Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2014 www.elsevier.com/locate/jbiosc
Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration Beom-Su Kim,1, 2 Hark-Mo Sung,2 Hyung-Keun You,3 and Jun Lee1, * Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea,1 Bonecell Biotech Inc., 77 Dunsan-dong, Seo-gu, Daejeon 302-830, Republic of Korea,2 and Department of Periodontology, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea3 Received 23 December 2013; accepted 27 March 2014 Available online xxx
Fibrin polymers are widely used in the tissue engineering field as biomaterials. Although numerous researchers have studied the fabrication of scaffolds using fibrin glue (FG) and bone powder, the effects of varied fibrinogen content during the fabrication of scaffolds on human mesenchymal stem cells (hMSCs) and bone regeneration remain poorly understood. In this study, we formulated scaffolds using demineralized bone powder and various fibrinogen concentrations and analyzed the microstructure and mechanical properties. Cell proliferation, cell viability, and osteoblast differentiation assays were performed. The ability of the scaffold to enhance bone regeneration was evaluated using a rabbit calvarial defect model. Micro-computed tomography (micro-CT) showed that bone powders were uniformly distributed on the scaffolds, and scanning electron microscopy (SEM) showed that the fibrin networks and flattened fibrin layers connected adjacent bone powder particles. When an 80 mg/mL fibrinogen solution was used to formulate scaffolds, the porosity decreased 41.6 ± 3.6%, while the compressive strength increased 1.16 ± 0.02 Mpa, when compared with the values for the 10 mg/mL fibrinogen solution. Proliferation assays and SEM showed that the scaffolds prepared using higher fibrinogen concentrations supported and enhanced cell adhesion and proliferation. In addition, mRNA expression of alkaline phosphatase and osteocalcin in cells grown on the scaffolds increased with increasing fibrinogen concentration. Micro-CT and histological analysis revealed that newly formed bone was stimulated in the scaffold implantation group. Our results demonstrate that optimization of the fibrinogen content of fibrin glue/bone powder scaffolds will be beneficial for bone tissue engineering. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Fibrin glue; Fibrinogen; Scaffold; Bone tissue engineering; Bone powder]
Bone defects are caused by a variety of conditions, such as tumors, trauma, disease, and bone fractures. Although small bone defects can heal themselves, large bone defects cannot and require bone graft replacements. In tissue engineering, reconstruction of bone often requires a biodegradable porous scaffold (1) because the porous scaffold provides necessary support for cell growth, adhesion, and differentiation (2). In addition, the selection of biomaterials with which to construct the scaffold is an important factor because their properties will determine scaffold properties like biocompatibility, osteoinductivity, and osteoconductivity (3,4). To construct a biocompatible scaffold for bone tissue engineering, several biomaterials, including hydroxyapatite (5), tricalcium phosphate (6), and allogeneic bone powder (7) were used. Among these, allogeneic bone powders have been frequently used to reconstruct bone defects. Because the molecular structure of bone is identical across species, it is possible to use bone from animal sources for dental implant bone grafts with good results (8). Furthermore, biodegradable natural polymers, such as a collagen (5), gelatin (9), chitosan (10), and fibrin (11), have been used to fabricate scaffolds. Fibrin polymers have been shown to have excellent adhesiveness and biocompatibility (12,13). Fibrin glues (FG) are mainly composed of fibrinogen and thrombin (14). Thrombin * Corresponding author. Tel.: þ82 42 341 2800; fax: þ82 42 341 2809. E-mail addresses: [email protected], [email protected] (J. Lee).
converts fibrin, a biopolymer, into fibrin monomers. Monomeric fibrin forms a fibrous clot that has biological adhesive properties, and because of these properties, these clots are widely used in various surgeries, including orthopedic surgery (15,16). In addition, fibrin structures function as a temporary matrix during the rebuilding and repair of tissues (17). Although some studies demonstrate that a composite biomaterial containing FG can exhibit increased biocompatibility and osteoconductivity when compared with the biomaterial alone (18,19), there is insufficient evidence for the utility of scaffolds composed of FG and bone powder in such applications. In an attempt to improve its utility for bone tissue engineering, we sought to optimize the composition of FG and bone powder scaffolds. In this study, FG and bone powder scaffolds were fabricated using various concentrations of fibrinogen, and their microstructural and mechanical properties were characterized. The in vitro biocompatibility of the scaffold was evaluated using human mesenchymal stem cells (hMSCs) in vitro, and their tissue response and ability to induce bone formation in vivo were evaluated using a rabbit calvarial defect model. MATERIALS AND METHODS Preparation of scaffolds FG from a Greenplast kit (Green Cross Corp., Seoul, Korea) and calcium-phosphate-coated bovine bone powder (Biocera; Oscotec, Chunan, Korea) were used in scaffold preparation. To construct the scaffolds, 450 mg of bone powder was placed in each hexahedron-shaped mold (10 mm 10 mm 5 mm). Next,
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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0.2 mL of fibrinogen solution (10, 20, 40, or 80 mg/mL in PBS) was added to the bone powder and mixed well. Then, 0.1 mL of thrombin solution (Greenplast Kit; 5 U/mL) was added, and the composites were rapidly blended. Polymerization of the resultant mixture was achieved at room temperature after 1 h of incubation. The blocks were then freeze-dried for 3 days to obtain FG/bone powder scaffolds.
analyzer (Sky-Scan). We additionally scanned scaffolds prior to implantation using a micro-CT, and bone volume (BV) was obtained from the data sets. Then, the percentage of bone volume was calculated using the following equation:
Micro-computerized tomography analysis To evaluate the entire scaffold structure, samples were scanned with an aluminum filter using micro-computerized tomography (Micro-CT; Sky-Scan 1172TM; Skyscan, Kontich, Belgium). Threedimensional and trans-sectional images were obtained, and the data were reconstructed using the CT-analyzer software (Sky-scan).
where BVpost is the bone volume 8 weeks after implantation, BVsc is the volume of scaffold prior to implantation, and TV is the total volume of the region of interest.
