組織工程的目的是為克服因捐贈者短缺的移植難題，及突破人工材料不能夠取代所 有受傷或缺損的器官或組織。人工合成支架材料必須具備生物相容性，且與骨組織相近 的結構及機械性。骨組織是一種複合材，主要是由有機相（膠原蛋白）與無機相
（HA）構成。是以成功的取代材設計應以骨組織本質為藍圖。本計劃的主要目標是結 合天然高分子及矽酸鈣，製造骨組織工程用之新式仿生支架，研究支架設計對仿生複合 材機械及生物性能之影響。成功製造出機械強度高達140 MPa與彈性係數2.3 GPa的仿生 矽酸鈣-明膠複合體（明膠含量10 wt%）。分析生物活性將材料浸泡到模擬體液之後，1 天內即形成似骨磷灰石，然複合體逐漸損失強度。180天之後，明膠含量5及10 wt%的 複合體分別損失30及47%的強度。僅管如此，明膠提供有利的細胞環境及減少免疫表 現。牙髓細胞培養於複合體後其增生、分化與礦化增加。整體而言，明膠含量10 wt%
的仿生矽酸鈣-明膠複合體具高強度、無毒性及骨形成性等特質，可作為承載負荷得骨 移植材。
關鍵詞：骨組織工程、矽酸鈣、明膠、支架 2. 英文摘要
Tissue engineering has emerged as a promising approach without the limitations of donor shortage and of synthetic prostheses that are not able to replace all the functions of a damaged or lost organ or tissue. A synthetic material used as scaffold must be biocompatible and it should exhibit some structural and mechanical equivalence to bone. Bone is a composite material and consists mainly of an organic matrix (collagen) and a mineral phase (hydroxyapatite). Thus, the successful design of bone substitute materials should be an analogue of the bone structure. In this project, we aim at fabricating an advanced biomimetic scaffold that combines natural polymers and calcium silicates (CaSi) for use in bone tissue engineering. The effect of design strategies on mechanical and biological performances of the hybrid scaffold is investigated. We successfully fabricated the biomimetic 10 wt% gelatin-containing CaSi composites with high compressive strength up to 140 MPa and elastic modulus of 2.3 GPa. The materialsprecipitated a‘bone-like”apatiteaftersoaking in SBF for 1 day. However, all bone grafts gradually lost their strengths with the increase in soaking time.
The composites containing 5 and 10 wt% gelatin lost 30 and 47% in compressive strength, respectively, after 180-day immersion. Nevertheless, the presence of gelatin promoted greater cell attachment and proliferation on the composite bone grafts. Pulp cells on the calcium silicate-gelatin bone grafts expressed higher levels of osteocalcin, osteopontin, and bone sialoprotein. The inhibition of inducible nitric oxide synthase and interleukin-1 expression and the activation of interleukin-10 were increased with increasing gelatin content. Overall, these findings provide evidence that composite bone grafts containing 10 wt % gelatin with a high initial strength were bioactive, nontoxic, and osteogenic and may be able to promote bone healing for load-bearing applications.
Keywords: Bone tissue engineering, calcium silicate, gelatin, scaffold 3. Introduction and purpose
A variety of bone replacement materials have been developed and the majority has been limited to non-load bearing applications. Bone grafts are necessary to provide support, fill voids, and enhance biologic repair of skeletal defects. Calcium phosphates, particularly
hydroxyapatite (HA), have been used as implant materials for bone repair and regeneration due to their chemical compositions being similar to the inorganic component of bone. However, the poorly compressive strength and fatigue failure limit its applicability to the low or non load-bearing sites in human body. On the other hand, it must be emphasized at this point that the successful design of a bone substitute material requires an appreciation of the structure of bone. Bone is a composite structure consisting of nanocrystals incorporated within a collagen matrix. Thus, the use of a hybrid composite that comprises polymer and ceramic most aptly resembles the morphology and properties of natural bone. This may be one way to solve the problem of ceramics, such as brittleness, without reducing mechanical properties, in addition to possessing good biocompatibility, high bioactivity, and great bone-bonding capability.
