골이식재로 Beta-tricalcium phosphate collagen block의 효능 연구

Abstract

1. 목적 발치, 치주질환, 노화, 외상 등 여러 원인에 의해서 치아 상실과 악골내에 골결손이 일어난 경우, 치조골의 재생이 이루어지지 않으면 기능적, 심미적 문제를 발생시킨다. 따라서 골재생을 위해 여러 가지 골이식재가 사용된다. 여러 이식재 중 이종골 이식재는 소나 돼지의 골에서 단백질을 제거하여 만든 골이식재로 자가골이나 동종골에 비해 사용량에 제한이 없지만 낮은 생분해성의 한계가 있다. 합성골 이식재는 대량 생산과 물성 조절이 가능하며 교차 감염의 우려가 없으나 재료에 따라 낮은 생분해성 등의 한계가 있다. 합성골 이식재 중 Beta-tricalcium phosphate (β-TCP)는 생체 내에서 분해되는 흡수성 재료로 골형성 능력은 증명 되었지만 생체 내 흡수 속도가 빠르고 기계적 강도가 약하다. 이에 본 연구에서는 β-TCP에 콜라겐을 혼합하여 만든 β-TCP Collagen을 기존 제품인 이종골 이식재 Bio-oss Collagen®과 비교하여 β-TCP Collagen의 골 재생 효과를 in vitro와 in vivo 실험을 통해 평가해 보고자 하였다. 2. 방법 In vitro에서의 실험군은 스캐폴드에 따라 아무 골이식재도 넣지 않은 대조군, Collagen plug, β-TCP Collagen, 및 Bio-oss Collagen® 실험군으로 각 군당 3개씩 실험하였다. 세포 부착 및 증식 연구에는 마우스 조골세포 전구체 세포주인 MC3T3 E1 subclone four cells (ATCC® CRL-2593™)를 사용하였다. ALP 활성은 7일, 14일 21일에 측정하였다. MC3T3 E1 세포에서 as alkaline phosphatase, collagen type I, osteocalcin 및 bone sialoprotein (BSP)을 골분화 마커로 사용하여 정량 PCR을 통해 이들 유전자의 발현을 분석하였다. In-vivo에서는 11주령의 백서 12마리의 두개부를 절개한 후 두개골 상면에 직경 8mm 크기의 골결손부를 형성하였다. β-TCP Collagen을 이식한 군, Bio-oss Collagen®을 이식한 군, Collagen plug만 이식한 군으로 분류하여 실험을 시행하였다. 실험동물들은 1주, 2주, 4주, 6주, 8주차에 마이크로컴퓨터단층촬영 (micro-CT) 되었으며 8주차에 희생되었다.

3. 결과 In vitro에서는 세포 부착과 증식 분석 평가를 보았을 때 세포 배양 시간이 증가함에 따라 대조군과 Collagen plug의 세포수가 증가하였으며, β-TCP Collagen, Bio-oss Collagen® 모두에서 명백한 세포 증식이 관찰되지 않았다. 골이식재를 활용한 MC3T3 E1 세포 배양의 ALP 활성 조사에서는 Collagen plug서 배양된 세포가 14일째에 다른 실험군과 비교하여 가장 높은 ALP 활성을 나타냈고 β-TCP Collagen에서만 21일째에 최대 ALP 활성을 나타내었다. 유전자 발현에서는 대조군과 Collagen plug에서 β-TCP Collagen, Bio-oss Collagen®보다 더 높은 수준의 골분화 마커 발현을 보였다. β-TCP Collagen에서 배양된 세포는 Bio-oss Collagen®에 비해 14일과 28일에 BSP는 4-5배, Col은 약 4배 및 1.5배 각각 증가된 발현 수준을 나타내었다. OCN의 경우 28일에 약 2배의 증가된 발현 수준을 나타내었고 그 외에는 유의한 차이를 나타내지 않았다. ALP 발현에서는 마찬가지로 유의한 차이를 나타내지는 않았다. In vivo에서는 모든 군에서 1주차에서는 명확한 골결손부가 관찰되었고 2주차부터 골결손부 경계선이 희미해지며 가장자리에서부터 중심부로의 골형성이 관찰되었다. Bone volume 백분율로 비교해보았을 때 Collagen plug군에서는 8주차에서 최대 11.47% 관찰되었으며 골전도성 골이식재로서의 역할을 하는 β-TCP Collagen군, Bio-oss Collagen®군에서는 β-TCP Collagen군은 8주차에 최대 69.54%, Bio-oss Collagen®군에서는 8주차에서 최대 67.08%까지 증가하였다.

