5. Results and Discussion
5.3. The results of In vitro study
5.3.3. Observation of cell morphology on calcium phosphate cements
It was very well-known that surface topography of cell attach region is performed very important role. One of the most important reason is that morphology of cells were very strongly influenced by surface topography.
Figure 9 and figure 10 was the FE-SEM image of MC3T3-E1 and hABMSCs cultured on the each CPCs. Cell morphology was analyzed by the FE-SEM images using Image J software. Each cell was regarded as an ellipse, and the major and minor axis of them were measured. Figure 13 shows those parameters, height length means major axis, width means minor axis. The cells cultured on the higher Rz value were more longer shape than others. The results showed that cells on calcium phosphate cements with smaller particles were more circular shapes. Additionally the results of morphology of cells using CLSM were observed similar with FE-SEM images.
Figure 11. FE-SEM images for the MC3T3-E1 on calcium phosphate cements with various horse bone powders: (1st line) cells on 5 μm CPC, (2nd line) cells on 50 μm CPC, and (3rd line) cells on 100 μm CPC.
Figure 12. FE-SEM images for the MC3T3-E1 on calcium phosphate cements with various horse bone powders: (1st line) cells on 5 μm CPC, (2nd line) cells on 50 μm CPC, and (3rd line) cells on 100 μm CPC.
Figure 13. Morphological parameter of cells on the each CPCs. (A, B) Height length means longer axis, and width means shorter axis. (C) Height/Width ratio. Error bars represent standard deviation, n=3.
Figure 14. CLSM(confocal laser scanning microscopy) images for the hABMSC on the calcium phosphate cements: blue colors was bone cements and red colors were cells.
It could be assumed that the shape of the cells was influenced on the cell behavior, such as osteogenesis and proliferation of cells. And the cellular
morphology was influenced by cell attach region, surface roughness was used to determine the degree of surface topography in this study. The cells with higher height/width ratio value was better cell growth, the cells with the lower value was good at osteogenic differentiation.
5.3.3. The results of western blot
Figure 15 showed the results of protein expression, in this study, osteocalcin (OCN) was used for the marker of osteoblasogenesis. Expression of OCN was differences between each samples and the expression band were appeared on 5M and 50M. It was very similar with alizarin red assay results.
Figure 15. Western blot assay of the cells cultured on each CPCs. The cells were cultured for two weeks with osteogenic media. The bands of OCN were appeared on 5 μm CPC and 50 μm CPC.
6. Conclusions
The objectives of this thesis were to develop calcium phosphate cements using different powder sizes and to evaluate the ability to enhance proliferation and differentiation. In this study, the followings are the main results:
1) There was no significant differences between result of FT-IR and XRD patterns with different particles sizes horse bone powder. It was signified that milling process could not influenced with crystal and functional groups.
2) The mechanical properties of calcium phosphate with horse bone powder.
The sintered horse bone powders could be controlled by grinding using two types of milling machine and sieving. Compressive strength of each bone powder samples were similar values each samples. The surface roughness of calcium phosphate cements were induced by horse bone particle sizes. The bigger sizes of particles led to rough surface.
3) It was assume that the Ca/P molar ratio could be affected by milling process. In milling process, all sources were same powder that be
conducted thermal treatment at 1300 degree Celsius using an electric furnace for 10 hours. After milling and sieving, the XRF results of horse bone powder were showed that smaller particles had higher Ca/P molar ratio. The phenomena could be made inferences two possible reasons.
First possible reason is that the heat generated caused milling was affected crystal of horse bone powder. Second possible reason is that the phosphate group was easily scattered than calcium during the collapse of amorphous region.
4) Cell behavior were influenced by surface roughness of calcium phosphate cements. Morphology of the cells were strongly influenced by surface roughness that could be expressed Rz value. Higher Rz led to longer shapes of cells and lower Rz led to circular shapes of cells.
Additionally, it could be expressed that higher Rz value had more 3D structure than lower Rz value. In this study, the calcium phosphate cement that had higher Rz value was investigated better proliferation ability than others. And, the calcium phosphate cement that had lower Rz value was investigated better differentiation ability than others.
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골 재생을 위한 입도 크기가 조절된 말뼈로 제조된
골시멘트의 개발
서울대학교 대학원
바이오시스템
‧소재학부 바이오시스템공학전공장 경 제
초 록
인산칼슘 시멘트(Calcium Phosphate Cements, CPCs)는 1980년대에 처음 개발된 이후 골 재생을 위한 임상 제품으로 가장 많이 사용되는 생체소재로 알려져 있다. 대부분의 시멘트는 인산칼슘계 물질들을 포함하는 분말과 고분 자를 포함하여 경화제의 역할을 하는 액상의 두 가지 재료로 이루어지며 최 근까지 분말의 입도 크기를 조절함으로써 시멘트의 강도를 강화시키는 연구 가 활발하게 이루어져왔다. 하지만 시멘트에 사용된 분말의 입도가 세포 기 능에 주는 영향에 대한 연구에 대한 사례는 보고된 바가 적고 분말의 입도에 따른 효과에 대한 고찰 역시 부족하다. 이에 본 연구에서는 인산칼슘 시멘트 의 분말의 입도 크기를 3종류로 분류하여 만들어진 인산칼슘 시멘트를 제조 하고, 세포를 배양시켜 그 기능을 조사하였다. 인산칼슘 시멘트는 말뼈를 소 결하여 크기별로 분류한 3종류의 분말과 키토산을 이용하여 제작되었다. 표 면 거칠기를 이용하여 각각의 시멘트의 표면의 형태를 수치화하여 나타냈을 때, 작은 입도를 가지는 분말을 이용하여 만든 시멘트보다 큰 입도를 가지는
분말을 이용하여 만든 시멘트가 큰 값의 표면 거칠기를 가지는 것을 확인하 였다. 각각의 시멘트 위에서의 세포의 기능에 대한 연구에서는 낮은 표면 거 칠기를 가지는 시멘트는 세포의 골분화에 유리하고, 높은 표면 거칠기를 가 지는 시멘트는 세포 증식에 유리한 결과를 확인하였다.
이 연구결과를 통해 인산칼슘 시멘트에 사용되는 분말의 입도 크기에 변화를 주어 표면의 거칠기를 조절하고, 이를 통해서 세포의 분화나 세포 증식과 같 은 세포의 기능성을 향상시킬 수 있음을 확인하였다. 이를 기반으로 인산칼 슘 시멘트에 사용되는 분말의 입도 크기를 조절하여 생체 내 골 재생 효과를 증진시키는데 기여할 수 있을 것으로 기대한다.