS, antibiotics (penicillin/streptomycin, PS), TrypLE ™ Express, PBS and Dubeccox phosphate buffered saline (DPBS) were purchased from Gibco (Thermo Fisher Scientific). Deionized distilled water was prepared using an ultrapure water system (Puris-Ro800, Bio Lab Tech, Republic of Korea).

1-2.분석장비1-2. Analysis equipment

수소 핵자기공명(1H NMR)스펙트럼은 Bruker Avance 400 (Bruker Corporation, Billerica, MA, USA)로 관찰하였다. 점탄성은 회전식 레오미터(AR-G2, TA Instruments, New Castle, DE, USA)로 측정하였다. 형광 표지된 세포는 공초점 레이저 스캐닝 현미경(CLSM, Eclipse E600W, Nikon Corporation, Tokyo, Japan)으로 관찰하였다. ELISA는 Benchmark Plus 마이크로 플레이트 스펙트로 포토미터 시스템(Bio-Rad Laboratories Inc, Hercules, CA, USA)으로 수행하였다. Dynamic light scattering(DLS)은 ELSZ-1000 (Photal, Otsuka Electronics, Osaka, Japan)을 사용하여 수행하였다. Thermogravimetric analysis(TGA)은 SDT Q600(TA Instruments)을 사용하여 수행하였다. dark-field(DF) 필터(Leica Microsystems, Wetzlar, Germany)를 갖는 DM2500 현미경을 사용하여 나노 입자의 세포 흡수량을 분석하였다.The hydrogen nuclear magnetic resonance ( 1 H NMR) spectrum was observed with Bruker Avance 400 (Bruker Corporation, Billerica, MA, USA). Viscoelasticity was measured with a rotary rheometer (AR-G2, TA Instruments, New Castle, DE, USA). Fluorescently labeled cells were observed with a confocal laser scanning microscope (CLSM, Eclipse E600W, Nikon Corporation, Tokyo, Japan). ELISA was performed with a Benchmark Plus micro plate spectrophotometer system (Bio-Rad Laboratories Inc, Hercules, CA, USA). Dynamic light scattering (DLS) was performed using ELSZ-1000 (Photal, Otsuka Electronics, Osaka, Japan). Thermogravimetric analysis (TGA) was performed using SDT Q600 (TA Instruments). The cellular uptake of nanoparticles was analyzed using a DM2500 microscope with a dark-field (DF) filter (Leica Microsystems, Wetzlar, Germany).

실시예 2. 금 나노입자(Gold Nano Particles, GNPs)의 제조Example 2. Preparation of Gold Nano Particles (GNPs)

금 나노입자(GNPs) 수용액은 HAuCl4 의 시트르산 환원으로 합성하였다. 0.5 mM의 HAuCl4(800 mL) 용액을 환류한 후, 2 % 시트르산삼나트륨(15 mL)을 빠르게 첨가하였다. 15 분 후, 용액의 색이 짙은 적색으로 변화하였고, 이어서 금 나노입자(GNPs)가 형성되었다.An aqueous solution of gold nanoparticles (GNPs) was synthesized by citric acid reduction of HAuCl 4 . After refluxing a 0.5 mM HAuCl 4 (800 mL) solution, 2% trisodium citrate (15 mL) was rapidly added. After 15 minutes, the color of the solution changed to dark red, and then gold nanoparticles (GNPs) were formed.

실시예 3. 금 나노입자(Gold Nano Particles, GNPs) 및 N-아세틸 시스테인(N-acyl cysteine, NAC)의 복합체(G-NAC)의 제조Example 3. Preparation of a complex (G-NAC) of gold nanoparticles (GNPs) and N-acetyl cysteine (N-acyl cysteine, NAC)

상기 실시예 2에서 제조된 금 나노입자(GNPs) 표면에 N-아세틸 시스테인(NAC)을 부착하기 위해 580 μM GNPs 용액을 29 mM NAC 용액과 반응시키고 실온에서 48 시간 동안 교반하였다. 혼합물을 3,500 Da 투석막을 사용하여 비공유 NAC를 제거하기 위해 5 일 동안 투석하였다.In order to attach N-acetyl cysteine (NAC) to the gold nanoparticles (GNPs) prepared in Example 2, a 580 μM GNPs solution was reacted with a 29 mM NAC solution and stirred at room temperature for 48 hours. The mixture was dialyzed for 5 days to remove non-covalent NAC using a 3,500 Da dialysis membrane.

상기와 같이 합성된 GNPs 및 G-NAC는 25 ℃, 200 μM에서 나노 입자의 크기를 확인하기 위해 DLS 분석을 수행하였다. UV-vis 스펙트럼 분석을 하기 전 G-NAC 용액의 농도가 생산 과정 중 투석될 때 감소하기 때문에 GNPs 용액의 농도를 농축하였다. 이후 13,000 rpm에서 15 분간 원심 분리 한 후 TGA로 동결 건조하였다. TGA는 100 mL/min의 고순도 질소 유량하에서 수행하였으며, 퍼니스(furnace)의 온도는 10 ℃/min에서 최대 800 ℃까지 증가하도록 설정하였다.GNPs and G-NAC synthesized as described above were subjected to DLS analysis to confirm the size of nanoparticles at 25° C. and 200 μM. Before the UV-vis spectrum analysis, the concentration of the GNPs solution was concentrated because the concentration of the G-NAC solution decreased when dialyzed during the production process. Then, after centrifugation at 13,000 rpm for 15 minutes, it was freeze-dried with TGA. TGA was performed under a high purity nitrogen flow rate of 100 mL/min, and the temperature of the furnace was set to increase from 10°C/min to a maximum of 800°C.