Microstructural analysis To observe the structure, the scaffolds were examined using a scanning electron microscope (SEM; EM-30; Coxem, Daejeon, Korea). Before observation, the samples were sputter-coated with gold for 120 s under vacuum. Porosity measurement Porosity was measured using a mercury intrusion porosimeter (AutoPore IV9500, Oak Ridge, TN, USA). Briefly, scaffolds were sealed in a penetrometer, weighed, and subjected to analysis (20). The porosity of 5 samples per scaffold was measured and reported as average percent porosity. Compressive strength analysis To evaluate the mechanical properties of the scaffolds, compressive strength was measured. The fabricated scaffold (10 mm 10 mm 5 mm) was subjected to a compression test using an Instron model 4505 universal test machine (Instron, Canton, MA, USA) by applying load via a 1 N load cell at a crosshead speed of 0.5 mm/min under ambient conditions. Cell culture Human bone marrow-derived mesenchymal stem cells (hMSCs) were obtained from Prof. HK You (Wonkwang University, Iksan, Korea). The cells were cultured in a-Minimal Essential Medium (a-MEM) (Gibco-BRL, Gaithersburg, MD, USA) with 1% penicillin/streptomycin and 10% fetal bovine serum (Gibco-BRL) at 37 C, 5% CO2, and 100% humidity. Passages 4e6 were used in each experiment. The differentiation of hMSCs into osteoblasts was induced by treatment with osteoblast stimulant solution (OS; 10 mM b-glycerophosphate, 0.05 mg/mL ascorbic acid, and 0.1 mM dexamethasone [SigmaeAldrich, St. Louis, MO, USA]). The culture medium and OS were replaced every 2 days during the experiment. Cell proliferation assay and evaluation of cytotoxicity CellTiter96 Aqueous One Solution (Invitrogen, Carlsbad, CA, USA) was used to measure cell proliferation. The hMSCs were seeded and cultured on the FG/bone powder scaffolds. After culture (1, 5, 10, or 15 days), 100 mL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent was mixed with 500 mL of culture media and added to each well. After incubation for 2 h, 200 mL of the supernatant was removed and its absorbance was measured at 490 nm using a microplate reader (SpectraMAX M3; Molecular Devices, Sunnyvale, CA, USA). In addition, a Live/Dead Viability/Cytotoxicity staining kit (Invitrogen) was used to evaluate the cytotoxicity of the produced scaffolds. According to the manufacturer’s protocol, cells were seeded and cultured for 3 days. Then, the sample was rinsed with PBS to remove the phenol red and serum, and reagent solution was added. After incubation for 30 min in a CO2 incubator, the samples were observed using an inverted fluorescence microscope (DM IL LED Fluo; Leica Microsystems, Wetzlar, Germany). Cell adhesion observation SEM was used to observe cell adhesion to the scaffold. After 5 days of culture, scaffolds were briefly washed with PBS. Then the samples were fixed with 2.5% glutaraldehyde, and postfixation was performed with 0.1% osmium tetroxide (OsO4, Sigma). The samples were dehydrated with a graded ethanol series (50%, 75%, 95%, 100%, and 100%), sputter-coated with gold, and visualized by SEM (EM-30). Real-time polymerase chain reaction To quantify osteoblast differentiation, cells were cultured on the FG/bone powder scaffolds for 10 days, and the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OC) marker genes was assessed using a quantitative real-time polymerase chain reaction (qRT-PCR) assay. To obtain total mRNA, the scaffolds were rinsed with PBS and chopped, and the cells were dissociated from the scaffold by sonication. Total mRNA was extracted using an RNA isolation kit (Ribospin; GeneAll, Seoul, Korea), according the manufacturer’s protocol. PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA), TaqMan Universal PCR Master Mix, TaqMan primers, and probe sets specifically targeting ALP (Hs01029144_m1), OC (Hs01587814_g1), and 18S rRNA (Hs99999901_s1; Applied Biosystems). The 18S rRNA gene was used as an internal standard. Animal experiments In this study, 3 months old New Zealand white rabbits, weighing 2.5e3.0 kg, were used. After the animal was anesthetized, the calvarium was exposed by making a skin incision. Circular calvarial defects were made using a trephine bur (8 mm in diameter), and FG/bone powder scaffolds were implanted in the induced calvarial defects. The animals were sacrificed at 8 weeks of age, and the bone tissue defects were dissected out from the host bone. The extracted bone tissue was fixed with 4% paraformaldehyde buffered with 0.1 M phosphate solution (pH 7.2) for 3e5 days before further experiments. All animal experiments were performed according to the guidelines of the Wonkwang University Institutional Animal Care and Use Committee. Micro-computed tomography The bone specimens were scanned using a micro-CT (Sky-Scan 1172TM). Then the image data were reconstructed using a CT-
ð%Þ New bone volume ¼ BVpost BVsc
TV 100
(1)
Histology After micro-CT scanning, the samples were dehydrated in a graded alcohol series (80e100%), decalcified in 8% formic acid/8% HCl, and embedded in paraffin. Sections of 5-mm thickness were prepared from the samples and mounted on slides. Following this, the samples were stained with hematoxylin/ eosin (H&E) and Goldner’s Masson trichrome (MT). Statistical analysis All experiments were performed in triplicate, and statistical analyses were performed using statistical analysis software (Origin 8.0; OriginLab, Northampton, MA, USA). Significant differences among groups were identified by ANOVA followed by Dunnett’s test. Values in the text are expressed as the means standard deviation (SD) and p values less than 0.05 were considered statistically significant.
