Immune response - 結果與討論 - 行政院國家科學委員會專題研究計畫成果報告

5. 結果與討論

5.13. Immune response

The role of gelatin on the immune expression of IL-1 and iNOS in human dental pulp cells was also exaimed. IL-1 (Fig. 17A) and iNOS (Fig. 17B) production on day 1 were higher in the pulp cells grown on the CSG0 control compared to all the other gelatin-containing composites. Significant differences (p < 0.05) in IL-1 and iNOS expression were detected between the bone grafts at all time points. With increasing incubation time, the two pro-inflammatory cytokines had a decreased expression. In contrast, pulp cells grown on CSG15 had the highest level of anti-inflammatory cytokine IL-10 expression among all of the specimens (Fig. 17C), resulting in a significant difference (p < 0.05) compared to the other specimens.

IL-1isthefirst“immune”cytokinepositively identified to be involved in the control of bone turnover and stimulates the proliferation of osteoclast precursors. It is also a typical example of multifunctional cytokines involved in the regulation of immune responses, hematopoiesis, and inflammation [32]. iNOS is only expressed in responses to inflammatory stimuli. IL-1 causes activation of the iNOS pathway in bone cells [33]. It was found that both IL-1 and iNOS expression of pulp cells on the bone grafts decreased with increasing incubation time. Most importantly, the lowest levels of the pro-inflammatory cytokine IL-1 and iNOS were expressed by pulp cells cultured on the specimen with the highest gelatin contentatallculturetimepoints.Interestingly,Scheffe’smultiplecomparison testing indicated that gelatin-containing specimens had significantly (p < 0.05) higher IL-10 values than the CSG0 control at all culture time points, with the exception of the 1-day expression of which there was no reaction. The greater the amount of gelatin in the bone grafts, the more iNOS and IL-1 expression levels were inhibited and the more IL-10 was activated.

6. Conclusions

Our principal objective was to develop synthetic biomimetic bone analogs with improved mechanical and osteoconductive properties based on the combination of bioactive calcium silicate and naturally polymeric gelatin. The hard calcium silicate ceramic matrix provides structural consistency and hardness, while the soft polymeric gelatin filler acts as a binder and provides a certain degree of ductility and fracture resistance. A suitable calcium silicate/gelatin ratio of 10% by mass-to-mass in the composite achieved the highest bending strength of 141.7 MPa, which is strong enough to be used in load-bearing sites of bone tissue. The pulp cells cultured on gelatin-enriched materials exhibited higher levels of osteogenic differentiation biomarkers, such as BSP, OPN, and OC. Moreover, gelatin effectively inhibited iNOS and IL-1 expression and activated IL-10 expression. Taking the high initial mechanical strength and biological functions into account, the 10 wt % gelatincalcium silicate composites appear to be promising for the use in load-bearing applications such as dental and orthopedic repair.

More importantly, it is more suitable for a small bone defect or fast healing trauma site.

The biomimetic composites with high-strength will open up the possibility of calcium silicate-type implant materials.

7. Evaluation

The three-year project has successfully developed biomimetic calcium silicate-based composite scaffolds for applications in load-bearing bone tissue engineering. Two SCI papers have been published and the others results have been prepared to submit to SCI journal. The application of patent to USA and Taiwan is pending.

本計畫目前衍生的成效

項目成果

專利已申請美國與台灣專利各 1 件：

1. 丁信智，“雙層骨移植裝置”，中華民國專利，100123987，July 7，

2011，申請。

2. 2. Ding SJ, “Bilayered bone graft device”, US PatentApplication 13/166,090, June 22, 2011.

SCI 論文已有 2 篇論文被接受，第 3 及 4 篇準備中

1. Ding SJ*, Shie MY, Wei CK. In vitro physicochemical properties, osteogenic activity, and immunocompatibility of calcium silicate-gelatin bone grafts for load-bearing applications. ACS Applied Materials &

Interfaces 2011;3(10):41424153. (Impact factor: 2.925-10; Ranking:

38/225)

2. Ding SJ*, Wei CK, Lai MH. Bio-inspired calcium silicate-gelatin bone grafts for load-bearing applications. Journal of Materials Chemistry 2011;21(34):1279312802.(Impact factor: 5.968-11; Ranking: 17/231),

國際會議 1. 2011 年第 11 屆亞洲生物陶瓷研討會邀請演講

2. 2012 年第 9 屆世界生物材料大會場次主持人 8. 參考文獻

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Table 1 Nominal composition, physicochemical properties, and Weibull parameters of various specimens CSG5 90:10 5 11.6±1.4^b 1.7±0.1^c 105.0±12.5^e 3.0±0.1^g 35.3±3.2^j 120.8±10.3^m CSG10 80:20 10 10.0±1.5^b 1.8±0.1^c 141.7±8.6^f 2.9±0.1^g 52.1±4.8^k 103.5±13.3^l CSG15 70:30 15 11.1±2.4^b 1.8±0.1^c 97.8±12.6^e 2.7±0.1^h 40.4±5.3^j 82.0±6.3ⁿ Values are mean ± standard deviation.