실시예 4. 젤라틴-티라민(Gel-Ty) 수화젤의 제조Example 4. Preparation of gelatin-tyramine (Gel-Ty) hydrogel

증류수에 50 mM 2-모르폴리노에탄술폰산(2-morpholinoethanesulfonic acid, MES) 및 0.5 M 염화나트륨을 혼합한 다음 pH를 6.0으로 조정하여 MES 완충액를 준비하였다. 이후 젤라틴을 2 % 농도의 60 ℃ MES 완충액에 용해시키고, 냉각 후, 25 mM N-히드록시석신이미드(N-Hydroxysuccinimide, NHS) 및 50 mM 1-에틸-3-(3-디메틸아미노프로필)-카르보디이미드히드로클로라이드(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochlorid, EDC)를 실온에서 첨가하였다.50 mM 2-morpholinoethanesulfonic acid (MES) and 0.5 M sodium chloride were mixed in distilled water, and the pH was adjusted to 6.0 to prepare a MES buffer. Thereafter, gelatin was dissolved in a 2% concentration of 60° C. MES buffer, and after cooling, 25 mM N-Hydroxysuccinimide (NHS) and 50 mM 1-ethyl-3-(3-dimethylaminopropyl) -Carbodiimide hydrochlorid (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochlorid, EDC) was added at room temperature.

1 시간 반응 후, 티라민(tyramin, Ty)을 젤라틴 양의 절반으로 첨가하고, 실온에서 24 시간 동안 교반하였다. 혼합물을 10 kDa 투석 튜브를 사용하여 증류수로 7 일 동안 투석한 후, 투석된 용액을 동결 건조하여 젤라틴-티라민(Gel-Ty)을 합성하였다. After the reaction for 1 hour, tyramine (tyramin, Ty) was added in half the amount of gelatin, and stirred at room temperature for 24 hours. The mixture was dialyzed with distilled water for 7 days using a 10 kDa dialysis tube, and then the dialyzed solution was freeze-dried to synthesize gelatin-tyramine (Gel-Ty).

합성된 Gel-Ty를 1H-NMR로 분석하였다. Gel-Ty 수화젤은 HRP(Horseradish peroxidase) 및 과산화수소(H2O2) 간의 반응으로 형성되었다. 구체적으로, HRP 5 단위(unit) 및 50 mM H2O2 25 ㎕를 동결 건조된 Gel-Ty가 필요한 농도로 용해된 PBS에서 혼합하였다. Gel-Ty 용액은 1 분 이내에 수화젤의 형태로 반응하였다. 수화젤을 1 mm 두께의 필름으로 준비하였고, 직경 8 mm 디스크로 성형하여 생체검사에 사용하였다.The synthesized Gel-Ty was analyzed by 1 H-NMR. Gel-Ty hydration gel was formed by a reaction between HRP (Horseradish peroxidase) and hydrogen peroxide (H 2 O 2 ). Specifically, 5 units of HRP and 25 μl of 50 mM H 2 O 2 were mixed in PBS in which freeze-dried Gel-Ty was dissolved at the required concentration. The Gel-Ty solution reacted in the form of a hydrogel within 1 minute. The hydration gel was prepared as a 1 mm thick film, and molded into an 8 mm diameter disk and used for biopsy.

상기 디스크는 레오미터 및 세포 실험에 사용하였다. Gel-Ty 수화젤의 점탄성은 회전식 레오미터를 사용하여 측정하였다. 유동학적(rheological) 분석을 위해, 샘플은 0.1 내지 100 Hz의 주파수에서 5 % strain으로 압축되었다. 점탄성 측정시 온도를 25 ℃로 조절하였다.The disk was used for rheometer and cell experiments. The viscoelasticity of Gel-Ty hydrogel was measured using a rotary rheometer. For rheological analysis, samples were compressed with 5% strain at a frequency of 0.1-100 Hz. When measuring viscoelasticity, the temperature was adjusted to 25°C.

실시예 5. Gel-Ty/GNPs 및 Gel-Ty/G-NAC복합 수화젤의 제조Example 5. Preparation of Gel-Ty/GNPs and Gel-Ty/G-NAC complex hydrogel

20, 100 및 200 μM GNPs 또는 G-NAC 용액을 각각 13,000 rpm으로 15 분간 원심 분리하여 입자를 침전시켰다. 상층액을 제거한 pellet을 5, 7.5 및 10 %의 Gel-Ty solution으로 혼합한 후 HRP, H2O2 및 지방유래 줄기세포와 반응시켰다. 상기 반응을 통해 각각 10, 100, 200 및 400 μM GNPs 또는 G-NAC가 포함된 Gel-Ty/GNPs 복합 수화젤, Gel-Ty/G-NAC복합 수화젤을 제조하였다.20, 100, and 200 μM GNPs or G-NAC solutions were centrifuged at 13,000 rpm for 15 minutes, respectively, to precipitate particles. The pellet from which the supernatant was removed was mixed with 5, 7.5 and 10% Gel-Ty solution and reacted with HRP, H 2 O 2 and fat-derived stem cells. Through the above reaction, 10, 100, 200 and 400 μM GNPs or Gel-Ty/GNPs complex hydration gel, and Gel-Ty/G-NAC complex hydration gel containing G-NAC, respectively, were prepared.

실험예 1. GNPs 및 G-NAC의 특성 분석Experimental Example 1. Characterization of GNPs and G-NAC

상기 실시예를 통해 합성된 GNPs 및 G-NAC는 25 ℃에서 200 μM의 농도에서 DLS 분석을 통해 나노입자의 크기를 측정하였다. 합성 중 투석 과정에서 G-NAC 용액 농도가 감소되기 때문에 자외선-가시광선(ultraviolet-visible, UV-vis) 스펙트럼 분석을 하기 전, GNPs 용액의 농도를 농축하였다. GNPs 및 G-NAC는 13,000 rpm에서 15 분간 원심 분리한 후 TGA 동결 건조하였다. TGA는 100 mL/min의 고순도 질소 유량 하에서 수행하였다. 퍼니스의 온도는 최대 800 ℃까지 10 ℃의 속도로 증가하도록 설정하였다.GNPs and G-NAC synthesized through the above example were measured for the size of nanoparticles through DLS analysis at a concentration of 200 μM at 25°C. Since the concentration of the G-NAC solution was decreased during the dialysis process during synthesis, the concentration of the GNPs solution was concentrated before performing ultraviolet-visible (UV-vis) spectrum analysis. GNPs and G-NAC were centrifuged at 13,000 rpm for 15 minutes and then freeze-dried by TGA. TGA was performed under a high purity nitrogen flow rate of 100 mL/min. The temperature of the furnace was set to increase at a rate of 10 °C up to 800 °C.