RESULTS Structural characterization of FG/bone powder scaffolds In the present study, the thrombin used had a concentration of 5 U/mL. Because this high concentration of thrombin accelerates crosslinking, making it difficult to handle, we used various concentrations (10e80 mg/mL) of fibrinogen to fabricate the scaffolds. In this study, we successfully fabricated porous scaffolds using bovine bone powder and FG (Fig. 1A). Micro-CT images of the scaffolds are shown in Fig. 1B. The micro-CT images showed that the bovine bone powders were uniformly distributed in all scaffolds. In addition, the trans-sectional layer micro-CT images indicated that several irregular and large pores were observed in the scaffolds fabricated with 10 and 20 mg/mL fibrinogen. SEM images of the fabricated scaffolds are shown in Fig. 1C. All of the scaffolds fabricated using fibrin glue contained fibrin networks and fibrin layers that branched among the bone powder particles. Fibril networks and fibrin layers were rarely observed in the scaffolds fabricated with 10 mg/mL fibrinogen, and more compact fibrin layers were observed in the scaffolds fabricated with 80 mg/mL fibrinogen when compared with those fabricated with 10 mg/mL fibrinogen. Furthermore, a fibrin layer closed the pores between adjacent bone powders in the scaffolds fabricated with 80 mg/mL fibrinogen. Measurement of porosity and compressive strength SEM showed that fibrin layer formation closed many pores (Fig. 1C). However, in bone tissue engineering, the porosity of the scaffold is important for cell migration, angiogenesis, and nutrient supplementation (21,22). Therefore, we assessed porosity as a function of fibrinogen concentration (Fig. 2A). The porosities of 10 mg/mL and 20 mg/mL fibrinogen-fabricated scaffolds were not statistically different, with 76.1% 2.9% and 69.2% 3.6% porosity, respectively. However, the porosity of the scaffolds fabricated with 80 mg/mL fibrinogen (41.6% 3.6%) was significantly higher those fabricated with 40 mg/mL (64.2% 2.9%; p < 0.05). These results demonstrate that the porosity of the scaffold decreased as the concentration of fibrinogen increased due to the formation of fibrin layers. In addition, we measured the compressive strength of the scaffolds because mechanical properties are also crucial for bone reconstruction. The compressive strength of the scaffolds fabricated with 20 mg/mL fibrinogen was 0.46 0.06 MPa and not significantly different when compared with those fabricated with 10 mg/mL fibrinogen (0.31 0.02 MPa). However, the compressive strengths of both the 40 mg/mL and 80 mg/mL fibrinogen-fabricated scaffolds were significantly higher, at 0.93 0.06 MPa and 1.16 0.02 MPa, respectively, than the 10 mg/mL fibrinogen-fabricated scaffold (Fig. 2B). Biocompatibility To evaluate the effects of fibrinogen concentration on cell proliferation, cytotoxicity, and cell adhesion, hMSCs were seeded and cultured on the scaffolds. As shown in
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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FIG. 1. A gross view (A) of FG/bone powder scaffolds that were successfully fabricated using various fibrinogen concentrations. Three-dimensional and trans-sectional microcomputed tomography (micro-CT) images (B) showed that the bone powders (white) were uniformly distributed, and an irregular pore (black) was formed in the scaffold. The scale bar is of 5 mm. Scanning electron microscopy (SEM) images (C) of scaffold show fibrin networks and fibrin layers branching among the bone powder particles. Fibril networks and fibrin layers were rarely observed on the surface of bone powders in the scaffold fabricated with 10 mg/mL fibrinogen. However, compact and flattened fibrin layers were observed in the scaffold fabricated with 80 mg/mL fibrinogen. To enhance the contrast between the fibrin and scaffold in the SEM image, fibrin networks and fibrin layers were manually false-colored in green using Adobe Photoshop. The pseudocolor high magnification SEM image (D) shows fibrin networks (white arrows) and fibrin layers (black arrows) formed on the surface the bone powder particles (asterisks).
Fig. 3A, the proliferation of hMSCs gradually increased with culture time. After 1 day of culture, the proliferation of cells cultured on the scaffold fabricated with 80 mg/mL fibrinogen was slightly higher than that of scaffolds fabricated with 10, 20, or 40 mg/mL fibrinogen. However, after 5 and 10 days of culture, the proliferation of cells cultured on the scaffolds fabricated with 80 mg/mL of fibrinogen was approximately 2-fold higher than that of cells cultured on scaffolds fabricated with 20 or 40 mg/mL fibrinogen. In contrast, at 15 days of culture, the proliferation was higher in cells cultured on the scaffolds fabricated with 20 or
40 mg/mL fibrinogen than cells cultured on the scaffolds fabricated with 80 mg/mL fibrinogen (Fig. 3A). To evaluate the cytotoxic effects of fibrinogen, hMSCs were cultured on scaffolds, and a live/dead staining assay was performed after 3 days. Fluorescence imaging showed that almost all hMSCs on all scaffolds were viable (Fig. 3B). Furthermore, the density of live green cells was consistent with the results of the MTS assay. Cellular adhesion observation To determine whether cells could attach to the scaffolds, hMSCs were cultured, and their morphologies were observed by SEM after 5 days of culture. Fig. 3C
FIG. 2. The effects of fibrinogen concentration on the porosity (A) and compressive strength (B) of fabricated scaffolds. Scaffolds were fabricated using various fibrinogen concentrations, and their porosity and compressive strength were measured. The porosity decreased, while compressive strength increased, when fibrinogen concentration was higher. Data shown represent the mean SD of 3 samples. The asterisks indicate significant differences compared to the scaffold fabricated using 10 mg/mL fibrinogen.
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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FIG. 3. The biocompatibility of hMSCs cultured on scaffolds fabricated using various fibrinogen concentrations. hMSCs were cultured on the scaffolds and proliferation was assessed by the MTS assay at 1, 5, 10, and 15 days (A). In addition, cytotoxicity was evaluated after 3 days of culture. Live cells were stained green with calcein acetoxymethyl (Calcein AM), while dead cells were stained red with ethidium homodimer-1 (EthD-1). The scale bar represents 500 mm. Data shown represent the mean SD of 3 samples. The asterisks indicate significant differences when compared with the scaffold fabricated using 10 mg/mL fibrinogen (p < 0.05). After 5 days of culture, the morphology of adherent and growing cells was observed by SEM (C). Attached hMSCs were more frequently observed on scaffolds constructed with increased fibrinogen concentrations. To enhance the contrast between the hMSCs and scaffold in the SEM image, the cells and scaffold were manually false-colored in green and brown, respectively, using Adobe Photoshop. The scale bar represents 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
shows that hMSCs adhered and grew on the scaffolds. Attached cells were rarely observed on the scaffolds fabricated with 10 mg/ mL fibrinogen, but they were more frequently observed on scaffolds constructed with increasing fibrinogen concentrations. These results are consistent with the findings of our MTS assay. Osteogenic differentiation of hMSCs To determine if the FG/ bone powder scaffolds could support an environment for hMSC differentiation into osteoblasts, cells were cultured on the scaffolds and treated with OS. Then, qRT-PCR was performed to detect osteoblast marker expression over time. ALP mRNA expression
increased in cultured cells as fibrinogen concentration increased. ALP mRNA expression peaked on day 6 and then decreased on day 12. Additionally, OC mRNA expression peaked on day 12 and increased as fibrinogen concentration increased (Fig. 4). Bone formation analysis in vivo For in vivo experiments, we used the scaffolds fabricated with 80 mg/mL fibrinogen due to their superior mechanical properties and biocompatibility. Histological assessment of the bone formation potential of FG/bone powder scaffolds was evaluated using H&E and MT staining. The newly formed bone stained a light pink color (in the web version),
FIG. 4. Relative ALP (A) and OC (B) mRNA expression. Total RNA was extracted from hMSCs cultured on fabricated FG/bone powder scaffolds for 10 days, and real-time PCR was performed to measure the expression of ALP and OC. The 18s rRNA gene was used as an internal control. The relative expression for all groups was normalized to the expression of cells cultured on scaffolds fabricated using 10 mg/mL fibrinogen. Data shown represent the mean SD of 3 samples. The asterisks indicate significant differences compared to the group cultured in the absence of OS (p < 0.05) when cultured on scaffolds fabricated using 10 mg/mL fibrinogen.