Extensive research has been carried out in this regard and composite materials based on calcium phosphate and a variety of polymers have been worked out. To design biomimetic materials, it is important to understand the mechanical properties of bone materials and the structural relationship between them at the various levels of hierarchical structural organization. At the macrostructure level, bone is distinguished into the cortical (or compact) and cancellous (or trabecular) types. The compressive strength and modulus of cortical bone are about 100–230 MPa and 7–30 GPa, respectively [1]. Cortical bone has a solid structure with a series of voids that result in 3–12% porosity [2]. Since the bone tissue is a two-phase composite comprised mainly of apatite and collagen, the amount of mineral is usually thought to determine the stiffness of the bone material. The properties and geometrical arrangement of the two components might have a much larger influence on the properties than traditionally assumed.
In previous studies [3−7], a fast-setting calcium silicate cement (CSC) consisting of powders containing one or more solid compounds of calcium silicate and water or a phosphate buffer solution have been developed. The cement, which was initially derived from an equimolar mixture of tetraethyl orthosilicate and calcium nitrate via a sol-gel method, hardened in about 20 min after mixing the powder with water. The purpose of this three-year project is to develop strong scaffolds by incorporating gelatin into calcium silicate. In particular, efforts have been oriented toward calcium silicates where we added gelatin to reinforce the mechanical properties. The appropriate amount of gelatin added to the composite cements provided the greater resistance to disintegration in simulated physiological solution.
Following the first year of this project, the biological properties of biomimetic gelatin-containing calcium silicate composites were evaluated in the 2nd-year progress.
4. Experimental
4.1. Preparation of the composite
The sol-gel method has been described elsewhere [7]. Reagent-grade tetraethyl orthosilicate (Si(OC2H5)4, TEOS, 98.0%) (Sigma-Aldrich, St. Louis, MO) and calcium nitrate (Ca(NO3)2·4H2O, 98.0%) (Showa, Tokyo, Japan) were used as precursors for SiO2 and CaO, respectively. Nitric acid was used as the catalyst and ethanol was used as the solvent. The general sol-gel procedure, including hydrolysis and aging, was adopted. Briefly, TEOS was hydrolyzed by the sequential addition of 2 M HNO3 and absolute ethanol, with 1 h of stirring after each addition. Ca(NO3)2·4H2O was added to the TEOS solution in an equimolar ratio, and the mixture was stirred for an additional 1 h. The molar ratio of (HNO3 + H2 O)-TEOS-ethanol was 10:1:10. The sol solution was sealed and aged at 60ºC for 1 day. After vaporization of the solvent in an oven at 120C, the dried gel was heated in air to 800C at a heating rate of 10C/min for 2 h using a high-temperature furnace and then cooled to room
temperature in the furnace to produce a powder. The sintered powders were then ball-milled for 12 h in ethyl alcohol using a Retsch S 100 centrifugal ball mill (Hann, Germany) and dried in an oven at 60C. Type B gelatin (isoelectric point at pH = 4.7−5.2) from bovine skin (Sigma-Aldrich) was weighed and dissolved in distilled water at 60C until a homogeneous gelatin solution was obtained. To fabricate the organic-inorganic composite, the calcium silicate (CaSi) powder was mixed with different gelatin solutions (10%, 20%, and 30%) at a powder-to-liquid ratio of 2 mg mL-1 using a conditioning mixer (ARE-250, Thinky, Tokyo, Japan), and then the mixture was dried at room temperature for 12 h. Herein, the ratios of gelatin to CaSi were approximately 5, 10, and 15% by weight (Table 1). After grinding the dried powders the dense bulks were obtained by molding the specimens with an aspect ratio of 2:1 (6 mm in diameter 12 mm in length) in a cylindrical stainless steel mold under the applied pressure of 500 MPa for 1 min using a uniaxial press, followed by being soaked in deionized water at 60C for 1 h for hydrothermal processing prior to being dried at 60C for 2 days in an oven, unless described otherwise. For comparison purposes, specimens without hydrothermal processing in water at 60C for 1 h were also prepared.