580 μM 농도의 GNPs에 대한 DLS 분석 결과, 입자의 직경은 28 내지 32 nm인 것으로 나타났다. 입자의 평균 직경은 35.83 ± 0.9 nm이고 다분산도(polydispersity, PDI)는 0.300 ± 0.026으로 측정되었다(도 2).DLS analysis of GNPs at a concentration of 580 μM showed that the particle diameter was 28 to 32 nm. The average diameter of the particles was 35.83 ± 0.9 nm and the polydispersity (PDI) was measured to be 0.300 ± 0.026 (Fig. 2).

또한, UV 분광 광도계를 사용하여 GNPs와 G-NAC의 흡광도를 측정하였다. 측정된 최대 흡광도 값은 GNPs 및 G-NAC 각각 522.5 및 527 nm로 나타났으며, G-NAC 및 GNPs의 그래프의 형태는 유사하지만 G-NAC의 그래프가 GNPs의 그래프 보다 더 오른쪽으로 이동하였음을 확인하였다(도 3).In addition, the absorbance of GNPs and G-NAC was measured using a UV spectrophotometer. The measured maximum absorbance values were 522.5 and 527 nm for GNPs and G-NAC, respectively, and although the shapes of the graphs of G-NAC and GNPs were similar, it was confirmed that the graph of G-NAC shifted more to the right than the graph of GNPs. (Fig. 3).

또한, TGA 그래프는 GNPs, G-NAC 및 NAC가 800 ℃로 가열되었을 때 열분해 특성을 나타내며, GNPs, G-NAC 및 NAC의 질량 감소율은 각각 2.177 3.088 및 82.58 %로 나타났다(도 4). In addition, the TGA graph shows pyrolysis properties when GNPs, G-NAC and NAC are heated to 800° C., and the mass reduction rates of GNPs, G-NAC and NAC are 2.177 3.088 and 82.58%, respectively (FIG. 4).

실험예 2. 지방유래 줄기세포(hADSC)의 생존능 및 골분화 관련 NAC, GNPs 및 G-NAC의 효과 확인Experimental Example 2. Confirmation of the effects of NAC, GNPs and G-NAC related to viability and bone differentiation of adipose-derived stem cells (hADSC)

2-1. 지방유래 줄기세포(human Adipose-Derived Stem Cells) 배양2-1. Human Adipose-Derived Stem Cells Cultivation

지방유래 줄기세포(hADSC)를 5 % CO2 배양기(Isotemp, Thermo Fisher Scientific) 내 37 ℃에서 성장 배지(GM) 또는 골 분화 배지 (OM)에서 배양 하였다. GM은 DMEM, 10 % FBS 및 1 % PS로 구성되었으며, OM은 GM, 10 mM β-glycerol phosphate disodium salt hydrate, 300 μM 아스코르브산 및 0.1 μM 덱사메타손으로 구성되었다. hADSC를 사용하는 모든 세포 실험에서, GM 및 OM은 3 일마다 교체하였다.Adipose-derived stem cells (hADSC) were cultured in growth medium (GM) or bone differentiation medium (OM) at 37° C. in a 5% CO 2 incubator (Isotemp, Thermo Fisher Scientific). GM consisted of DMEM, 10% FBS and 1% PS, and OM consisted of GM, 10 mM β-glycerol phosphate disodium salt hydrate, 300 μM ascorbic acid and 0.1 μM dexamethasone. In all cell experiments using hADSC, GM and OM were replaced every 3 days.

2-2. 생존능 확인2-2. Viability check

지방유래 줄기세포의 생존능에 대하여 NAC, GNPs및 G-NAC가 각각 어떤 영향을 미치는지 확인하였다. 구체적으로, 지방유래 줄기세포의 생존능은 형광 염색 및 세포 생존능 분석 키트(EZ-Cytox, Dogne, Republic of Korea)를 사용하여 평가하였다.The effects of NAC, GNPs and G-NAC on the viability of adipose-derived stem cells were confirmed. Specifically, the viability of adipose-derived stem cells was evaluated using a fluorescent staining and cell viability assay kit (EZ-Cytox, Dogne, Republic of Korea).

GNPs, NAC 및 G-NAC의 평가를 위해, 지방유래 줄기세포는 성장배지에 1x104 cell/well의 밀도로 분주하였다. 1 일 후, GNPs, NAC 및 G-NAC의 다양한 농도(GNPs : 1, 10, 20, 40, 100 및 200 μM; NAC : 0.01, 0.05, 0.1, 1, 5, 10 및 50 mM; G-NAC : 40, 100 및 200 μM)가 포함된 성장배지로 교체한 후, 10 배 희석한 EZ-Cytox 용액으로 90 분간 반응시키고, 450 nm 파장에서 ELISA 판독기를 사용하여 반응 용액을 측정하였다. NAC 또는 GNPs를 처리하지 않은 군을 대조군(100 %)으로 하고 나머지 군을 대조군과 비교하였다. For the evaluation of GNPs, NAC and G-NAC, adipose-derived stem cells were dispensed in growth medium at a density of 1×10 4 cells/well. After 1 day, various concentrations of GNPs, NAC and G-NAC (GNPs: 1, 10, 20, 40, 100 and 200 μM; NAC: 0.01, 0.05, 0.1, 1, 5, 10 and 50 mM; G-NAC : 40, 100 and 200 μM) was replaced with a growth medium containing, and then reacted for 90 minutes with a 10-fold diluted EZ-Cytox solution, and the reaction solution was measured using an ELISA reader at 450 nm wavelength. The group not treated with NAC or GNPs was used as a control (100%) and the rest of the groups were compared with the control group.