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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FIG. 5. Histological analysis of the new bones 8 weeks after implantation. The new bone (arrows) formed from the marginal defect site (arrowheads). Further bone formation was higher in the FG/bone powder scaffold implanted group than in the empty group. Asterisks highlight the remaining bone powder particles of scaffold. Hb: host bone. The specimens were stained with H&E. The scale bar represents 500 mm.
and bone powder particles of the scaffold stained a wine color (in the web version) in the H&E stain. At 8 weeks post-implantation, new bone formation was observed around the margin of the defective site in the non-implanted (empty) group. However, in the FG/bone powder-implanted group, new bone formation was observed not only around margin of the defective site but also around bone powder of the defective site. Furthermore, new bone formation was higher in the FG/bone powder-implanted group than in the empty group (Fig. 5). Images of the central area of the calvarial defect collected after MT staining showed large amounts of fibroblastic connective tissue in the empty group. In the MTstained image, newly formed bone stained a deep red color (in the web version), and bone powder particles stained a light red color (in the web version). Furthermore, the newly formed bone
contained lacunae, which each contained an osteocyte. However, the bone powder particles from scaffold only contained lacunae, but without osteocytes. The new mature bone was clearly observed in the FG/bone powder scaffold implanted group (Fig. 6A). The reconstructed three-dimensional micro-CT images of the empty and FG/bone powder-implanted scaffolds are shown in Fig. 6B. In all groups, the regeneration of bone occurred from the margins of the defect site. Quantification of bone regeneration revealed significant differences between the empty and FG/bone powder implanted groups. The % BVpost and BVpre for the FG/bone powder-implanted group were w52% and w32%, respectively. Therefore, the percentage of newly formed bone volume was w21%, and the values were significantly higher than those for the empty group (w9%, Fig. 6C). The results of the
FIG. 6. Goldner’s Masson trichrome stained histological image (A) of regenerated bones in the central area of calvarial defects at 1 week and 8 weeks post-implantation. The amount of regenerated bone in the FG/bone powder scaffold group was more than that in the empty group. Asterisks highlight the remaining bone powder particles of scaffold. Nb: newly formed bone. The scale bar represents 250 mm. Three-dimensional (3D) reconstructed micro-CT images were obtained from the empty and scaffold-implanted groups with calvarial defect at 8 weeks. Scaffold image (pre-implantation) were also obtained from micro-CT scanning of the FG/bone powder scaffold alone before implantation. The scale bar represents 4 mm (B). Quantitative analysis of newly formed bone calculated from micro-CT, following the equation described in Materials and methods (C).
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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micro-CT analysis were similar to those found by histological analysis. DISCUSSION In the present study, a porous scaffold was produced using bone powder and various concentrations of fibrinogen. When FG is used as a cross-linking agent, the clotting time of FG is dependent on the concentration of thrombin. Micro-CT showed a uniform distribution of bone powder during scaffold fabrication. These results suggested that 5 U/mL thrombin facilitated scaffold fabrication when 10e80 mg/mL fibrinogen solutions were used. Furthermore, SEM showed formation of a thin fibrin network at low fibrinogen concentrations and an extensive fibrin network and flattened fibrin layer at increased fibrinogen concentrations. The properties of the fibrin network and fibrin layers, including the fiber thickness and degree of branching, are affected by several factors, such as the fibrinogen and thrombin concentrations, method of mixing, and cross-linking time (23). In this study, the thrombin concentration was constant in our experiments. Therefore, the fibrin network thickness and layer structure were mainly affected by the fibrinogen concentration. Porosity measurements of the scaffold indicated that the percent porosity decreased as the fibrinogen concentration increased. These results are consistent with a previous report (24), where it was suggested that the porosity reduction might have been due to the filling in and subsequent closure of porous structures. Therefore, porosity was likely reduced because the spaces between each granule were closed by fibrin layer formation due to the high fibrinogen concentration. In contrast, compressive strength increased as fibrinogen concentration increased. Compressive strength is closely related to porosity (25). The SEM images (Fig. 1C) showed several fibrin networks and layers bridging between the bone granules. Furthermore, a more extensive fibrin network and thicker fibrin layers were formed as the fibrinogen concentration increased. Therefore, the degree of fibrin fiber formation and the thickness and branches of the fibrin layer may have affected the compressive strength of the scaffolds. In the proliferation assay, after 1 day of culture on the scaffold fabricated with 80 mg/mL fibrinogen, the OD was significantly higher than that of cultured cells on the scaffold fabricated with 10 mg/mL fibrinogen. These results suggest that cells might have been more adherent initially to the scaffold fabricated with 80 mg/ mL fibrinogen than to the other scaffolds. Scaffold surface area has a major influence on cell attachment, as higher surface area enhances cell attachment, and the initial cell attachment influences the proliferation rate (26). As shown by SEM (Fig. 1C), many more fibrin layers were observed as fibrinogen concentration increased. This may be because the fibrin network and fibrin layers eventually increased the surface area. However, the growth rate of cells cultured on the scaffold fabricated with 80 mg/mL fibrinogen slowed at 15 days. This may be due to the reduction in scaffold porosity, resulting in a reduction in the surface area available for cell adhesion and growth (27). Some studies have reported that cell proliferation decreases with increasing fibrinogen concentration when the cells are embedded in three-dimensional fibrinogen gels (23,28). Bensaid et al. reported that, at a fibrinogen concentration of 18 mg/mL, cells could not spread or proliferate well (28). They demonstrated that the fibrinogen concentration could affect cell behavior when cells were embedded into FG as the cell carrier. In contrast, in our study, we used FG as the cross-linking agent to fabricate a porous scaffold. Therefore, the higher fibrinogen concentration may not have directly inhibited cell spreading and proliferation because the cells were not directly embedded in the fibrin clot. Previous studies may explain our proliferation results. Natural fibrinogen has a b 15e42
