추간판 세포에서 Transforming Growth Factor-β1 및 Bone Morphogenetic Protein-2에 의한 기질 생성 및 연골성 표현형 발현

김동준,문성환,김향,권언혜,한수봉,천용민,김학선,한경진,박문수,이환모 대한척추외과학회 2002 대한척추외과학회지 Vol.9 No.3

척추 황색 인대 세포에서 Bone Morphogenetic Protein-2 cDNA 전달에 의한 골형성 유도 : 조직 공학적 접근의 가능성

문성환,김향,권언혜,원정훈,김학선,한수봉,이환모 대한척추외과학회 2002 대한척추외과학회지 Vol.9 No.4

아데노바이러스 벡터를 이용한 BMP-2와 FGF-2의 동시 발현이 골형성에 미치는 영향

송상훈,장원구,김선헌,국민석,김옥준,김인애,박인규,고정태 한국조직공학과 재생의학회 2009 조직공학과 재생의학 Vol.6 No.13

FGF-2 and BMPs are expressed simultaneously during the embryonic bone development and repair of fractured bone. Delivery of BMP-2 peptide and gene has been evaluated for a therapeutic bone regeneration. However, low efficacy and short half -life of BMP-2 peptide have limited their clinical uses. This study was to determine the effects of BMP-2 and FGF-2 combination on bone formation with the adenoviral BMP-2 and FGF-2 gene delivery. Adenoviruses (Ad) expressing BMP-2 or FGF-2 were constructed under the CMV promoter, and characterized. AdBMP-2 dose-dependantly induced BMP-2 peptide expression in BLK cells, and increased alkaline phosphatase (ALP) activity in C2C12. AdFGF-2 also induced FGF-2 peptide expression in a dose-dependent manner. Proliferation of C3H10T1/2, ST-2 and MC3T3-E1 cell was stimulated by AdFGF-2 alone treatment, but ALP activity of the cells was inhibited. To evaluate the effect of BMP-2 and FGF-2 combination on bone formation, AdBMP-2 (100 moi) and/or AdFGF-2 (100 or 200 moi) were transduced into BLK fibroblast cells, and then the cells were transferred into subcutaneous spaces with collagen hydrogel in mice. Microradiographic analysis, biochemical assays and histology showed AdBMP-2 and AdFGF-2 combination produced less bone than AdBMP-2 alone at 2 weeks after implantation. In addition, AdBMP-2/AdFGF-2 combination group had less ALP activity and concentrations of inorganic phosphate and calcium in implants than AdBMP-2 treated group. These results suggest that FGF-2 may stimulate the proliferation of osteoblastic cells, but inhibit the BMP-2 induction of osteoblast differentiation and bone formation. FGF-2 and BMPs are expressed simultaneously during the embryonic bone development and repair of fractured bone. Delivery of BMP-2 peptide and gene has been evaluated for a therapeutic bone regeneration. However, low efficacy and short half -life of BMP-2 peptide have limited their clinical uses. This study was to determine the effects of BMP-2 and FGF-2 combination on bone formation with the adenoviral BMP-2 and FGF-2 gene delivery. Adenoviruses (Ad) expressing BMP-2 or FGF-2 were constructed under the CMV promoter, and characterized. AdBMP-2 dose-dependantly induced BMP-2 peptide expression in BLK cells, and increased alkaline phosphatase (ALP) activity in C2C12. AdFGF-2 also induced FGF-2 peptide expression in a dose-dependent manner. Proliferation of C3H10T1/2, ST-2 and MC3T3-E1 cell was stimulated by AdFGF-2 alone treatment, but ALP activity of the cells was inhibited. To evaluate the effect of BMP-2 and FGF-2 combination on bone formation, AdBMP-2 (100 moi) and/or AdFGF-2 (100 or 200 moi) were transduced into BLK fibroblast cells, and then the cells were transferred into subcutaneous spaces with collagen hydrogel in mice. Microradiographic analysis, biochemical assays and histology showed AdBMP-2 and AdFGF-2 combination produced less bone than AdBMP-2 alone at 2 weeks after implantation. In addition, AdBMP-2/AdFGF-2 combination group had less ALP activity and concentrations of inorganic phosphate and calcium in implants than AdBMP-2 treated group. These results suggest that FGF-2 may stimulate the proliferation of osteoblastic cells, but inhibit the BMP-2 induction of osteoblast differentiation and bone formation.