4.2. Preparation of SBF
The simulated body fluid (SBF) solution, an extracellular solution with an ionic composition similar to that of human blood plasma, was used as the supporting solution for the compressive property measurement and fatigue tests. It consists of 7.9949 g of NaCl, 0.3528 g of NaHCO3, 0.2235 g of KCl, 0.147 g of K2HPO4, 0.305 g of MgCl2·6H2O, 0.2775 g of CaCl2, and 0.071 g of Na2SO4 in 1000 mL of distilled H2O and was buffered to pH 7.4 with hydrochloric acid (HCl) and trishydroxymethyl amino methane (Tris, CH2OH)3CNH2). All of the chemicals used were of reagent grade and used as obtained.
4.3. Evaluation of physicochemical properties
The uniformity of the gelatin distribution in the composites was measured by burnout in air at 600ºC for 2 h. Each composite bulk was cut into five parts using a low-speed diamond saw (Isomet, Buehler, Lake Bluff, IL). The specimens were dried at 120ºC for 2 h and then were weighed before and after burnout using a four-digital balance (AE 240S, Mettler-Toledo AG, Greifensee, Switzerland). Five samples were tested for each condition. The measurement of the density and porosity was conducted using a liquid displacement technique. In this method, ethanol was used as the displacement liquid because water was the setting liquid. A total of five specimens were used in each measurement to enhance the precision. The average value of six measurements was taken as the porosity and density of the specimens. Before the measurement, the set specimens were dried in an oven at 120C for 2 h. The specimen was immersed in a graduated cylinder containing a known volume (V1) of ethanol. Afterwards, it was ultrasonically stirred for at least 3 min to force the ethanol into the pores of the specimens until no air bubbles were observed emerging from the specimen. The total volume of ethanol and the ethanol-impregnated specimen was then recorded as V2. The ethanol-impregnated specimen was removed from the cylinder, and the residual ethanol volume was recorded as V3.
The volume differences, (V1 –V3) and (V2 −V3), were the pore volume and total volume of the specimen, respectively. Thus, the porosity of the specimen was obtained by the following equation:
Porosity = (V1 − V3)/(V2 − V3) (1)
The density can be expressed by the equation: d = W/(V2 − V3), where W is the weight of the specimen. Phase analysis of the specimens was performed using an X-ray diffractometer
(XRD, Shimadzu XD-D1, Kyoto, Japan) operated at 30 kV and 30 mA at a scanning speed of 1/min. Micro-Raman measurements were taken with a DXR instrument (Thermo Scientific, Waltham, MA) equipped with a laser at 780 nm, a microscope with ×10 magnification, and an electrically refrigerated CCD camera. The laser spot size was approximately 5 μm. The specimens for the interior structure examination were prepared by cutting with a diamond saw.
The interiors and surfaces of the specimens were dried with liquid CO2 using a critical point dryer device (LADD 28000, LADD, Williston, VT) and then coated with gold using a JFC-1600 (JEOL, Tokyo, Japan) coater and examined under a scanning electron microscope (SEM, JSM-7401F, JEOL) operated in the lower secondary electron image (LEI) mode at 3 kV of accelerating voltage.