epitope region (29) that promotes cell spreading and proliferation. This epitope is normally hidden, but it becomes exposed after thrombin converts fibrinogen to fibrin (30,31). Exposure of the b 15e42 epitope has been shown to promote cell spreading and proliferation on a fibrin matrix (32). In the present study, FG/bone powder scaffolds were fabricated through fibrinogen cross-linking by thrombin. Therefore, the b 15e42 epitope might be exposed during scaffold fabrication, and this region could then aid in cell proliferation and spreading. Our cell adhesion and proliferation results suggest that osteoblast differentiation is dependent on fibrinogen concentration. To address this issue, the expression of ALP and OC mRNA was evaluated in hMSCs cultured on each FG/bone powder scaffold type. ALP is one of the most commonly used intermediate markers of osteogenesis because it reflects the proportion of cells undergoing osteogenic differentiation (33). OC is frequently used as a late marker of osteoblast differentiation (34). When cells were cultured on the scaffolds in OS-containing medium, the expression of both ALP and OC mRNA increased as fibrinogen concentration increased. A previous study demonstrated that the effect of fibrinogen on osteogenic differentiation is concentration-dependent. In their study, higher fibrinogen concentrations induced higher ALP activity. However, in their experiments, a commercial fibrin glue product (Tisseel) was used, and they thought that the ALP induction was due to the growth factors, such as TGF-b1 and b-FGF, that were contained in the fibrinogen complex of Tisseel (35). In contrast, in the present study, we used a Greenplast fibrin glue product that does not contain any growth factors in the fibrinogen component. This finding suggests that osteoblast differentiation is not influenced by the growth factors because ALP and OC mRNA expression increased as the concentration of fibrinogen, without growth factors, increased. Li et al. (36) reported that cell confluence affects intracellular signaling and cell differentiation. Our results showed that cells rapidly adhered, proliferated, and reached confluence when the fibrinogen concentration used to fabricate the scaffold was increased. In the in vivo experiment, we did not observe any remaining fibrin glue in the defects (data not shown). New bone formation typically occurred in the margins of the defect. Furthermore, the FG/bone powder scaffold implanted groups exhibited significantly more bone regeneration as compared with the empty group after 8 weeks of implantation. This suggests that the fibrin polymers could affect fibroblast and vascular cell migration into mature collagenous connective tissue (37,38), and the bone powder could affect osteoinductivity. These results suggest that in the stage of bone healing, the FG/bone powder scaffold supported an environment favorable for cell adhesion, which leads to the growth of bone tissue. In summary, the FG/bone powder three-dimensional scaffolds were fabricated using various fibrinogen concentrations. The fabricated FG/bone powder scaffolds had numerous fibrous fibrils and fibrin layers on the scaffolds. Higher concentrations of fibrinogen provided increased compressive strength. In contrast, porosity decreased with increasing fibrinogen concentrations. Furthermore, the FG/bone powder scaffold provides a suitable environment for the adherence, proliferation, and differentiation of hMSCs into osteoblasts and stimulates bone formation in vivo. Our findings demonstrate that the properties of FG/bone powder scaffolds can be optimized for applications in bone tissue regeneration. ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korean Ministry of Education, Science and Technology (2013-0731).
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration Beom-Su Kim,1, 2 Hark-Mo Sung,2 Hyung-Keun You,3 and Jun Lee1, * Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea,1 Bonecell Biotech Inc., 77 Dunsan-dong, Seo-gu, Daejeon 302-830, Republic of Korea,2 and Department of Periodontology, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea3 Received 23 December 2013; accepted 27 March 2014 Available online xxx
Fibrin polymers are widely used in the tissue engineering field as biomaterials. Although numerous researchers have studied the fabrication of scaffolds using fibrin glue (FG) and bone powder, the effects of varied fibrinogen content during the fabrication of scaffolds on human mesenchymal stem cells (hMSCs) and bone regeneration remain poorly understood. In this study, we formulated scaffolds using demineralized bone powder and various fibrinogen concentrations and analyzed the microstructure and mechanical properties. Cell proliferation, cell viability, and osteoblast differentiation assays were performed. The ability of the scaffold to enhance bone regeneration was evaluated using a rabbit calvarial defect model. Micro-computed tomography (micro-CT) showed that bone powders were uniformly distributed on the scaffolds, and scanning electron microscopy (SEM) showed that the fibrin networks and flattened fibrin layers connected adjacent bone powder particles. When an 80 mg/mL fibrinogen solution was used to formulate scaffolds, the porosity decreased 41.6 ± 3.6%, while the compressive strength increased 1.16 ± 0.02 Mpa, when compared with the values for the 10 mg/mL fibrinogen solution. Proliferation assays and SEM showed that the scaffolds prepared using higher fibrinogen concentrations supported and enhanced cell adhesion and proliferation. In addition, mRNA expression of alkaline phosphatase and osteocalcin in cells grown on the scaffolds increased with increasing fibrinogen concentration. Micro-CT and histological analysis revealed that newly formed bone was stimulated in the scaffold implantation group. Our results demonstrate that optimization of the fibrinogen content of fibrin glue/bone powder scaffolds will be beneficial for bone tissue engineering. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Fibrin glue; Fibrinogen; Scaffold; Bone tissue engineering; Bone powder]
Bone defects are caused by a variety of conditions, such as tumors, trauma, disease, and bone fractures. Although small bone defects can heal themselves, large bone defects cannot and require bone graft replacements. In tissue engineering, reconstruction of bone often requires a biodegradable porous scaffold (1) because the porous scaffold provides necessary support for cell growth, adhesion, and differentiation (2). In addition, the selection of biomaterials with which to construct the scaffold is an important factor because their properties will determine scaffold properties like biocompatibility, osteoinductivity, and osteoconductivity (3,4). To construct a biocompatible scaffold for bone tissue engineering, several biomaterials, including hydroxyapatite (5), tricalcium phosphate (6), and allogeneic bone powder (7) were used. Among these, allogeneic bone powders have been frequently used to reconstruct bone defects. Because the molecular structure of bone is identical across species, it is possible to use bone from animal sources for dental implant bone grafts with good results (8). Furthermore, biodegradable natural polymers, such as a collagen (5), gelatin (9), chitosan (10), and fibrin (11), have been used to fabricate scaffolds. Fibrin polymers have been shown to have excellent adhesiveness and biocompatibility (12,13). Fibrin glues (FG) are mainly composed of fibrinogen and thrombin (14). Thrombin * Corresponding author. Tel.: þ82 42 341 2800; fax: þ82 42 341 2809. E-mail addresses: [email protected], [email protected] (J. Lee).