Mean values followed by the same superscript letter were not significantly different (p >

0.05) according to Scheffé post hoc multiple comparisons.

Fig. 1 The actual gelatin content and distribution of the composites.

(a) (b)

Fig. 2 (a) XRD patterns of CaSi powder and composites with and without gelatin; (b) Raman spectra for various specimens.

Fig. 3 Surface SEM micrographs of various specimens. (a) CSG0 control, (b) CSG5, (c) CSG10, and (d) CSG15.

-4 -3 -2 -1 0 1 2

4.0 4.2 4.4 4.6 4.8 5.0 5.2

lnσ

lnln(1/(1-F))

CSG0 CSG5 CSG10 CSG15

Fig. 4 SEM micrographs of the interior surface of the specimens. (a) CSG0 control, (b) CSG5, (c) CSG10, and (d) CSG15. The right pictures are at a higher magnitude.

Fig. 5 Weibull strength distribution plots of various specimens. F is the failure probability and σ isthestrength.The solid line represents the regression line.

10 20 30 40 50 60

1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 Number of cycles

Maximumappliedstress(MPa)

CSG0 CSG5 CSG10 CSG15

d c

Fig. 6 SEM micrographs of indented specimens. (a) CSG0 control, (b) CSG5, (c) CSG10, and (d) CSG15. Arrows indicate the indentation-induced crack.

Fig. 7 Maximum stress applied in compressive stress cyclic fatigue versus the number of cycles to failure for various specimens in an SBF solution at 37ºC.

Fig. 8 Surface (a-d) and cross-sectional (e-h) SEM micrographs of various bone grafts after soaking in an SBF solution for 1 day. (a, e) CSG0 control, (b, f) CSG5, (c, g) CSG10, and (d, h) CSG15. The arrows indicate the precipitated apatite spherulites or layer.

(a)

(d)

(g) (c)

(f) (b)

(e)

(h)

Fig. 9 Surface (ad) and cross-sectional (eh) SEM micrographs of various bone grafts after soaking in an SBF solution for 180 days. (a, e) CSG0 control, (b, f) CSG5, (c, g) CSG10, and (d, h) CSG15.

Fig. 10 Compressive strength of the specimens before and after immersion in an SBF solution for predetermined periods of time.

Fig. 11 Weight loss of the specimens before and after soaking in an SBF solution for predetermined time durations.

Fig. 12 Variations in the pH of the SBF solution during soaking.

Fig. 13 Cytotoxicity of various test samples seeded with L929 cells at various time points.

RM-A and RM-C were used as the positive control and negative control, respectively.

Fig. 14 AlamarBlue assay of dental pulp cells cultured on the specimens to reveal cell attachment and proliferation at various time points.

Fig. 15 (A) Variations of the pH in culture medium and (B) ALP assay on the pulp cells presented as optical density for cell differentiation on various test groups.

0.0

Fig. 16 Osteogenic expression levels of the pulp cells cultured on the specimens at various time points. The intensity of each amplified cDNA band was semiquantified and normalized to that of actin. COL I = collagen I, ALP = alkaline phosphatase, BSP = bone sialoprotein, OPN

= osteopontin, and OC = osteocalcin. (A) COL I, (B) ALP, (C) BSP, (D) OPN, and (E) OC.

0.0 0.2 0.4 0.6 0.8 1.0 1.2

1 3 7 15

Incubation time (day)

Normalizedtoactin

CSG0 CSG5

CSG10 CSG15

(A)

0.0 0.5 1.0 1.5 2.0 2.5

1 3 7 15

Incubation time (day)

Normalizedtoactin

CSG0 CSG5

CSG10 CSG15

(B)

0.0 0.1 0.2 0.3 0.4 0.5

1 3 7 15

Incubation time (day)

Normalizedtoactin

CSG0 CSG5

CSG10 CSG15

(C)

Fig. 17 (A) IL-1, (B) iNOS, and (C) IL-10 responses in the pulp cells cultured on the specimens. The mRNA levels of IL-1, iNOS, and IL-10 were determined using RT-PCR and normalized to the corresponding actin mRNA levels.