NAC와 GNPs 처리에 따른 지방유래 줄기세포의 생존능을 확인한 결과, 처리 기간이 길어질수록 NAC 농도가 높은 군에서 세포 증식률이 감소하였다. 7 일째, 0.01 mM NAC 군 (265.8 %)을 제외한 모든 NAC 처리 군은 대조군 (255.1 %)보다 22 % 낮은 생존능을 나타내었다(도 5).As a result of confirming the viability of adipose-derived stem cells according to NAC and GNPs treatment, the longer the treatment period, the lower the cell proliferation rate in the group with high NAC concentration. On the 7th day, all NAC-treated groups except for the 0.01 mM NAC group (265.8%) showed 22% lower viability than the control group (255.1%) (FIG. 5).

GNPs가 처리된 모든 군은 대조군보다 생존능이 높게 나타났으며, 특히 40 μM GNPs로 7 일간 처리한 군에서 생존능은 대조군보다 1.73 배 높은 결과를 나타내었다(도 6).All groups treated with GNPs showed higher viability than the control group, and in particular, in the group treated with 40 μM GNPs for 7 days, the viability was 1.73 times higher than that of the control group (FIG. 6 ).

G-NAC는 상기 GNPs와 동일한 농도를 첨가하여 생존능을 확인하였으며, 지방유래 줄기세포의 생존능은 40, 100 및200 μM 의 G-NAC를 3일 간 투여했을 때 약 300%에 가까운 생존능을 나타내어 대조군(140.6%)과 유의한 차이를 나타내었다(도 7). 또한, 100 μM 의 G-NAC을 처리 후 7일 째에는 대조군에 비해 1.65배 높은 생존능을 나타내었다(도 7). The viability of G-NAC was confirmed by adding the same concentration as the GNPs, and the viability of adipose-derived stem cells was approximately 300% when 40, 100 and 200 μM of G-NAC was administered for 3 days. (140.6%) showed a significant difference (Fig. 7). In addition, on the 7th day after treatment with 100 μM of G-NAC, the viability was 1.65 times higher than that of the control group (FIG. 7).

또한, 상기 NAC, GNPs및 G-NAC 각각을 처리한 군들의 생존능을 대비하였다. GNPs및 G-NAC는 20 μM로, NAC는 2.2 μM로 처리하였다. 대비 결과, NAC 투여군은 7 일째 대조군에 비해 0.85 배의 생존능을 보였으나 GNPs 및 G-NAC는 대조군에 비해 높은 생존능을 나타냄을 확인하였다(도 8).In addition, the viability of the groups treated with each of the NAC, GNPs and G-NAC was compared. GNPs and G-NAC were treated with 20 μM and NAC with 2.2 μM. As a result of comparison, it was confirmed that the NAC-administered group showed 0.85 times more viability than the control group on the 7th day, but GNPs and G-NAC showed higher viability than the control group (FIG. 8 ).

상기와 같은 결과로부터 본 발명의 GNPs 및 G-NAC를 처리한 경우 대조군 및 NAC 처리군에 비해 세포 생존능이 높게 나타남을 확인하였다. 이는 독성 문제없이 생체에 적용될 수 있음을 나타내는 것이다.From the above results, it was confirmed that when the GNPs and G-NAC of the present invention were treated, cell viability was higher than that of the control group and the NAC-treated group. This indicates that it can be applied to a living body without a toxicity problem.

2-3. 알칼리 포스파타아제(Alkaline phosphatase, ALP) 활성 확인2-3. Alkaline phosphatase (ALP) activity check

NAC, GNPs및 G-NAC의 골 분화 효과를 확인하기 위해 세포 독성을 띄지 않는 범위에서 NAC, GNPs및 G-NAC의 ALP 활성을 확인하였다. In order to confirm the bone differentiation effect of NAC, GNPs and G-NAC, the ALP activities of NAC, GNPs and G-NAC were confirmed in a range that did not show cytotoxicity.

구체적으로, 지방유래 줄기세포를 웰당 또는 샘플 당 2x104 세포의 밀도로 48-웰 세포 배양 플레이트에 분주하였다. 1 일 후 성장배지는 각각 GNPs, NAC 또는 G-NAC가 포함된 분화배지 또는 일반 분화배지로 변경하였다. 이어서 세포를 5, 10 및 15 일 동안 배양하였다. 골분화 된 지방유래 줄기세포를 DPBS로 세척하고 4 ℃에서 1 시간 동안 3X RIPA 버퍼로 용해시켰다. 용해된 지방유래 줄기세포를 10,000 rpm에서 10 분간 원심 분리하고, 상등액을 37 ℃ 항온기에서 p-니트로페놀 포스페이트(Sigma-Aldrich)와 30 분 동안 반응시켰다. p- 니트로페놀 생산 수준은 405 nm 파장에서 ELISA 판독기로 측정하였다.Specifically, adipose-derived stem cells were dispensed into 48-well cell culture plates at a density of 2×10 4 cells per well or per sample. After 1 day, the growth medium was changed to a differentiation medium or general differentiation medium containing GNPs, NAC or G-NAC, respectively. The cells were then cultured for 5, 10 and 15 days. The osteodifferentiated adipose-derived stem cells were washed with DPBS and lysed with 3X RIPA buffer for 1 hour at 4°C. The dissolved adipose-derived stem cells were centrifuged at 10,000 rpm for 10 minutes, and the supernatant was reacted with p-nitrophenol phosphate (Sigma-Aldrich) for 30 minutes in an incubator at 37°C. The level of p-nitrophenol production was measured with an ELISA reader at a wavelength of 405 nm.

NAC는 0.005, 0.01, 0.1, 1, 5 및 10 mM, GNPs는 10, 50, 100 및 200 μM 농도에서 평가하였으며, G-NAC는 GNPs와 동일한 농도를 처리하였다.NAC was evaluated at 0.005, 0.01, 0.1, 1, 5 and 10 mM, GNPs at 10, 50, 100 and 200 μM concentrations, and G-NAC was treated with the same concentration as GNPs.

그 결과, NAC처리군의 경우 상대적으로 저농도인 0.005, 0.01 및 0.1 mM 의 농도에서 대조군 대비 높은 ALP 활성이 나타남을 확인하였다(도 9). As a result, it was confirmed that the NAC-treated group exhibited higher ALP activity compared to the control group at relatively low concentrations of 0.005, 0.01 and 0.1 mM (FIG. 9).