4.4. Measurement of mechanical properties
The compressive strength (CS) was measured in an SBF at a crosshead speed of 1 mm min-1 using a static mechanical testing machine AG-1000E (Shimadzu, Kyoto, Japan) with a 10 kN load cell. The CS value of each specimen was calculated using the relationship defined in the equation CS = P/πr2, where P is the peak load (Newtons, N) and r is the radius (mm) of the specimen. The maximal compression load at failure was obtained from the recorded load-deflection curves.Young’smodulusofthespecimens was determined from the slope of the initial linear elastic portion of the load-deflection curve. The work-of-fracture (toughness, kJ m-2) was calculated by taking the area under the load-deflection curve. For the statistical significance of the two-parameter Weibull analysis, twenty specimens from each group were tested. A digital microhardness tester (HMV-2000, Shimaduz, Kyoto, Japan) with a four-sided diamond pyramid was used to evaluate the hardness of the various specimens. A load of 4.9 N for 15 s in an air atmosphere was used. The indentation impressions were also examined using SEM to observe the change in the damage associated with indentation. The average value was determined from twenty collections. For in vitro fatique test the specimen was placed in a polystyrene container, to which SBF in a dynamic (continuous exchange of solution) condition using a peristaltic pump at 37ºC was added to completely cover the specimen. A fatigue cyclic loading lower than the maximal compression loading at failure with a stress ratio of Smin/Smax
= 0.1 was imposed at 5 Hz until fracture was achieved using a Shimadzu servopulser 48000 system (Kyoto, Japan). The number of cycles to failure under a cyclic compression condition was promptly recorded upon the specimen rupture. Three specimens from each group were tested.
4.5 In vitro bioactivity and degradation
To evaluate the in vitro physicochemical activity, the specimens were immersed in a 37 ºC 30 mL SBF solution, equivalent to a specimen surface-to-volume ratio of 0.1 cm-1, which was arbitrarily chosen to completely cover each surface of the specimen. After soaking for specific time duration (15, 30, 90, and 180 days), specimens were removed from the vials and their properties were evaluated. Samples were removed from the vials and evaluated for CS or were dried in an oven at 60 ºC for the analysis of weight loss, phase composition, and morphology. To monitor the weight change of the samples, the dried specimens were weighed until reaching a constant weight before (day 0) and after immersion using a four-digit balance (AE 240S, Mettler-Toledo AG, Greifensee, Switzerland). Eight repeated specimens were examined for each of the materials investigated at each time point. At each time point, the pH values of the SBF solutions were measured using a pH meter (SP-701, Suntex Instruments, Taipei, Taiwan). Eight measurements were used.
4.6. Cytotoxicity
The cytotoxicity of the bone grafts were evaluated by incubating the specimens with L929 mouse fibroblast cells (BCRC 60279, Hsinchu, Taiwan) for 12, 24, and 48 h. Prior to cell incubation, the specimens were sterilized by soaking each in a 75% ethanol solution and exposing to UV lightfor2 h.TheL929 cellsweresuspended in Dulbecco’sModified Eagle’s medium (DMEM; Gibco, Langley, OK) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin solution (Gibco). Cell suspensions (5 103 cells per well) were directly seeded over each of the specimens in a 96-well plate. The segmented polyurethane films containing 0.1% zinc diethyldithiocarbamate (RM-A) were used as positive standard reference materials, and a high-density polyethylene sheet (RM-C) was used as the negative standard reference material, based on ISO 10993-5. The two standard reference materials were purchased from Hatano Research Institute, Food and Drug Safety Center (Kanagawa, Japan).
After the established L929-cell incubation period, the cytotoxicity was examined using the alamarBlue assay (Invitrogen, Grand Island, NY), which is based on the detection of mitochondrial activity. One microliter of alamarBluesolution and 100 μL of DMEM were added to each well followed by 2 h of incubation. After incubation, the solution in each well was transferred to a new 96-well ELISA plate. Plates were read in a Sunrise microtiter plate reader (Tecan Austria Gesell- schaft, Salzburg, Austria) at 570 nm with a reference wavelength of 600 nm. Optical density (OD) results were obtained from three separate experiments.
4.7. Primary cell culture
Human dental pulp cells were freshly derived from an intact caries-free premolar that was extracted for orthodontic treatment purposes. The tooth was split sagittally with a chisel, and the periodontal ligament tissue was then immersed in phosphate-buffered saline (PBS) solution. The pulp tissue was cut into fragments and immersed in DMEM containing 0.1%
collagenase (Sigma) and 0.1% Dispase (Sigma) for 1 h. The pulp cells were collected from the medium by centrifugation at 1500 rpm for 5 min. The cell pellet was resuspended in DMEM containing 20% FBS, 100 units/mL penicillin G, and 100 mg/mL streptomycin in 5% CO2 at 37 ºC. Cells were subcultured by successive passaging at a 1:3 ratio. Cell cultures between the fourth and eighth passages were used. Pulp cell suspensions (2 104 cells per well) were directly seeded over each sterilized specimen, which was placed in a 24-well plate.