converts fibrin, a biopolymer, into fibrin monomers. Monomeric fibrin forms a fibrous clot that has biological adhesive properties, and because of these properties, these clots are widely used in various surgeries, including orthopedic surgery (15,16). In addition, fibrin structures function as a temporary matrix during the rebuilding and repair of tissues (17). Although some studies demonstrate that a composite biomaterial containing FG can exhibit increased biocompatibility and osteoconductivity when compared with the biomaterial alone (18,19), there is insufficient evidence for the utility of scaffolds composed of FG and bone powder in such applications. In an attempt to improve its utility for bone tissue engineering, we sought to optimize the composition of FG and bone powder scaffolds. In this study, FG and bone powder scaffolds were fabricated using various concentrations of fibrinogen, and their microstructural and mechanical properties were characterized. The in vitro biocompatibility of the scaffold was evaluated using human mesenchymal stem cells (hMSCs) in vitro, and their tissue response and ability to induce bone formation in vivo were evaluated using a rabbit calvarial defect model. MATERIALS AND METHODS Preparation of scaffolds FG from a Greenplast kit (Green Cross Corp., Seoul, Korea) and calcium-phosphate-coated bovine bone powder (Biocera; Oscotec, Chunan, Korea) were used in scaffold preparation. To construct the scaffolds, 450 mg of bone powder was placed in each hexahedron-shaped mold (10 mm 10 mm 5 mm). Next,
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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0.2 mL of fibrinogen solution (10, 20, 40, or 80 mg/mL in PBS) was added to the bone powder and mixed well. Then, 0.1 mL of thrombin solution (Greenplast Kit; 5 U/mL) was added, and the composites were rapidly blended. Polymerization of the resultant mixture was achieved at room temperature after 1 h of incubation. The blocks were then freeze-dried for 3 days to obtain FG/bone powder scaffolds.
analyzer (Sky-Scan). We additionally scanned scaffolds prior to implantation using a micro-CT, and bone volume (BV) was obtained from the data sets. Then, the percentage of bone volume was calculated using the following equation:
Micro-computerized tomography analysis To evaluate the entire scaffold structure, samples were scanned with an aluminum filter using micro-computerized tomography (Micro-CT; Sky-Scan 1172TM; Skyscan, Kontich, Belgium). Threedimensional and trans-sectional images were obtained, and the data were reconstructed using the CT-analyzer software (Sky-scan).
where BVpost is the bone volume 8 weeks after implantation, BVsc is the volume of scaffold prior to implantation, and TV is the total volume of the region of interest.
Microstructural analysis To observe the structure, the scaffolds were examined using a scanning electron microscope (SEM; EM-30; Coxem, Daejeon, Korea). Before observation, the samples were sputter-coated with gold for 120 s under vacuum. Porosity measurement Porosity was measured using a mercury intrusion porosimeter (AutoPore IV9500, Oak Ridge, TN, USA). Briefly, scaffolds were sealed in a penetrometer, weighed, and subjected to analysis (20). The porosity of 5 samples per scaffold was measured and reported as average percent porosity. Compressive strength analysis To evaluate the mechanical properties of the scaffolds, compressive strength was measured. The fabricated scaffold (10 mm 10 mm 5 mm) was subjected to a compression test using an Instron model 4505 universal test machine (Instron, Canton, MA, USA) by applying load via a 1 N load cell at a crosshead speed of 0.5 mm/min under ambient conditions. Cell culture Human bone marrow-derived mesenchymal stem cells (hMSCs) were obtained from Prof. HK You (Wonkwang University, Iksan, Korea). The cells were cultured in a-Minimal Essential Medium (a-MEM) (Gibco-BRL, Gaithersburg, MD, USA) with 1% penicillin/streptomycin and 10% fetal bovine serum (Gibco-BRL) at 37 C, 5% CO2, and 100% humidity. Passages 4e6 were used in each experiment. The differentiation of hMSCs into osteoblasts was induced by treatment with osteoblast stimulant solution (OS; 10 mM b-glycerophosphate, 0.05 mg/mL ascorbic acid, and 0.1 mM dexamethasone [SigmaeAldrich, St. Louis, MO, USA]). The culture medium and OS were replaced every 2 days during the experiment. Cell proliferation assay and evaluation of cytotoxicity CellTiter96 Aqueous One Solution (Invitrogen, Carlsbad, CA, USA) was used to measure cell proliferation. The hMSCs were seeded and cultured on the FG/bone powder scaffolds. After culture (1, 5, 10, or 15 days), 100 mL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent was mixed with 500 mL of culture media and added to each well. After incubation for 2 h, 200 mL of the supernatant was removed and its absorbance was measured at 490 nm using a microplate reader (SpectraMAX M3; Molecular Devices, Sunnyvale, CA, USA). In addition, a Live/Dead Viability/Cytotoxicity staining kit (Invitrogen) was used to evaluate the cytotoxicity of the produced scaffolds. According to the manufacturer’s protocol, cells were seeded and cultured for 3 days. Then, the sample was rinsed with PBS to remove the phenol red and serum, and reagent solution was added. After incubation for 30 min in a CO2 incubator, the samples were observed using an inverted fluorescence microscope (DM IL LED Fluo; Leica Microsystems, Wetzlar, Germany). Cell adhesion observation SEM was used to observe cell adhesion to the scaffold. After 5 days of culture, scaffolds were briefly washed with PBS. Then the samples were fixed with 2.5% glutaraldehyde, and postfixation was performed with 0.1% osmium tetroxide (OsO4, Sigma). The samples were dehydrated with a graded ethanol series (50%, 75%, 95%, 100%, and 100%), sputter-coated with gold, and visualized by SEM (EM-30). Real-time polymerase chain reaction To quantify osteoblast differentiation, cells were cultured on the FG/bone powder scaffolds for 10 days, and the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OC) marker genes was assessed using a quantitative real-time polymerase chain reaction (qRT-PCR) assay. To obtain total mRNA, the scaffolds were rinsed with PBS and chopped, and the cells were dissociated from the scaffold by sonication. Total mRNA was extracted using an RNA isolation kit (Ribospin; GeneAll, Seoul, Korea), according the manufacturer’s protocol. PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA), TaqMan Universal PCR Master Mix, TaqMan primers, and probe sets specifically targeting ALP (Hs01029144_m1), OC (Hs01587814_g1), and 18S rRNA (Hs99999901_s1; Applied Biosystems). The 18S rRNA gene was used as an internal standard. Animal experiments In this study, 3 months old New Zealand white rabbits, weighing 2.5e3.0 kg, were used. After the animal was anesthetized, the calvarium was exposed by making a skin incision. Circular calvarial defects were made using a trephine bur (8 mm in diameter), and FG/bone powder scaffolds were implanted in the induced calvarial defects. The animals were sacrificed at 8 weeks of age, and the bone tissue defects were dissected out from the host bone. The extracted bone tissue was fixed with 4% paraformaldehyde buffered with 0.1 M phosphate solution (pH 7.2) for 3e5 days before further experiments. All animal experiments were performed according to the guidelines of the Wonkwang University Institutional Animal Care and Use Committee. Micro-computed tomography The bone specimens were scanned using a micro-CT (Sky-Scan 1172TM). Then the image data were reconstructed using a CT-
ð%Þ New bone volume ¼ BVpost BVsc
TV 100
(1)
Histology After micro-CT scanning, the samples were dehydrated in a graded alcohol series (80e100%), decalcified in 8% formic acid/8% HCl, and embedded in paraffin. Sections of 5-mm thickness were prepared from the samples and mounted on slides. Following this, the samples were stained with hematoxylin/ eosin (H&E) and Goldner’s Masson trichrome (MT). Statistical analysis All experiments were performed in triplicate, and statistical analyses were performed using statistical analysis software (Origin 8.0; OriginLab, Northampton, MA, USA). Significant differences among groups were identified by ANOVA followed by Dunnett’s test. Values in the text are expressed as the means standard deviation (SD) and p values less than 0.05 were considered statistically significant.