1. Purpose In case of bone loss due to tooth loss, periodontal disease, aging, trauma, etc. if the alveolar bone is not regenerated, functional and aesthetic problems occur. Therefore, various bone graft materials are used for bone regeneration. Autograft is ideal, but have limited drawbacks and can cause secondary deficiencies in donors after harvest. Allograft does not require surgery, but there may be differences in bone formation depending on the condition of the bone donor. Xenograft is a bone graft made by removing protein from bovine or swine bone. It is not limited in amount compared to autograft or allograft, but have a low limit of biodegradability. Synthetic bone grafts can be mass-produced and controlled in physical properties and there is no risk of cross-infection, but it has limitations such as low biodegradability depending on the material. Beta-tricalcium phosphate (β-TCP), a synthetic bone graft, is an absorbent material that decomposes in vivo and its bone formation ability has been demonstrated. However, the biodegradability in vivo is high and the mechanical strength is weak. The purpose of this study is to evaluate the effect of bone regeneration compared by β-TCP Collagen with Bio-oss Collagen® in vitro and in vivo. 2. Methods In vitro experimental groups were divided into 4 groups depending on scaffolds (control without any bone graft material, Collagen plug, β-TCP Collagen, and Bio-oss Collagen® (Col), n=3 per group). The biocompatibility of the scaffolds was evaluated via a cell adhesion and growth assay. MC3T3 E1 cells were seeded and cultured on the scaffold (Collagen plug, β- TCP Collagen, and Bio-oss Collagen®) and on the bare plate without scaffold as control, and proliferation of the adhered cells was assessed via MTS assay for 1, 3 and 7 days. MC3T3 E1 cells were cultured in GM for 2 days under the same conditions as mentioned above, the medium was changed to BM, and ALP activity was measured at 7, 14 and 21 days. Quantitative PCR (qPCR) was used for evaluating the expression of osteogenic differentiation markers in MC-3T3 E1 such as alkaline phosphatase (ALP), collagen type I (Col), osteocalcin (OCN) and bone sialoprotein (BSP). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. Twelve 9-week-old Sprague-Dawley rats with an average weight of 250g ~ 300g were used for in vivo. A dentaltrephine bur was used under copious saline solution irrigation to form a full-thickness calvarial defect. An 8-mm-diameter defect was created on the rat parietal bone, and the control graft or experimental graft was placed in the defect. The experiments were classified into 3 groups: β-TCP Collagen implanted group, Bio-oss Collagen® implanted group, and Collagen plug implanted group. The animals were micro-CT at 1, 2, 4, 6, and 8 weeks and sacrificed at 8 weeks before histological analysis. 3. Results The biocompatibility of the scaffolds was evaluated via a cell adhesion and growth assay. As the culture time increased, there was an increase in the number of cells on control and Collagen plug (p<0.05). Obvious cell proliferation was not seen both in β-TCP Collagen and Bio-oss Collagen® with prolonged incubation time. The cells cultured on Collagen scaffold exhibited the highest ALP activity at Day 14 as compared with others. The cells cultured on other scaffolds showed maximal ALP activity at Day 14, whereas on β-TCP Collagen they gradually increased with time, showing maximum activity at Day 21. To investigate the effects of scaffolds on osteogenic differentiation, the expression of osteogenic genes, including Aalkaline phosphatase (ALP), Collagen type I (Col), Bone sialoprotein (BSP) and osteocalcin (OCN), was verified by RTqPCR at Day 14, Day 21 and Day 28. Control and Collagen scaffold showed higher expression levels of bone differentiation markers as compared with mineral/Collagen composites scaffolds. In comparison of β-TCP Collagen and Bio-oss Collagen®, the cells cultured in the former showed 4 to 5-fold superior expression levels of Col and BSP over those in the latter. In vivo, clear bone defects were observed at week 1 in all groups. From week 2, the borderline of the bone defect was blurred and bone formation from the edge to the center was observed. In comparison with the percentage of bone volume, Collagen plug group showed up to 11.47% at week 8, β-TCP Collagen group and Bio-oss Collagen® group showed up to 69.54% at week 8, Bio-oss Collagen® group, it increased up to 67.08% at week 8.