GNPs를 처리한 경우, 10 μM를 처리하였을 때 모든 처리 기간에서 가장 높은 ALP 활성을 나타내었으나, 50 μM 이상의 농도에서 ALP 활성은 생존능과 달리 GNPs의 농도가 증가함에 따라 감소하는 경향을 나타내었다(도 10). In the case of treatment with GNPs, when 10 μM was treated, the highest ALP activity was shown in all treatment periods, but at a concentration of 50 μM or more, ALP activity showed a tendency to decrease as the concentration of GNPs increased, unlike viability (Fig. 10).

또한, 상대적으로 저농도인 0.1, 1, 10, 20 및 40 μM의 GNPs를 처리하고 5일 및 10일 째의 ALP 활성을 측정하였으며, 모든 농도 군에서 대조군 대비 우수한 ALP 활성을 나타냄을 확인하였다. 특히, 20 μM에서의 ALP 활성이 가장 높게 나타남을 확인하였다(도 11).In addition, relatively low concentrations of 0.1, 1, 10, 20 and 40 μM of GNPs were treated, and ALP activity was measured on days 5 and 10, and it was confirmed that ALP activity was superior to that of the control group in all concentration groups. In particular, it was confirmed that the ALP activity was highest at 20 μM (FIG. 11).

G-NAC를 처리한 경우, 10 μM를 처리하였을 때 모든 처리 기간에서 가장 높은 ALP 활성을 나타내었으며, GNPs를 처리한 경우와 마찬가지로 50 μM 이상의 농도에서 ALP 활성은 다소 감소되는 경향을 나타내었으나 여전히 대조군에 비해 높은 활성을 나타냄을 확인하였다(도 12).In the case of G-NAC treatment, when 10 μM was treated, the highest ALP activity was displayed in all treatment periods, and as in the case of GNPs treatment, ALP activity showed a tendency to decrease slightly at a concentration of 50 μM or more, but still control It was confirmed that it shows higher activity compared to (Fig. 12).

또한, 상기 NAC, GNPs및 G-NAC 각각을 처리한 군들의 ALP 활성을 대비하였다. GNPs및 G-NAC는 20 μM로, NAC는 2.2 μM로 처리하였다. 대비 결과, NAC 투여군은 5, 10 및 15 일째 ALP 활성도가 대조군에 비해 각각 1.17, 1.16 및 1.09 배 높게 나타났다. 또한, G-NAC 처리군의 경우 GNPs 처리군에 비해 최소 1.08배, 최대 1.18배 높은 유의미한 차이를 나타내었다(도 13). In addition, the ALP activity of the groups treated with each of the NAC, GNPs and G-NAC was compared. GNPs and G-NAC were treated with 20 μM and NAC with 2.2 μM. As a result of comparison, the NAC-administered group showed higher ALP activity on days 5, 10 and 15 than the control group, 1.17, 1.16 and 1.09 times, respectively. In addition, the G-NAC treatment group showed a significant difference, which is at least 1.08 times and at most 1.18 times higher than that of the GNPs treatment group (Fig. 13).

상기와 같은 결과로부터 NAC 를 처리한 경우 대조군에 비해 높은 ALP 활성을 나타내며, 특히 본 발명의 G-NAC를 처리한 경우 GNPs 처리군을 비롯하여 모든 군 대비 높은 ALP 활성을 나타냄을 확인하였다. From the above results, it was confirmed that the NAC treatment showed higher ALP activity compared to the control group, and in particular, the G-NAC treatment of the present invention exhibited higher ALP activity compared to all groups including the GNPs treatment group.

실험예 3. GNPs 및 G-NAC의 세포 내 흡수능력 확인Experimental Example 3. Confirmation of intracellular absorption capacity of GNPs and G-NAC

GNPs 및 G-NAC의 세포 내 흡수는 dark-field(DF) 분석으로 평가하였다. 지방유래 줄기세포를 confocal dish(SPL, 포천, 대한민국)에 배양한 뒤, 20 μM GNPs 및 G-NAC 용액을 처리하였다. 12 시간 후, 이들을 3.7 % 포름알데히드 용액을 사용하여 고정시키고, DF 필터가 장착 된 DM2500 현미경으로 관찰하였다. DF 이미지는 ImageJ 프로그램을 통해 분석하였다.Intracellular uptake of GNPs and G-NAC was evaluated by dark-field (DF) analysis. Adipose-derived stem cells were cultured in confocal dishes (SPL, Pocheon, Korea), and then treated with 20 μM GNPs and G-NAC solution. After 12 hours, they were fixed using a 3.7% formaldehyde solution and observed under a DM2500 microscope equipped with a DF filter. DF images were analyzed through the ImageJ program.

그 결과, 도 14에 나타난 바와 같이 Bright field(BF) 이미지에서 GNPs(A) 및 G-NAC(C)의 세포 내 흡수가 관찰되었다. 또한, Dark field(DF) 이미지에서도 흡수된 입자가 흰색으로 관찰되었으며 이로부터 DF에서도 GNPs(B) 및 G-NAC(D) 모두 흡수된 입자가 나타남을 확인하였다. As a result, as shown in FIG. 14, intracellular absorption of GNPs (A) and G-NAC (C) was observed in the Bright field (BF) image. In addition, the absorbed particles were observed as white in the dark field (DF) image, and from this, it was confirmed that both GNPs (B) and G-NAC (D) absorbed particles appeared in DF.

또한, ImageJ 프로그램을 사용하여 DF 이미지를 정량적으로 비교하였으며, 도 15에 나타난 바와 같이, 세포 내 흡수에 있어서는GNPs(2.38 %)와 G-NAC(2.27 %)간에 큰 차이가 나타나지 않음을 확인하였다.In addition, DF images were quantitatively compared using the ImageJ program, and as shown in FIG. 15, it was confirmed that there was no significant difference between GNPs (2.38%) and G-NAC (2.27%) in intracellular absorption.

