Effect of recombinant human Bone Morphogenetic Protein- 4 with carriers in rat calvarial defects

Abstract

[한글]

골형성 유도 단백질(bone morphogenetic protein, BMP)은 치주 치료와 골 재생 치료를 위한 골 대체 물질의 하나로 연구, 평가되어 지고 있다. rhBMP에 대한 연구 중 rhBMP-4의 골 형성 유도 효과와 운반체에 대한 연구가 아직 부족한 편이다. 이에 본 연구에서는 흡수성 콜라겐 스폰지(ACS)와 베타 삼화인산칼슘염(β-TCP)을 운반체로 하여 백서 두개골 결손부에서의 rhBMP-4의 골형성 유도 효과를 평가하고, rhBMP-4의 운반체로서 ACS와 β-TCP를 비교하고자 한다. 본 연구에서 100마리의 웅성 백서를 5개의 그룹으로 나누고, 8 mm 백서 두개골 결손부에 rhBMP-4/ACS, rhBMP-4/β-TCP, ACS, β-TCP를 이식하고, 수술 대조군에는 아무것도 이식하지 않았다. 술후 2, 8 주에 실험 동물을 희생하고, 치유 결과를 방사선밀도계측학적으로, 조직학적으로, 조직계측학적으로 비교 관찰하였다.

방사선밀도계측학적 결과로는, 술후 2주에 rhBMP-4/ACS, rhBMP-4/β-TCP, ACS 대조군, β-TCP 대조군, 그리고 수술 대조군의 신생골 밀도(%) 계측 결과는 각각 55.0±7.2%, 56.7±7.3%, 37.9±7.0%, 39.5±5.1%, 15.3±2.9%와 같이 나타났으며, 술후 8주에는 각각 61.4±12.6%, 76.1±5.8%, 34.0±3.4%, 42.7±7.9%, 17.2±3.7%와 같이 나타났다. 2주와 8주에서 rhBMP-4/ACS와 rhBMP-4/β-TCP 는 다른 실험군에 비해 신생골 밀도가 더 높게 나타났다(P<0.01). 8주에서 rhBMP-4/β-TCP는 rhBMP-4/ACS보다 더 높은 신생골 밀도를 보였다(P<0.01).

조직학적 관찰 결과, rhBMP-4/ACS 군에서는, 술후 2주에 신생골 내에 ACS 분절이 묻혀 있는 양상을 보였으며, 술후 8주에 결손부가 신생골로 가득 메워졌으며, ACS는 관찰되지 않았다. rhBMP-4/β-TCP 군에서는, 술후 2주에 다수의 잔존 β-TCP와 신생골이 관찰되었으며 술후 8주에는 다소 흡수된 양상의 잔존 β-TCP와 신생골이 관찰되었다.

조직계측학적 결과로는, 술후 2주에 rhBMP-4/ACS, rhBMP-4/β-TCP, ACS 대조군, β-TCP 대조군, 그리고 수술 대조군의 신생골/결손부(%) 계측 결과는 각각 71.7±2.8%, 60.8±.2.0%, 13.6±2.3%, 15.3±.2.0%, 4.8±0.1%와 같이 나타났으며, 술후 8주에는 각각 91.9±2.6%, 75.9±2.3%, 17.4±2.5%, 20.2±3.0%, 8.2±0.0%와 같이 나타났다. 2주와 8주에서 rhBMP-4/ACS와 rhBMP-4/β-TCP는 다른 실험군에 비해 신생골/결손부(%)가 더 높게 나타났으며(P<0.01), rhBMP-4/ACS는 rhBMP-4/β-TCP보다 더 높은 신생골/결손부(%)를 보였다(P<0.05).

이상의 결과에서 볼 때, 백서 두개골 결손부에서 rhBMP-4/ACS, rhBMP-4/β-TCP를 적용한 경우, 신생골/결손부(%)와 신생골 밀도에 있어서, rhBMP-4의 골 형성 유도 능력을 확인할 수 있었으며, 또한 ACS와 β-TCP는 rhBMP-4의 운반체로서 효과적이라 사료된다.