出席國際學術會議心得報告

計畫編號 NSC 98-2221-E-040-006-MY3

計畫名稱雙層仿生骨組織支架材之設計及分析

出國人員姓名服務機關及職稱

丁信智

中山醫學大學口腔科學研究所教授

會議時間地點 2011/11/30-12/3 日本筑波

會議名稱 2011 年第 11 屆亞洲生物陶瓷研討會

發表論文題目 Novel bone graft substitutes for load-bearing applications.

一、參加會議經過

第 11 屆亞洲生物陶瓷研討會由日本陶瓷學會及物質材料研究機構主辦，地點在科學城筑波的物質材料研究機構內的國際會議廳舉行，此屬於小而美且領域專門的國際會議，參與的學者皆是 SCI 論文上常見的人物。有四場邀請演講，另有口頭及海報貼示，並有 Prof. M. Filiaggi, Prof. T. Hanawa, Prof. R. Pilliar, Prof. J. Dennis, Prof. Ohgushi, Prof. K.S. Cho, Prof. S. Y. Kim, Prof. H.S.Fan, Prof. R. LeGeros 等知名學者的專題演講。整個會議研討涵蓋與生醫陶瓷材料相關的各種不同議題，如磷酸鈣陶瓷、氧化鋁/氧化鋯、生物活性玻璃、骨取代材、生物活性鈦金屬、組織工程、生物相容性研究、藥物制放載體等。會場討論氣氛十分熱絡。本人並代表台灣爭取及宣告 2012 在台南成功大學舉辦。

二、與會心得

亞洲生物陶瓷研討會主要由日本陶瓷學會發起，每年在日本舉辦 1 次，隔年在亞洲其他國家。此次主要是亞洲生醫材料學者參加，來自台灣的有成功大學與長庚大學學者參與。生醫（陶瓷）材料研究為一跨領域且理論、應用並重的學門，從與會中所發表的論文可知仍有相當大的研究空間，但有待臨床醫師與生醫材料研究者雙向交流與合作，才能更加突破目前所面臨之瓶頸。從國外學者的研究趨勢及發表主題，顯示台灣生醫材料界研究方向與世界並進、並未偏離。此會議中正式下一屆在台灣舉辦，台灣已將醫療器材產業規劃為重點科技，

目前有更多的產業與學者投入生醫陶瓷研發，因此實有必要強化國際交流與曝光度，有利於醫療器材產業輸出。

NOVEL BONE GRAFT SUBSTITUTES FOR LOAD-BEARING APPLICATIONS

S.J. Ding^*and C.K. Wei

Institute of Oral Biology and Biomaterials Science, Chung Shan Medical University, Taichung City 402, Taiwan

*Corresponding Author: [email protected]

Introduction

Calcium silicate-based materials have been found to foster osteoblast adhesion, growth, and differentiation and have been used as implant materials for bone repair and regeneration [1].

However, their inherent brittleness and fatigue failure limits their application to the low- or non-load-bearing sites in the human body. Although conventional high-temperature solid-state sintering could be used to fabricate compact high-strength ceramic materials, it is not suitable for the preparation of materials containing polymers, drugs, and other bioactive molecules. Naturally polymeric gelatin has been used as a scaffold material in tissue engineering and as a drug carrier for controlled delivery because of its biocompatibility, biodegradability, and nontoxicity. The use of a bio-inspired composite composed of a polymer and ceramic not only most aptly resembles the morphology and properties of natural bone but also solves the brittleness of bioceramics. Here we prepared novel bone analogs consisting of calcium silicate and gelatin that could match both the mechanical and osteogenic properties of bone by a simple pressing-hydrothermal method.

Physicochemical and biocompable properties of the composites were assayed.

Materials and Methods

The sol-gel method for the preparation of calcium silicate (CaSi) powder has been described elsewhere [2]. Type B gelatin was weighed and dissolved in distilled water at 60C until a homogeneous gelatin solution was obtained. To fabricate the organic-inorganic composite, the calcium silicate powder was mixed with different gelatin solutions (10%, 20%, and 30%) at a powder-to-liquid ratio of 2 mg mL^-1, and then the mixture was dried at room temperature. Herein, the ratios of gelatin to CaSi were approximately 5, 10, and 15% by weight. After grinding the dried powders the dense bulks were obtained by molding the specimens under the applied pressure of 500 MPa for 1 min, followed by being soaked in water at 60C for 1 h.