실험예 4. Gel-Ty 수화젤의 특성 분석Experimental Example 4. Characterization of Gel-Ty Hydrogel

합성된 Gel-Ty 및 젤라틴의 1H NMR 분석을 진행하였으며, Gel-Ty의 경우 7.2 및 6.9 ppm에서 고유한 피크가 검출되었다(도 16A). 1 H NMR analysis of the synthesized Gel-Ty and gelatin was performed, and in the case of Gel-Ty, unique peaks were detected at 7.2 and 6.9 ppm (FIG. 16A).

또한, 합성된 Gel-Ty 수화젤을 레오미터(rheometer)를 사용하여 실온에서 5 % 스트레스를 가해 고정시키고, 진동수를 0.1 Hz 에서 40 Hz로 올려 그에 따른 탄성률을 측정하였다. 그 결과, 도 16B에 나타난 바와 같이, 5, 6.5 및 8 %의 Gel-Ty 수화젤의 손실 탄성률(G')은 각각 4.32 ± 0.86, 5.32 ± 0.96 및 6.56 ± 0.71 이었며, 저장 탄성률(G'')은 Gel-Ty 농도에 비례하여 증가하였다.In addition, the synthesized Gel-Ty hydration gel was fixed by applying 5% stress at room temperature using a rheometer, and the modulus of elasticity was measured by raising the frequency from 0.1 Hz to 40 Hz. As a result, as shown in FIG. 16B, the loss modulus (G') of 5, 6.5 and 8% Gel-Ty hydration gel was 4.32 ± 0.86, 5.32 ± 0.96 and 6.56 ± 0.71, respectively, and the storage modulus (G' ') increased in proportion to the Gel-Ty concentration.

또한, 합성된 Gel-Ty는 PBS에 용해되었을 때 액체 상태 였으나 HRP 및 H2O2가 첨가된 후에 점탄성 고체 형태를 유지함을 확인하였다(도 16C).In addition, it was confirmed that the synthesized Gel-Ty was in a liquid state when dissolved in PBS, but maintained a viscoelastic solid form after HRP and H 2 O 2 were added (FIG. 16C).

실험예 5. 지방유래 줄기세포의 생존능 및 골분화 관련 Gel-Ty 수화젤 및 Gel-Ty/GNPs 복합 수화젤의 효과 확인Experimental Example 5. Effect of Gel-Ty hydration gel and Gel-Ty/GNPs complex hydration gel related to viability and bone differentiation of adipose-derived stem cells

5-1. 생존능 확인5-1. Viability check

Gel-Ty 수화젤 및 Gel-Ty/GNPs 복합 수화젤 적용시의 지방유래 줄기세포의 생존능을 측정하였다. 구체적으로, 수화젤의 표면 및 내부 특성을 확인하기 위해 2가지 방법을 이용하였다. 첫 번째로, 직경 8 ㎜의 디스크 형상으로 준비된 수화젤을 48 웰의 세포 배양플레이트에 놓고, 1 시료 당 1×104 세포의 농도로 드롭 시딩하였다. 두 번째로, 두 종류의 Gel-Ty 수화젤 용액을 수화젤 가교 결합 전 준비하였다. The viability of adipose-derived stem cells was measured when Gel-Ty hydration gel and Gel-Ty/GNPs complex hydration gel were applied. Specifically, two methods were used to check the surface and internal characteristics of the hydrogel. First, a hydration gel prepared in the shape of a disk having a diameter of 8 mm was placed on a 48-well cell culture plate, and seeded by drop at a concentration of 1×10 4 cells per sample. Second, two types of Gel-Ty hydration gel solutions were prepared before hydration gel crosslinking.

가교 결합제 H2O2를 10 % Gel-Ty 용액에 첨가하고, HRP가 있는 다른 10 % Gel-Ty 용액에 2×106 cell/mL의 지방유래 줄기세포를 분산시켰다. 상기 두 용액을 혼합한 후, 이중 주사기(dual syringe)를 이용하여 쏟아부었다. 이후 상기 가교 결합된 수화젤을 디스크로 절단한 다음 48 웰 세포 배양 플레이트로 옮겼다. A crosslinking agent H 2 O 2 was added to a 10% Gel-Ty solution, and 2×10 6 cells/mL of adipose-derived stem cells were dispersed in another 10% Gel-Ty solution with HRP. After mixing the two solutions, it was poured using a dual syringe. Thereafter, the cross-linked hydrogel was cut into a disk and then transferred to a 48-well cell culture plate.

이후 모든 수화젤 샘플을 세포 생존능 분석 키트를 사용하여 평가하였다. 또한, 수화젤에서 배양된 지방유래 줄기세포를 3.7 % 포름알데히드 용액을 사용하여 고정시킨 다음, Alexa Fluor 488 phalloidin 및 DAPI를 사용하여 염색하였다. 상기와 같이 준비된 샘플을 CLSM으로 관찰하였다.Thereafter, all hydrogel samples were evaluated using a cell viability assay kit. In addition, adipose-derived stem cells cultured in hydration gel were fixed using a 3.7% formaldehyde solution, and then stained using Alexa Fluor 488 phalloidin and DAPI. The sample prepared as described above was observed with CLSM.

그 결과, 도 17에 나타난 바와 같이 5 내지 8%의 Gel-Ty 수화젤 표면에서 배양된 세포들은 모두 형광 염색되었으며, CLSM을 통해 모든 그룹에서 잘 증식되었음을 확인하였다. As a result, as shown in FIG. 17, cells cultured on the surface of 5 to 8% Gel-Ty hydration gel were all fluorescently stained, and it was confirmed that the cells proliferated well in all groups through CLSM.

또한, 도 18에 나타난 바와 같이 Gel-Ty 수화젤의 농도 차이에 따른 효과 차이는 거의 나타나지 않았으며, 시간이 경과할수록 생존능이 증대되었다. 특히, 7일 째, 모든 Gel-Ty 수화젤 군에서 대조군에 비해 1.5배 이상의 생존능을 나타냄을 확인하였다.In addition, as shown in FIG. 18, the difference in effect according to the difference in the concentration of the Gel-Ty hydrated gel was hardly observed, and the viability increased as time passed. In particular, on the 7th day, it was confirmed that all Gel-Ty hydrogel groups showed 1.5 times more viability than the control group.