RESULTS Structural characterization of FG/bone powder scaffolds In the present study, the thrombin used had a concentration of 5 U/mL. Because this high concentration of thrombin accelerates crosslinking, making it difficult to handle, we used various concentrations (10e80 mg/mL) of fibrinogen to fabricate the scaffolds. In this study, we successfully fabricated porous scaffolds using bovine bone powder and FG (Fig. 1A). Micro-CT images of the scaffolds are shown in Fig. 1B. The micro-CT images showed that the bovine bone powders were uniformly distributed in all scaffolds. In addition, the trans-sectional layer micro-CT images indicated that several irregular and large pores were observed in the scaffolds fabricated with 10 and 20 mg/mL fibrinogen. SEM images of the fabricated scaffolds are shown in Fig. 1C. All of the scaffolds fabricated using fibrin glue contained fibrin networks and fibrin layers that branched among the bone powder particles. Fibril networks and fibrin layers were rarely observed in the scaffolds fabricated with 10 mg/mL fibrinogen, and more compact fibrin layers were observed in the scaffolds fabricated with 80 mg/mL fibrinogen when compared with those fabricated with 10 mg/mL fibrinogen. Furthermore, a fibrin layer closed the pores between adjacent bone powders in the scaffolds fabricated with 80 mg/mL fibrinogen. Measurement of porosity and compressive strength SEM showed that fibrin layer formation closed many pores (Fig. 1C). However, in bone tissue engineering, the porosity of the scaffold is important for cell migration, angiogenesis, and nutrient supplementation (21,22). Therefore, we assessed porosity as a function of fibrinogen concentration (Fig. 2A). The porosities of 10 mg/mL and 20 mg/mL fibrinogen-fabricated scaffolds were not statistically different, with 76.1% 2.9% and 69.2% 3.6% porosity, respectively. However, the porosity of the scaffolds fabricated with 80 mg/mL fibrinogen (41.6% 3.6%) was significantly higher those fabricated with 40 mg/mL (64.2% 2.9%; p < 0.05). These results demonstrate that the porosity of the scaffold decreased as the concentration of fibrinogen increased due to the formation of fibrin layers. In addition, we measured the compressive strength of the scaffolds because mechanical properties are also crucial for bone reconstruction. The compressive strength of the scaffolds fabricated with 20 mg/mL fibrinogen was 0.46 0.06 MPa and not significantly different when compared with those fabricated with 10 mg/mL fibrinogen (0.31 0.02 MPa). However, the compressive strengths of both the 40 mg/mL and 80 mg/mL fibrinogen-fabricated scaffolds were significantly higher, at 0.93 0.06 MPa and 1.16 0.02 MPa, respectively, than the 10 mg/mL fibrinogen-fabricated scaffold (Fig. 2B). Biocompatibility To evaluate the effects of fibrinogen concentration on cell proliferation, cytotoxicity, and cell adhesion, hMSCs were seeded and cultured on the scaffolds. As shown in
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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FIG. 1. A gross view (A) of FG/bone powder scaffolds that were successfully fabricated using various fibrinogen concentrations. Three-dimensional and trans-sectional microcomputed tomography (micro-CT) images (B) showed that the bone powders (white) were uniformly distributed, and an irregular pore (black) was formed in the scaffold. The scale bar is of 5 mm. Scanning electron microscopy (SEM) images (C) of scaffold show fibrin networks and fibrin layers branching among the bone powder particles. Fibril networks and fibrin layers were rarely observed on the surface of bone powders in the scaffold fabricated with 10 mg/mL fibrinogen. However, compact and flattened fibrin layers were observed in the scaffold fabricated with 80 mg/mL fibrinogen. To enhance the contrast between the fibrin and scaffold in the SEM image, fibrin networks and fibrin layers were manually false-colored in green using Adobe Photoshop. The pseudocolor high magnification SEM image (D) shows fibrin networks (white arrows) and fibrin layers (black arrows) formed on the surface the bone powder particles (asterisks).
Fig. 3A, the proliferation of hMSCs gradually increased with culture time. After 1 day of culture, the proliferation of cells cultured on the scaffold fabricated with 80 mg/mL fibrinogen was slightly higher than that of scaffolds fabricated with 10, 20, or 40 mg/mL fibrinogen. However, after 5 and 10 days of culture, the proliferation of cells cultured on the scaffolds fabricated with 80 mg/mL of fibrinogen was approximately 2-fold higher than that of cells cultured on scaffolds fabricated with 20 or 40 mg/mL fibrinogen. In contrast, at 15 days of culture, the proliferation was higher in cells cultured on the scaffolds fabricated with 20 or
40 mg/mL fibrinogen than cells cultured on the scaffolds fabricated with 80 mg/mL fibrinogen (Fig. 3A). To evaluate the cytotoxic effects of fibrinogen, hMSCs were cultured on scaffolds, and a live/dead staining assay was performed after 3 days. Fluorescence imaging showed that almost all hMSCs on all scaffolds were viable (Fig. 3B). Furthermore, the density of live green cells was consistent with the results of the MTS assay. Cellular adhesion observation To determine whether cells could attach to the scaffolds, hMSCs were cultured, and their morphologies were observed by SEM after 5 days of culture. Fig. 3C
FIG. 2. The effects of fibrinogen concentration on the porosity (A) and compressive strength (B) of fabricated scaffolds. Scaffolds were fabricated using various fibrinogen concentrations, and their porosity and compressive strength were measured. The porosity decreased, while compressive strength increased, when fibrinogen concentration was higher. Data shown represent the mean SD of 3 samples. The asterisks indicate significant differences compared to the scaffold fabricated using 10 mg/mL fibrinogen.