[영문]

Bone morphogenetic proteins (BMPs) are being evaluated as a candidate for periodontal and bone regenerative therapy. However, the research of recombinant human bone morphogenetic protein-4 (rhBMP-4) has been insufficient to evaluate its capacity to enhance bone formation and its carrier system. The purpose of this study was to evaluate the bone regenerative effect of rhBMP-4 delivered with absorbable collagen sponge (ACS) or β-tricalcium phosphate (β-TCP). In addition, we compared the potential of β- TCP to that of ACS as a carrier system for

rhBMP-4./8 mm calvarial critical-sized defects were created in one hundred male Sprague-Dawley rats. The animals were divided into 5 groups of 20 animals each. The defects were treated with rhBMP-4(rhBMP-4 at 0.05 ㎎/㎖)/ACS, rhBMP-4/β-TCP, ACS alone, or β-TCP alone, or were left untreated for surgical control. The rats were sacrificed at 2 or 8 weeks postsugery, and the outcomes were evaluated radiodensitometrically, histologically, and histomorphometrically.

The results of radiodesitometric analysis were as follows; at 2 weeks postsurgery, mean radiodensity (±SD) for the rhBMP-4/ACS group, the rhBMP-4/β-TCP group, the ACS alone group, the β-TCP alone group, and the surgical control group amounted to 55.0±7.2%, 56.7±7.3%, 37.9±7.0%, 39.5±5.1%, and 15.3±2.9%, respectively, and, 61.4±12.6%, 76.1±5.8%, 34.0±3.4%, 42.7±7.9%, and 17.2±3.7%, respectively, at 8 weeks. The rhBMP-4/ACS group and the rhBMP-4/β-TCP group were significantly

different from the other groups (P<0.01) at both 2 and 8 weeks, and the rhBMP-4/β-TCP group had a significantly greater radiodensity than the rhBMP-4/ACS group at 8 weeks (P<0.01).

The histologic observations were as follows; in the rhBMP-4/ACS group, some degraded ACS fragments were embedded within the new bone at 2 weeks. At 8 weeks, the defect was almost completely filled with the new bone and remnant of ACS could not be detected. In the rhBMP-4/β-TCP group, a lot of residual β-TCP particles and woven bone were evident at the defect sites at 2 weeks. At 8 weeks, residual β-TCP particles were less, and woven bone was greater than at 2 weeks. The results of histomorphometric analysis were as follows; at 2 weeks postsurgery, mean bone fill (±SD) for the rhBMP-4/ACS group, the rhBMP-4/β-TCP group, the ACS alone group, the β-TCP alone group, and the surgical control group amounted to 71.7±2.8%, 60.8±.2.0%, 13.6±2.3%, 15.3±.2.0%, and 4.8±0.1%, respectively, and, 91.9±2.6%, 75.9±2.3%, 17.4±2.5%, 20.2±3.0%, and 8.2±0.0%, respectively, at 8 weeks. The rhBMP-4/ACS group and the rhBMP-4/β-TCP group were significantly

different from the other groups (P<0.01) and the rhBMP-4/ACS group had a significantly greater bone fill than the rhBMP-4/β-TCP group at both 2 and 8 weeks (P<0.05).

In conclusion, the bone regenerative effect of rhBMP-4/ACS was superior to that of rhBMP-4/β-TCP in the rat calvarial critical-sized defect. Surgical implantation of rhBMP-4/ACS may be used to support bone regeneration in the rat calvarial critical sized defect without complication, also rhBMP-4/β-TCP may be able to regenerate bone in the rat calvarial critical sized defect without any side effects occurring.

In addition, both ACS and β-TCP may be considered as effective carriers for rhBMP-4.