Kisspeptin-10 (KP-10) has been reported to act as a tumor metastasis suppressor via its receptor, G protein-coupled receptor 54 (GRP54). The KP-10/GPR54/BMPs signaling pathway plays an important role in embryonic kidney development. However, its function in osteoblast differentiation is unknown. The aim of this study was to confirm the molecular mechanism for the action of KP-10 on osteoblast differentiation. Expression of the Bone morphogenetic protein-2 (BMP2) and osteogenic genes were determined by RT-PCR and real-time PCR analysis in C3H10T1/2 cells. Transient transfection assays were performed to confirm the effects of KP-10 on BMP2-Luc activity. BMP2 and phospho-Smad1/5/9 protein levels were determined by Western blot analysis. Alkaline phosphatase (ALP) staining experiment was performed to evaluate ALP activity. To further confirm the effect of KP-10-induced GPR54, we used GPR54 Knock out (KO) C3H10T1/2 cells. KP-10 significantly increased osteogenic gene such as Runx2, ALP and Dlx5 in C3H10T1/2 cells. The ALP staining levels were also increased by KP-10. Interestingly, BMP2 mRNA, protein expression and promoter activity were also increased by KP-10. However, KP-10-induced BMP2 expressions were not increased in GPR54 KO cells. These results suggest that KP-10 increases BMP2 expression through GPR54. Next, Western blot analysis shown Smad1/5/9 phosphorylation were enhanced in a time-dependent manner by KP-10 treatment. It is well known that BMP2 increased phosphorylation of Smad1/5/9 via BMP2 receptor. In addition, KP-10 increased NFATc4 mRNA levels and NFATc4 overexpression enhance BMP2 mRNA levels. To confirm the KP-10-induced BMP2 action, we used KP-10-treated medium in wild type cells and GPR54 KO cells. The osteogenic genes were not elevated by KP-10-treated medium (GPR54 KO cells) whereas increased expression levels by KP-10 medium (wild type cells). These data indicate that KP-10 induced osteoblast differentiation through NFATc4-mediated BMP2 signaling.

In this study, we fabricated a novel micro porous hybrid scaffold of biphasic calcium phosphate (BCP) and a polylectrolyte complex (PEC) of chitosan (CS) and hyaluronic acid (HA). The fabrication process included loading of CS-HA PEC in a bare BCP scaffold followed by lypophilization. SEM observation and porosimetry revealed that the scaffold was full of micro and macro pores with total porosity of more than 60 % and pore size in the range of 20~200μm. The composite scaffold was mechanically stronger than the bare BCP scaffold and was significantly stronger than the CS-HA PEC polymer scaffold. Bone morphogenetic growth factor (BMP-2) was immobilized in CS-HA PEC in order to integrate the osteoinductive potentiality required for osteogenesis. The BCP frame, prepared by sponge replica, worked as a physical barrier that prolonged the BMP-2 release significantly. The preliminary biocompatibility data show improved biological performance of the BMP-2 immobilized hybrid scaffold in the presence of rabbit bone marrow stem cells (rBMSC).

This study evaluated the possibility of clinical application using matrigel-based bioceramic/polymer scaffolds treated with bone morphogenetic protein, angiogenic factor, and mesenchymal stem cells (MSCs) for new bone formation. In the in vitro study, bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) containing matrigel, which is a basement membrane gel, was injected into HA/PCL scaffolds to estimate the release rates of growth factors. In the in vivo study, BMP-2, VEGF, and MSCs with matrigel-based scaffolds were implanted into rat femoral segmental defects, and new bone formation was evaluated at 4 and 8 weeks. In the results, the release rates of BMP-2 and VEGF explosively increased by day 5. For the in vivo study results, radiological evaluation revealed that the matrigel-based HA/PCL scaffolds with BMP-2 and VEGF grafted (M+B+V) and matrigel-based HA/PCL scaffolds with BMP-2, VEGF, and MSC grafted (MSC) groups showed increased bone volume and bone mineral density. Moreover, in the histological evaluation, large new bone formation was observed in the M+B+V group, and high cellularity in the scaffold was observed in the MSC group. In conclusion, grafted matrigel-based HA/PCL scaffolds with BMP-2, angiogenic factor, and MSCs increased new bone formation, and in clinical cases, it may be effective and useful to enhance healing of delayed fractures.

Recently, extensive research has been performed in the field of orthopedic medicine to develop cell-based therapies for the restoration of injured bone tissue. But there has been rarely reported about rehabilitaton of oral and maxillofacial bone defect using self-derived osteoblasts. Normal human osteoblast cell(NHost) was previously established into marrow-derived human mesenchymal stem cells for their capacity to proliferate and differentiate into osteoblasts under various culture conditions. The purpose of this study was to examine proliferation and differentiation of NHosts effected by growth factors with ALP activity and RT-PCR. After NHosts were cultured under basal and osteogenic medium at 37℃ and 5% CO2, they were analyzed by ALP activity and RT-PCR. BMP-2 under osteogenic medium decreased growth rate of NHosts compared to under osteogenic medium. BMP-2 under osteogenic medium induced osteoblastic differentiation in NHosts by increased ALP activity. The differentiating capacity of NHosts under osteogenic medium showed that NHosts expressed higher mRNA expression levels of OSX and OCN, while that of RUNX2 decreased after BMP-2 treatment. It suggested that NHosts having characteristics of osteoprecursor cells might be more advanced in their osteogenesis development by BMP-2, making NHosts an interesting biological tool for treatment of skeletal defects and diseases of oral and maxillofacial bone.

암 환자의 방사선 치료는 환자의 생존 수명을 연장하기 위해 사용한다. 그러나 방사선 조사 후 피폭으 로 인해 정상적인 조직 재생은 치명적인 장해를 수반한다. 방사선 치료 후 발생 할 수 있는 장해는 골세포와 골대사 메커니즘에 결정적인 방사선 장해를 수반하여 방사성골괴사가 발생된다. 따라서 방사성골괴사를 극복하기 위해 방사선 장해의 극복을 위한 조직재생의 연구가 필수적으로 대두된다. 이번 연구에서는 골재생의 대표적인 사이토카인 단백질인 BMP-2가 방사성조사 쥐 두개골 모델에서 골재생의 효과가 있는 지 또한 몇 주부터 골재생 효과가 많이 발생되는지를 검증하고자 하였다. 결론적으로 방사선이 조사된 쥐의 두개골 결손모델에서는 8주가 지나야 BMP-2의 효과가 더욱더 효과적으로 발생하는 결과를 얻을 수 있었다. 만일 BMP-2 가 처리된 지지체를 방사선골괴사 치료제로 사용한다면 단시간에 골 재생 효과를 기대 하는 것보다는 8주 이상의 시간이 경과 후 골재생 효과를 기대해야 할 것으로 사료된다.

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.