또한, 도 19에 나타난 바와 같이, Gel-Ty 수화젤에서 배양하였을 때, 5 내지 10 % 각 농도의 Gel-Ty 수화젤 간에 생존 세포수가 유사하였으며, 3 일 배양 후, Gel-Ty 수화젤에서의 생존능은 더욱 증가되어 있음을 확인하였다.In addition, as shown in FIG. 19, when cultured in Gel-Ty hydration gel, the number of viable cells was similar between Gel-Ty hydration gels at each concentration of 5 to 10%, and after 3 days of cultivation, in Gel-Ty hydration gel It was confirmed that the viability was further increased.

아울러, 5 내지 10 % 각 Gel-Ty 수화젤에서 배양된 형태를 CLSM을 통해 형광 이미지로 확인하였으며, 도 20에 나타난 바와 같이, 10 % Gel-Ty 수화젤의 경우 3일 배양 후에도 특히 형태가 잘 유지됨을 확인하였다. In addition, the form cultured in each of 5 to 10% Gel-Ty hydration gel was confirmed by a fluorescence image through CLSM, and as shown in FIG. 20, in the case of 10% Gel-Ty hydration gel, the shape is particularly well after 3 days incubation. It was confirmed that it was maintained.

또한, GNPs의 Gel-Ty 수화젤에 대한 적용 가능성을 평가하기 위해 다양한 농도(10, 100, 200 및 400 μM)의 GNPs를 가진 10%의 Gel-Ty/GNPs 복합 수화젤을 합성하여 생존능을 확인하였다. 그 결과, 도 21에 나타난 바와 같이, GNPs농도가 증가함에 따라 세포 생존능이 증가하는 것을 확인하였다. 특히 200 μM 이상의 GNPs 농도에서는 대조군에 비해 생존능이 현저히 증가되어 있음을 확인하였다.In addition, in order to evaluate the applicability of GNPs to Gel-Ty hydration gel, a 10% Gel-Ty/GNPs complex hydration gel with various concentrations (10, 100, 200 and 400 μM) of GNPs was synthesized to confirm the viability. I did. As a result, as shown in Fig. 21, it was confirmed that the cell viability increased as the concentration of GNPs increased. In particular, it was confirmed that the viability was significantly increased compared to the control group at a concentration of GNPs of 200 μM or more.

상기와 같은 결과로부터 본 발명의 Gel-Ty 수화젤 및 Gel-Ty/GNPs 복합 수화젤의 세포 생존능은 모두 높게 나타났는바, 독성 문제없이 생체에 적용될 수 있음을 확인하였다.From the above results, it was confirmed that the cell viability of both the Gel-Ty hydration gel and the Gel-Ty/GNPs complex hydration gel of the present invention were high, and could be applied to a living body without a toxicity problem.

5-2. 알칼리 포스파타아제(Alkaline phosphatase, ALP) 활성 확인5-2. Alkaline phosphatase (ALP) activity check

20, 100 및 200 μM 각각의 GNPs를 포함하는 Gel-Ty/GNPs 복합 수화젤의ALP 활성을 확인하였다. ALP 활성 측정 방법은 상기 실험예 2-3과 동일하다.The ALP activity of the Gel-Ty/GNPs composite hydration gel containing 20, 100 and 200 μM GNPs was confirmed. The method for measuring ALP activity is the same as in Experimental Example 2-3.

그 결과, 도 22에 나타난 바와 같이, 20, 100 및 200 μM 전체 농도에서 GNPs가 포함되어 있지 않은 대조군과 비교하여 우수한 ALP 활성을 나타내었다. 특히, 100 μ의 GNPs를 포함한 Gel-Ty 수화젤의 경우 5, 10 및 15 일 배양 전체 기간 동안 20및 200 μM의 GNPs를 포함하는 Gel-Ty 수화젤에 비해 높은 ALP 활성을 나타내었으며, GNPs가 포함되어 있지 않은 대조군과 비교하여 최소 1.33 내지 최대 1.62배 더 높은 활성을 나타냄을 확인하였다.As a result, as shown in FIG. 22, it exhibited excellent ALP activity compared to the control group not containing GNPs at the total concentration of 20, 100 and 200 μM. In particular, Gel-Ty hydration gel containing 100 μ GNPs showed higher ALP activity than Gel-Ty hydration gel containing 20 and 200 μM GNPs for the entire 5, 10 and 15 day culture period. It was confirmed that the activity was at least 1.33 to 1.62 times higher than that of the control group not included.

또한, 100 μM의 GNPs를 포함하는 Gel-Ty/GNPs 복합 수화젤 및 Gel-Ty/G-NAC복합 수화젤의 ALP 활성을 측정하였다. 그 결과 도 23에 나타난 바와 같이, Gel-Ty/G-NAC복합 수화젤의 ALP 활성은 Gel-Ty 및 Gel-Ty/GNPs 보다 최소 1.41 내지 최대 1.85배 더 높은 활성을 나타냄을 확인하였다.In addition, the ALP activity of the Gel-Ty/GNPs composite hydration gel and the Gel-Ty/G-NAC composite hydration gel containing 100 μM GNPs were measured. As a result, as shown in FIG. 23, it was confirmed that the ALP activity of the Gel-Ty/G-NAC complex hydration gel exhibited a minimum of 1.41 to a maximum of 1.85 times higher than that of Gel-Ty and Gel-Ty/GNPs.

상기와 같은 결과로부터 본 발명의 Gel-Ty/GNPs 복합 수화젤 및 Gel-Ty/G-NAC복합 수화젤은 모두 대조군에 비해 매우 우수한 ALP 활성을 나타내는 바, 우수한 골 분화 효과를 나타냄을 확인하였다.From the above results, it was confirmed that both the Gel-Ty/GNPs composite hydration gel and the Gel-Ty/G-NAC composite hydration gel of the present invention exhibited very excellent ALP activity compared to the control group, thus exhibiting excellent bone differentiation effect.

