(The) role of polaprezinc in fracture healing by differentiation of both osteoclasts and osteoblasts

다국어 초록 (Multilingual Abstract)

Fractures and fracture related complications are common causes of morbidity and mortality nowadays. According to the data from National Health Insurance and Assessment Service, over 2,200,000 fractures are sustained every year in South Korea of all ages and sexes. Although the majority heal satisfactorily, 5% to 10% go on to delayed union or non-union. Vitamin D and calcium are the broadly available medications for fracture healing, while zinc has been recognized as a nutritional supplement for healthy bones. We aimed to use polaprezinc, an anti-ulcer drug and a chelate form of zinc and L-carnosine, as a supplement for fracture healing.

To investigate whether polaprezinc could be a candidate small molecule for drug repurposing to bone-related diseases, we used hBMSCs and mBMMs to determine whether polaprezinc acts as a positive or negative modulator during osteoblast and osteoclast differentiation. Next, we confirmed the effects of polaprezinc on hBMSC differentiation to osteogenic lineage at the early and late stages of differentiation. We compared the mRNA levels of osteogenic-related genes between vehicle- and polaprezinc-treated hBMSCs. To clarify the effect of polaprezinc on osteoclast differentiation, mBMMs were induced by treatment with mRANKL in the presence of polaprezinc dose dependently. To evaluate whether polaprezinc may serve as a supplement for enhancing the treatment of fracture healing, mice with femoral fractures were employed for animal studies in vivo.

Polaprezinc enhanced expression of osteogenesis-related genes and enhanced osteogenic potential of human bone marrow-derived mesenchymal stem cells such as RUNX2 protein level and the osteoclast differentiation potential of mouse bone marrow-derived monocytes such as TRAP activities induced by RANKL. In mouse experimental models with bone fractures, oral administration of polaprezinc accelerated fracture healing and maintained a high number of both osteoblasts and osteoclasts in the fracture areas. μCT image showed polaprezinc group had lower remaining callus volume and callus BMD was significantly increased in orally administered polaprezinc mice.

In conclusion, polaprezinc promotes the fracture healing process efficiently by enhancing the activity of both osteoblasts and osteoclasts. Therefore, these data suggest that drug repositioning of polaprezinc as a fracture healing therapy would be helpful for patients with fractures.

국문 초록 (Abstract)

서론: 골절과 골절 관련 합병증은 최근 가장 흔한 질병 및 사망의 원인중 하나이다. 국민건강보험공단 자료에 따르면 한국에서는 남녀노소를 불문하고 매년 220만 건 이상의 골절이 발생하고 있다. 대다수가 만족스럽게 치유되지만 5~10%는 지연유합 또는 불유합으로 진행된다. 비타민 D와 칼슘은 골절 치료에 널리 사용되는 보충제이며, 아연 역시 건강한 뼈를 위한 영양 보충제로 인식되어 왔다. 본 연구에서는 골절 치유를 위한 치료제로서 아연과 L-카르노신의 킬레이트 형태의 약물인 폴라프레징크를 골절의 치료제로서 사용 가능한지를 알아보고자 하였다.
방법: 폴라프레징크가 골절의 치료제로서 약물 재배치의 좋은 후보가 될 수 있는지를 알아보기 위해, 인간 골수 유래 중간엽 줄기세포와 생쥐 골수 유래 단핵구를 사용하여 폴라프레징크가 조골세포와 파골세포분화 동안 양의 조절제 또는 음의 조절제로 작용하는지 여부를 관찰하였다. 다음으로, 분화 초기 및 후기 단계에서 인간 골수 유래 중간엽줄기세포 분화에서 골형성 계통으로의 폴라프레징크 영향을 관찰하였다. 폴라프레징크로 처리된 인간 골수 유래 중간엽 줄기세포와 대조군 간의 골형성 관련 유전자의 mRNA 수준을 비교했다. 파골세포 분화에 대한 폴라프레징크의 영향을 명확히 하기 위해 생쥐 골수 유래 단핵구는 mRANKL로 처리하여 폴라프레징크의 양을 다르게 하여 유도하였다. 폴라프레징크가 골절 치료제로서의 역할을 할 수 있는지를 평가하기 위해 대퇴골 골절이 있는 쥐를 동물 연구에 사용하였다.
결과: 폴라프레징크는 인간 골수 유래 중간엽 줄기세포에서 조골세포분화를 촉진하고, 생쥐 골수 유래 단핵구에서 파골세포 분화를 촉진하였다. 즉, 조골세포와 파골세포의 분화에 이중양성 효과를 보였는데, 이는 일반 아연의 역할과는 차이가 있었다. 폴라프레징크의 조골세포 및 파골세포 분화는 특히 전사 활성에서 YAP의 존재에 따라 달라졌다. 폴라프레징크를 경구 투여하면 다수의 조골세포와 파골세포에 의한 능동적인 뼈 항상성을 통해 대퇴골 골절이 있는 쥐에게 신속하고 성공적인 골절 치유를 유도할 수 있었다.
결론: 폴라프레징크는 조골세포와 파골세포의 활성을 향상시켜 골절 치유 과정을 효율적으로 촉진한다. 따라서, 우리는 폴라프레징크가 성공적인 골절 치유를 위한 치료제일 뿐만 아니라 약물 재배치를 통해 임상적용에 좋은 후보가 될 수 있다고 생각한다.

목차 (Table of Contents)

• ABSTRACT iv
• I. INTRODUCTION 1
• II. MATERIALS AND METHODS 3
• 1. Cell culture and reagents 3
• 2. Cell viability measurement 4
• 3. In vitro osteogenic differentiation 4
• 4. Alkaline phosphatase and alizarin red S staining 4
• 5. In vitro osteoclastogenic differentiation 5
• 6. TRAP activity and TRAP staining 5
• 7. Quantitative real-time polymerase chain reaction 6
• 8. Western blot analysis 8
• 9. Intracellular zinc measurement 8
• 10. Luciferase assay 9
• 11. siRNA-mediated knockdown 9
• 12. Mouse femoral fracture model 9
• 13. Micro-computed tomography 10
• 14. Immunohistochemical analysis 11
• 15. Immunocytochemistry 11
• 16. Protein fractionation 12
• 17. Statistics and reproducibility 12
• III. RESULTS 13
• 1. Polaprezinc promotes osteoblast differentiation in hBMSCs 13
• 2. Polaprezinc accelerates osteoclast differentiation in mBMMs 22
• 3. Different effects of polaprezinc and zinc on osteoblast and osteoclast differentiation 25
• 4. YAP may be as responsible molecular mediator of polaprezinc-induced osteoblast and osteoclast activity 28
• 5. Oral administration of polaprezinc accelerates bone remodeling in a mouse fracture model 35
• IV. DISCUSSION 42
• V. CONCLUSION 44
• REFERENCES 45
• ABSTRACT (IN KOREAN) 53
• PUBLICATION LIST 55

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