생쥐의 oviduct에서 estrogen에 의한 bone morphogenetic protein 2, 4 (BMP2, BMP4)의 발현 조절 및 기능

조남희,계명찬 한국발생생물학회 2017 한국발생생물학회 학술발표대회 Vol.2017 No.8

골 형성 단백질(Bone morphogenetic protein, BMP)은 TGF-β superfamily의 구성원 중에 하나이며, 이들은 원래 뼈 형성을 유도하는 능력에 의해 발견되었지만, 뼈 외에도 다른 세포 및 기관의 성장과 분화를 조절하는 것으로 밝혀졌다. 다기능적인 골 형성 단백질은 포유동물의 reproduction에도 매우 중요한 역할을 하는데, 예를 들어 BMP8A와 BMP8B는 생쥐의 정자 형성과 부고환에 일정한 기능을 하는 것으로 보고되었고 난소에서의 BMP15는 난포성장을 자극하고, 과립막세포(granulosa cell)의 증식시키는데 관여하는 단백질로 알려져 있다. 하지만 난관(oviduct)에서의 BMP 역할은 알려진 바가 적다. 그래서 본 연구에서는 골 형성 단백질이 착상 전 oviductal environment에 어떠한 영향을 미치는지 밝히고자 하였다. 8주령 생쥐의 Estrous cyclic oviduct을 가지고 RT-PCR과 Immunohistochemistry(IHC)를 통해 BMP2, 4의 mRNA와 단백질 발현을 확인하였다. Estrogen에 의한 BMP2, 4의 영향을 확인하고자 난소절제술을 시행한 생쥐와 Estrogen receptor alpha(ERα) Knockout 생쥐를 통해 mRNA와 단백질 발현을 확인하였다. Oviduct의 ciliated cell을 가지고 BMP2, 4의 기능을 밝히고자 siRNA실험을 진행 하였다. Estrous cyclic oviduct cDNA를 통해 RT-PCR한 결과, 이 중 Estrus 시기에 가장 높은 발현을 보인 BMP2, 4의 mRNA level은 Isthmus보다 Infundibulum과 Ampulla에 증가하였고, Immunohistochemistry (IHC)를 통해 BMP2와 BMP4 단백질은 난관 상피세포 중에서 ciliated cell에 발현되었다. 이를 ciliated cell marker인 β-tubulin과 함께 Immunofluorescence(IF)를 진행한 결과, 주로 β-tubulin-positive ciliated cell에서 발현됨을 확인하였다. 난소 절제술을 시행한 생쥐의 난관에서는 E2와 DPN (ERβ agonist)에 의해 BMP2, 4 mRNA 발현이 증가하고, ERαKO 난관의 경우 WT(Die)에 비해 BMP2, 4 mRNA, 단백질의 발현 모두 증가하였다. BMP2와 BMP4의 기능을 밝히고자 Ciliated cell line인 OA-6b를 가지고 siRNA 실험을 진행한 결과, Bmp2, Bmp4 siRNA 처리군에서 Ciliated cell marker인 FOXJ1의 발현이 줄어들고 Proliferation marker인 Ki67, Pcna의 발현이 낮아졌다. 이로써, Oviduct 내 BMP2와 BMP4 발현은 Estrogen과 양의 상관성이 있으며, Ciliated cell에서 Estrogen - ER β signaling을 통해 조절된다. Estrogen에 의해 유도되는 BMP2와 BMP4는 ciliated cell에서 Autocrine, Paracrine factor로 작용할 가능성이 있다. 더 나아가 Oviduct의 Infundibulum 및 Ampulla에 강하게 발현되는 BMP2, BMP4는 난자 및 배아에 autocrine, paracrine factor로 작용하여 oviductal cell proliferation을 조절하고, 수정을 위한 oviductal environment 조성에 중요한 역할을 하는 것으로 사료된다.

Sustained BMP-2 delivery and injectable bone regeneration using thermosensitive polymeric nanoparticle hydrogel bearing dual interactions with BMP-2

Seo, B.B.,Choi, H.,Koh, J.T.,Song, S.C. Elsevier Science Publishers 2015 Journal of controlled release Vol.209 No.-