Phase analysis and morphology of the specimens were performed using an X-ray diffractometer (XRD) and scanning electron microscope (SEM). The simulated body fluid (SBF) solution was used as the supporting solution for the compressive property measurement and fatigue tests. MG63 human osteoblast-like cells were used to evaluate the biocompatibility of the specimens.

Results

The XRD patterns of the bulk composites with and without gelatin indicated an obvious diffraction peak near 2θ = 29.4, corresponding to the calcium silicate hydrate (C–S–H) gel overlapped with calcite, and incompletely reacted inorganic componentphasesofβ-Ca2SiO4. The hybrid composites were a heterogeneous structure with a homogenous distribution of gelatin within

d c

the CaSi matrix, and there were many filament crystals surrounding the CaSi body, which became more obvious with increasing gelatin content.

Composites containing 5 wt% gelatin had a compressive strength value of 105.0 MPa on average, significantly higher than the control without gelatin (86.1 MPa). The addition of gelatin up to 10 wt% achieved a significantly increased compressive strength of up to 141.7 MPa, which is within the reported compressive strength for cortical bone [3]. However, higher gelatin content at 15 wt% adversely affected the mechanical strength with a reduction of up to 31% of the highest value. The modulus decreased somewhat after the incorporation of either 5 wt% or 10 wt% gelatin to the control, but there was no significant difference.

The results of fatigue experiments showed that the stability of the composites was apparently affected by the cyclic loading with a remarkable decrease in the strength as the number of cycles increased. For example, the control fatigued in SBF for 2 ×10³cycles had a significant degradation down to 35% of the original strength. When a loading stress of 37 MPa was applied, the specimen containing 10 wt% gelatin lasted approximately 40 min in the in vitro fatigue test until failure occurred.

The seeded MG63s were adhered and spread on the specimen surfaces in a gelatin-dependent manner (Fig. 1). On the 15-wt%-containing composite surfaces, cell spreading became more prominent, and the cells were in a flat morphology.

Figure 1 SEM images of MG63s attached on the surfaces of various specimens after 6 h of incubation. (a) CSG0 control, (b) CSG5, (c) CSG10, and (d) CSG15.

The cell proliferation steadily increased in all of the specimens on days 1 through 7, indicating the increasing number of viable cells. Interestingly, cells became attached to the 15 wt% gelatin at a rapid rate of more than 100% binding on day 1 compared to the control in terms of optical density, indicating that gelatin could be regarded as a cell- promoter. Moreover, the optical density values for the 15 wt% gelatin-containing composite were significantly higher than those obtained for the control on days 3 and 7. The alkaline phosphatase (ALP) activity from the MG63 cells was enhanced by the gelatin compared to the control on day 14, particularly for the 15 wt% gelatin, showing a significant difference (p < 0.05). Notably, the higher gelatin content in the composites led to improved differentiation. For calcium deposits in mineralized tissues and cultures, clearly calcified tissue formation was seen in the cultures grown in the presence of the CSG on day 14. The mineralized matrix synthesis was most evident in the composite specimens containing 10 or 15 wt%

gelatin.

Discussion

The added gelatin, which was examined in this study, may play a crucial role in the properties of the bio-inspired composites. The dense structures of gelatin-containing composites might be due to the existence of negatively charged gelatin. One concern is that the incorporation of gelatin into

in this study, this concern did not occur. When increasing the gelatin, to the result was an increase in the compressive strength; however, once it reached the maximum value (i.e., 10% of gelatin), the compressive strength decreased drastically. The resulting high strength of the hybrid composites were due to a combination between the progressive hardening originating from the main CaSi reactant and reinforcement effect filling the defects by the gelatin phase, which might serve as a

‘glue’to fusetheparticlestogether,asconfirmed by SEM. It is also possible that the gelatin in the composites reduced the effect of the flaws and the porosity on the mechanical behavior of these hybrid composites. The decrease in strength of the bio-inspired materials was also caused by environmental factors, such as the penetration of water/ions. Water/ions can easily infiltrate the inner portion of the specimens through structural imperfections, particularly under applied stress, resulting in weakened bond strength due to particle dissolution. The addition of gelatin can also increase the ratio of water absorption of the composite, which reduced the retention of the mechanical properties of the CaSi-gelatin composite fatigued under moisture conditions.

The increased proliferation, differentiation, and mineralization of the MG63 cells showed that gelatin could provide a favorable environment for osteoblasts to function normally, promoting the biological activity of CaSi. Biomolecules with OH, COOH, and NH2 functional groups, such as

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