The reagent alamarBlue was used for real-time and repeated monitoring of cell attachment and proliferation. To assess the attachment, the cells were cultured for 3, 6, and 12 h. Cell proliferation was assessed on days 1, 3, and 7. Briefly, at the end of the culture period, the medium was discarded and the wells were washed twice with PBS. Each well was filled with 100 µL of solution at a ratio 1:100 of alamarBlue to fresh medium and were incubated at 37 °C for 2 h. The solution in each well was transferred to a new 96-well tissue culture plate.
Plates were read in a Sunrise Microtiter reader at 570 nm with a reference wavelength of 600 nm. The OD results were obtained in six independent measurements.
To evaluate the effect of gelatin amount on earlier cell differentiation, the alkaline phosphatase (ALP) activity assay was performed using a TRACP & ALP assay kit (Takara, Shiga, Japan) according to the manufacturer’sinstructions.TheALP catalyzes the hydrolysis of the colorless organic phosphate ester substrate, nitrophenyl phosphate (pNPP), to p-nitrophenol, a yellow product, and phosphate. To perform the assay, after incubation, the cells were washed with physiological saline (150 mM NaCl) and lysed in 50 μL oflysisbuffer(1%
NP40 in 150 mM NaCl). For measurement purposes, 50 μL ofthesubstratesolution (20 mM Tris−HCl,1 mM MgCl2, 12.5 mM p-nitrophenyl phosphate, pH = 9.5) was added to each well and allowed to react at 37 ºC for 30 min. The reaction was stopped by theaddition of50 μL of 0.9 N NaOH and read at 405 nm using a Sunrise Microplate reader. The experiments were carried out in triplicate. The pH of the media was also monitored with an IQ120 miniLab pH meter (IQ Scientific Instruments, San Diego, CA). Triplicate measurements were used.
Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine gene expression in the human dental pulp cells cultured on the various specimens. The matrixes were washed with PBS on days 1, 3, 7, and 15, and the total RNA from the cells was isolated using Trizol (Invitrogen) according to the manufacturer’s protocol. The total RNA concentration was quantified by the OD at 260 nm, and the OD260/OD280 ratio was calculated using a Beckman DU640B spectrophotometer (Fullerton, CA). PCR primers were designed for various bone-formation genes: osteocalcin (OC), collagen type I (COL I), alkaline phosphatase (ALP), bone sialoprotein (BSP), and actin (AC). These primers were designed on the basis of published gene sequences (NCBI and PubMed). The mRNA was converted to cDNA using a thermal cycler (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA). Each PCR product was analysed by separation with 2% agarose (in TrisacetateEDTA buffer) gel electrophoresis and visualized after ethidium bromide staining. The stained bands
Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine gene expression in the human dental pulp cells cultured on the various specimens. The matrixes were washed with PBS on days 1, 3, 7, and 15, and the total RNA from the cells was isolated using Trizol (Invitrogen) according to the manufacturer’s protocol. The total RNA concentration was quantified by the OD at 260 nm, and the OD260/OD280 ratio was calculated using a Beckman DU640B spectrophotometer (Fullerton, CA). PCR primers were designed for various bone-formation genes: osteocalcin (OC), collagen type I (COL I), alkaline phosphatase (ALP), bone sialoprotein (BSP), and actin (AC). These primers were designed on the basis of published gene sequences (NCBI and PubMed). The mRNA was converted to cDNA using a thermal cycler (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA). Each PCR product was analysed by separation with 2% agarose (in TrisacetateEDTA buffer) gel electrophoresis and visualized after ethidium bromide staining. The stained bands