Localized and continuous osteogenic stimulation to defected sites is required for effective bone regeneration. Here, we suggest an injectable and sustained bone morphogenetic protein-2 (BMP-2) release system using thermosensitive polymeric nanoparticles bearing dual interacting forces with BMP-2. For sustained BMP-2 release, hydrophobic and ionic interactions were introduced to thermosensitive poly(phosphazene). Hydrophobic isoleucine ethyl ester and hydrophilic poly-ethylene glycol were mainly substituted to the poly(phosphazene) back bone for amphiphilicity and hydrophobic interaction with BMP-2. Carboxylic acid moiety was additionally substituted to the back bone for ionic interaction with BMP-2. These dual interacting polymeric nanoparticles (D-NPs) formed compact nanocomplexes with BMP-2. The aqueous solution of BMP-2/D-NP nanocomplexes was transformed to hydrogel when the temperature of the solution increased. Loaded BMP-2 was sustain-released for three weeks from the BMP-2/D-NP nanocomplex hydrogel. The extended BMP-2 exposure caused higher osteocalcin secretion in C2C12 cells. Significant bone generations were observed at the target site by single injection of BMP-2/D-NP nanocomplexes in vivo.

척추 골유합에 있어 황색 인대의 이중 역할: 골형성 유도 기능 및 체외 유전자 전달 매개체

문성환,김향,권언혜,김경희,윤홍기,김학선,한수봉,이환모 대한척추외과학회 2003 대한척추외과학회지 Vol.10 No.1

아텔로콜라겐 지지체, 유전자 치료, 배양된 수핵세포를 이용한 조직 공학적 추간판의 재생

김학선,이광일,김향,권언혜,남미란,장주웅,조인제,김보람,이환모,문성환 대한척추외과학회 2010 대한척추외과학회지 Vol.17 No.2

Study Design: This is an in-vitro experiment using rabbit intervertebral disc (IVD) cells and growth factors. Objectives: We wanted to determine the effect of types I,and II atelocollagen and growth factor gene therapy for matrix regeneration of rabbit IVD cells. Summary of the Literature Review: Adenovirus-medicated growth factor gene therapy is efficient for matrix regeneration of the IVD. Atellocollagen has provided a favorable environment for matrix synthesis. However, a combined approach using gene and cell therapy in an atelocollagen scaffold has not yet been attempted. Materials and Methods: Rabbit IVD cells were transduced with Ad/TGF-β1 and Ad/BMP-2. The cells were then implanted to the atelocollagen scaffold. The [methyl-3H]thymidine incorporation for DNA synthesis and the [35S]sulfur incorporation for proteoglycan synthesis were measured. RT-PCR was performed for assessing the aggrecan, collagen type I, collagen type II and osteocalcin mRNA expressions.  Results: The rabbit IVD cells with Ad/TGF-β1 and that were cultured in type I atelocollagen showed a 130% increase in new proteoglycan synthesis, while the rabbit IVD cells with Ad/TGF-β1 and that were cultured in type II atelocollagen showed a 180% increase in new proteoglycan synthesis (p<0.05). The rabbit IVD cells with Ad/BMP-2 and that were cultured in type I atelocollagen showed a 70% increase in new proteoglycan synthesis, while the rabbit IVD cells with Ad/BMP-2 and that were cultured in type II atelocollagen showed a 95% increase (p<0.05). Rabbit IVD cells with Ad/TGF-β1 and Ad/BMP-2 and that were cultured in type I and II atelocollagen demonstrated increased collagen type I and II mRNA expressions without an osteocalcin mRNA expression (p<0.05). Conclusion: Cell and gene therapy in an atelocollagen scaffold provided a efficient mechanism for chondrogenic matrix regeneration of rabbit IVD cells. 연구 계획: 토끼의 추간판 세포와 제5형 아데노바이러스(Ad) transforming growth factor beta-1(TGF-β1), 아데노바이러스 bone morphogenetic protein-2 (BMP-2), 아텔로콜라젠을 이용한 실험적 연구목적: 제1, 2형 아텔로콜라젠과 성장 인자 유전자 치료의 토끼 추간판 세포의 기질 생성 반응을 알아보았다. 선행문헌의 요약: 아데노바이러스를 이용한 성장 인자 유전자 치료는 추간판 세포의 기질 재생에 효과적이다. 아텔로콜라젠은 추간판 세포의 기질 합성에 유리한 환경을 제공한다. 하지만 아텔로콜라젠 지지체에 유전자 치료를 병행하는 방법은 시도된 바가 없었다. 대상 및 방법: 토끼의 추간판 세포를 배양하였다. Ad/TGF-β1, Ad/BMP-2 아텔로콜라젠을 생산하였다. 추간판 세포를 Ad/TGF-β1, Ad/BMP-2에 감염시키고 이 세포를 아텔로콜라젠 지지체에 배양하였다. DNA생성은 [methyl-3H] Thymidine을 이용하였고 새로운 당단백 생성은 [35S]Sulfur를 이용하였다. 또한 전사 수준에서의 추간판세포 관련 유전자의 변화를 확인하고자 RT-PCR을 통한 aggrecan, 제 1, 2형 교원질, osteocalcin mRNA발현을 조사하였다. 결과: 제1형 아텔로콜라젠에 배양되고 Ad/TGF-β1로 유전자 전달된 추간판 세포군은 대조군에 비해 130% 신생 당단벡 생성증가가 있었고 제 2형 아텔로콜라젠에 배양되고 Ad/TGF-β1로 유전자 전달된 세포군은 180%의 신생 당단백 생성 증가가 있었다. (p<0.05) 제 1형 아텔로콜라젠에 배양되고 Ad/BMP-2로 유전자 전달된 추간판 세포군은 대조군에 비해 70% 신생 당단벡 생성증가가 있었고 제2형 아텔로콜라젠에 배양되고 Ad/BMP-2로 유전자 전달된 세포군은 95%의 신생 당단백 생성 증가가 있었다. (p<0.05) 제 1, 2형 아텔로콜라젠에 배양되고 Ad/TGF-β1, Ad/BMP-2로 유전자 전달된 세포군은 제 1, 2형 교원질 mRNA발현증가가 있었으나 (p<0.05) 어떠한 경우에도 osteocalcin mRNA발현은 없었다. 결론: 추간판 세포를 아텔로콜라젠 지지체에서 배양하고, Ad/TGF-β1, Ad/BMP-2으로 유전자 치료를 시행하여 효과적으로 연골형 기질을 생성을 유도할 수 있었다.