즉, 본 발명의 조성물은 세포의 생존능을 유지하여 독성 문제가 없으면서도 우수한 골 분화 효과를 나타내는 바, 골 재생용, 골 질환 치료용으로 널리 활용될 수 있다.That is, the composition of the present invention can be widely used for bone regeneration and for treating bone diseases, as the composition of the present invention maintains the viability of cells and exhibits excellent bone differentiation effect without toxicity problems.

상기 모든 실험예의 통계 분석은 Tukey의 다중 비교 사후 테스트를 이용한 양방향 ANOVA를 사용하여 수행하였다. 모든 값은 평균 ± 표준 편차로 나타내었고, 모든 실험군을 서로 비교하였다. 유의성은 * P<0.05, ** P<0.01 및 *** P<0.001로 정의하였다.Statistical analysis of all the experimental examples was performed using a two-way ANOVA using Tukey's multiple comparison post test. All values were expressed as mean ± standard deviation, and all experimental groups were compared with each other. Significance was defined as *P<0.05, **P<0.01 and ***P<0.001.

전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다. The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included in the scope of the present invention.

Claims (9)

i) 금 나노입자 및 항산화 물질의 결합체 또는
ii) 금 나노입자 또는 금 나노입자 및 항산화 물질의 결합체를 포함하는 수화젤을 포함하는, 골 재생 또는 골 형성 촉진용 조성물. i) a combination of gold nanoparticles and antioxidants, or
ii) A composition for promoting bone regeneration or bone formation, comprising a hydrogel containing gold nanoparticles or a combination of gold nanoparticles and antioxidants. 제1항에 있어서,
상기 수화젤은 젤라틴 및 티라민의 결합체를 포함하는 것인, 골 재생 또는 골 형성 촉진용 조성물. The method of claim 1,
The hydrogel is a composition for promoting bone regeneration or bone formation comprising a combination of gelatin and tyramine. 제1항에 있어서,
상기 항산화 물질은 N-아세틸 시스테인(N-acetyl cysteine)인, 골 재생 또는 골 형성 촉진용 조성물.The method of claim 1,
The antioxidant is N-acetyl cysteine (N-acetyl cysteine), a composition for promoting bone regeneration or bone formation. 제1항에 있어서,
상기 금 나노입자는 10 μM 내지 400 μM 의 농도로 포함되는 것인, 골 재생 또는 골 형성 촉진용 조성물.The method of claim 1,
The gold nanoparticles will be contained in a concentration of 10 μM to 400 μM, a composition for promoting bone regeneration or bone formation. 제1항에 있어서,
상기 금 나노입자 및 항산화 물질의 결합체는 10 μM 내지 400 μM 의 농도로 포함되는 것인, 골 재생 또는 골 형성 촉진용 조성물. The method of claim 1,
The combination of the gold nanoparticles and the antioxidant is contained in a concentration of 10 μM to 400 μM, bone regeneration or bone formation promoting composition. 제1항 내지 제5항 중 어느 한 항의 조성물을 포함하는,
골다공증, 암세포의 골전이에 의해 초래되는 뼈의 손상, 골연화증, 구루병, 섬유성 골염, 무형성 골질환, 대사성 골질환, 골용해, 백혈구 감소증, 뼈의 기형, 고칼슘혈증, 류마티스성 관절염, 골관절염, 골수염, 변성 관절증 및 골종양으로 구성되는 군으로부터 선택되는 하나 이상의 질환의 골 재생 또는 골 형성 촉진용 약학적 조성물.Comprising the composition of any one of claims 1 to 5,
Osteoporosis, bone damage caused by bone metastasis of cancer cells, osteomalacia, rickets, fibrotic osteopathy, amorphous bone disease, metabolic bone disease, osteolysis, leukopenia, bone deformity, hypercalcemia, rheumatoid arthritis, osteoarthritis, osteomyelitis , A pharmaceutical composition for promoting bone regeneration or bone formation of one or more diseases selected from the group consisting of degenerative arthrosis and bone tumors. 제6항에 있어서,
상기 조성물은 기형 교정, 치과 교정, 골절 치료, 골접합술, 가관절에서의 골 재생, 골 형성 또는 골 이식에 사용되는 것인, 골 재생 또는 골 형성 촉진용 약학적 조성물.The method of claim 6,
The composition is used for deformity correction, dental orthodontics, fracture treatment, bone splicing, bone regeneration in the knee joint, bone formation or bone graft, a pharmaceutical composition for promoting bone regeneration or bone formation. a) 금 나노입자, 항산화 물질, 또는 금 나노입자 및 항산화 물질의 결합체를 준비하는 단계;
b) 젤라틴 및 티라민을 혼합하여 수화젤을 제조하는 단계; 및
c) 상기 a) 단계의 금 나노입자, 항산화 물질, 또는 금 나노입자 및 항산화 물질의 결합체와 상기 b) 단계에서 제조된 수화젤을 혼합하는 단계를 포함하는, 골 재생 또는 골 형성 촉진용 수화젤의 제조방법.a) preparing gold nanoparticles, antioxidants, or a combination of gold nanoparticles and antioxidants;
b) preparing a hydrogel by mixing gelatin and tyramine; And
c) a hydration gel for promoting bone regeneration or bone formation, comprising mixing the gold nanoparticles, antioxidants, or a combination of gold nanoparticles and antioxidants in step a) with the hydration gel prepared in step b) Method of manufacturing. 제8항에 있어서,
상기 항산화 물질은 N-아세틸 시스테인(N-acetyl cysteine)인, 골 재생 또는 골 형성 촉진용 수화젤의 제조방법.The method of claim 8,
The antioxidant is N-acetyl cysteine (N-acetyl cysteine), a method for producing a hydration gel for promoting bone regeneration or bone formation.
KR1020190028576A 2019-03-13 2019-03-13 Hydrogel for bone regeneration and preparation method thereof KR20200110495A (en)

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Non-Patent Citations (1)

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Title
O. J. Sul, J. C. Kim, T. W. Kyung, H. J. Kim, Y. Y. Kim, S. H. Kim, J. S. Kim and H. S. Choi, Biosci., Biotechnol., Biochem., 2010, 74, 2209-2213

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