Zinc upregulates bone-specific transcription factor Runx2 expression via BMP-2 signaling and Smad-1 phosphorylation in osteoblasts

조영은,권인숙 한국영양학회 2018 Journal of Nutrition and Health Vol.51 No.1

Purpose: Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression. Methods: Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn- (1 μM Zn) or Zn+ (15 μM Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed. Results: In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn-. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn-. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn-, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn-, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn-. Conclusion: The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.

Zinc upregulates bone-specific transcription factor Runx2 expression via BMP-2 signaling and Smad-1 phosphorylation in osteoblasts

Cho, Young-Eun,Kwun, In-Sook 한국영양학회 2018 Journal of Nutrition and Health Vol.51 No.1

Purpose: Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression. Methods: Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn- (1 μM Zn) or Zn+ (15 μM Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed. Results: In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn-. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn-. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn-, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn-, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn-. Conclusion: The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.

Adenovirus에 의해서 발현된 BMP-2가 치주인대세포의 분화에 미치는 영향

김경화,박윤정,이상철,김태일,설양조,이용무,구영,한수부,정종평,류인철,Kim, Kyoung-Hwa,Park, Yoon-Jeong,Lee, Sang-Cheul,Kim, Tae-Il,Seol, Yang-Jo,Lee, Yong-Moo,Ku, Young,Han, Soo-Boo,Chung, Chong-Pyoung,Rhyu, In-Chul 대한치주과학회 2005 Journal of Periodontal & Implant Science Vol.35 No.2

The regeneration of lost periodontal tissue is a major goal of therapy. Periodontal ligament cell(PDL) is a specialized connective tissue that connects cementum and alveolar bone to maintain and support teeth in situ and preserve tissue homoeostasis. Bone morphogenetic proteins(BMPs) have shown much potential in the reconstruction of the periodontum by stimulate new bone and new cementum formation. Limitiations of BMP administration to periodontal lesions is high dose delivery, BMP transient biological activity, and low bioavailability of factors at the wound site. Gene delivery method can be alternative treatment strategy to deliver BMPs to periodontal tissue. The purpose of this study is to investigate efficiency of BMP-2 gene delivery with cell-based therapy using PDL cells. PDL cell were transduced with adenoviruses encoding either BMP-2 or Lac-Z gene. To evaluate osteogenic activity of expressed BMP-2 on PDL cells, we investigated secreted BMP-2, cellular activity, ALPase, produced mineralized nodules. To evaluate collagen scaffold as carrier for transduced cell delivery, we examined morphology and secreted BMP-2 of transducd PDL cells on it. BMP-2 transducd PDL cells produced higher levels of BMP-2, ALPase, mineralized nodules than non transduced cells. Cellular activity of transduced cells was showed similar activity to non transduced cells. Transduce cells attached on collagen scaffold secreted BMP-2 at 7day and was showed similar morphology to non transduced cells. These results demonstrated that transduced PDL cells produced biologically active BMP-2 and collagen scaffold could be carrier of transducd cells.