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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FIG. 3. The biocompatibility of hMSCs cultured on scaffolds fabricated using various fibrinogen concentrations. hMSCs were cultured on the scaffolds and proliferation was assessed by the MTS assay at 1, 5, 10, and 15 days (A). In addition, cytotoxicity was evaluated after 3 days of culture. Live cells were stained green with calcein acetoxymethyl (Calcein AM), while dead cells were stained red with ethidium homodimer-1 (EthD-1). The scale bar represents 500 mm. Data shown represent the mean SD of 3 samples. The asterisks indicate significant differences when compared with the scaffold fabricated using 10 mg/mL fibrinogen (p < 0.05). After 5 days of culture, the morphology of adherent and growing cells was observed by SEM (C). Attached hMSCs were more frequently observed on scaffolds constructed with increased fibrinogen concentrations. To enhance the contrast between the hMSCs and scaffold in the SEM image, the cells and scaffold were manually false-colored in green and brown, respectively, using Adobe Photoshop. The scale bar represents 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
shows that hMSCs adhered and grew on the scaffolds. Attached cells were rarely observed on the scaffolds fabricated with 10 mg/ mL fibrinogen, but they were more frequently observed on scaffolds constructed with increasing fibrinogen concentrations. These results are consistent with the findings of our MTS assay. Osteogenic differentiation of hMSCs To determine if the FG/ bone powder scaffolds could support an environment for hMSC differentiation into osteoblasts, cells were cultured on the scaffolds and treated with OS. Then, qRT-PCR was performed to detect osteoblast marker expression over time. ALP mRNA expression
increased in cultured cells as fibrinogen concentration increased. ALP mRNA expression peaked on day 6 and then decreased on day 12. Additionally, OC mRNA expression peaked on day 12 and increased as fibrinogen concentration increased (Fig. 4). Bone formation analysis in vivo For in vivo experiments, we used the scaffolds fabricated with 80 mg/mL fibrinogen due to their superior mechanical properties and biocompatibility. Histological assessment of the bone formation potential of FG/bone powder scaffolds was evaluated using H&E and MT staining. The newly formed bone stained a light pink color (in the web version),
FIG. 4. Relative ALP (A) and OC (B) mRNA expression. Total RNA was extracted from hMSCs cultured on fabricated FG/bone powder scaffolds for 10 days, and real-time PCR was performed to measure the expression of ALP and OC. The 18s rRNA gene was used as an internal control. The relative expression for all groups was normalized to the expression of cells cultured on scaffolds fabricated using 10 mg/mL fibrinogen. Data shown represent the mean SD of 3 samples. The asterisks indicate significant differences compared to the group cultured in the absence of OS (p < 0.05) when cultured on scaffolds fabricated using 10 mg/mL fibrinogen.
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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FIG. 5. Histological analysis of the new bones 8 weeks after implantation. The new bone (arrows) formed from the marginal defect site (arrowheads). Further bone formation was higher in the FG/bone powder scaffold implanted group than in the empty group. Asterisks highlight the remaining bone powder particles of scaffold. Hb: host bone. The specimens were stained with H&E. The scale bar represents 500 mm.
and bone powder particles of the scaffold stained a wine color (in the web version) in the H&E stain. At 8 weeks post-implantation, new bone formation was observed around the margin of the defective site in the non-implanted (empty) group. However, in the FG/bone powder-implanted group, new bone formation was observed not only around margin of the defective site but also around bone powder of the defective site. Furthermore, new bone formation was higher in the FG/bone powder-implanted group than in the empty group (Fig. 5). Images of the central area of the calvarial defect collected after MT staining showed large amounts of fibroblastic connective tissue in the empty group. In the MTstained image, newly formed bone stained a deep red color (in the web version), and bone powder particles stained a light red color (in the web version). Furthermore, the newly formed bone
contained lacunae, which each contained an osteocyte. However, the bone powder particles from scaffold only contained lacunae, but without osteocytes. The new mature bone was clearly observed in the FG/bone powder scaffold implanted group (Fig. 6A). The reconstructed three-dimensional micro-CT images of the empty and FG/bone powder-implanted scaffolds are shown in Fig. 6B. In all groups, the regeneration of bone occurred from the margins of the defect site. Quantification of bone regeneration revealed significant differences between the empty and FG/bone powder implanted groups. The % BVpost and BVpre for the FG/bone powder-implanted group were w52% and w32%, respectively. Therefore, the percentage of newly formed bone volume was w21%, and the values were significantly higher than those for the empty group (w9%, Fig. 6C). The results of the
FIG. 6. Goldner’s Masson trichrome stained histological image (A) of regenerated bones in the central area of calvarial defects at 1 week and 8 weeks post-implantation. The amount of regenerated bone in the FG/bone powder scaffold group was more than that in the empty group. Asterisks highlight the remaining bone powder particles of scaffold. Nb: newly formed bone. The scale bar represents 250 mm. Three-dimensional (3D) reconstructed micro-CT images were obtained from the empty and scaffold-implanted groups with calvarial defect at 8 weeks. Scaffold image (pre-implantation) were also obtained from micro-CT scanning of the FG/bone powder scaffold alone before implantation. The scale bar represents 4 mm (B). Quantitative analysis of newly formed bone calculated from micro-CT, following the equation described in Materials and methods (C).
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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micro-CT analysis were similar to those found by histological analysis. DISCUSSION In the present study, a porous scaffold was produced using bone powder and various concentrations of fibrinogen. When FG is used as a cross-linking agent, the clotting time of FG is dependent on the concentration of thrombin. Micro-CT showed a uniform distribution of bone powder during scaffold fabrication. These results suggested that 5 U/mL thrombin facilitated scaffold fabrication when 10e80 mg/mL fibrinogen solutions were used. Furthermore, SEM showed formation of a thin fibrin network at low fibrinogen concentrations and an extensive fibrin network and flattened fibrin layer at increased fibrinogen concentrations. The properties of the fibrin network and fibrin layers, including the fiber thickness and degree of branching, are affected by several factors, such as the fibrinogen and thrombin concentrations, method of mixing, and cross-linking time (23). In this study, the thrombin concentration was constant in our experiments. Therefore, the fibrin network thickness and layer structure were mainly affected by the fibrinogen concentration. Porosity measurements of the scaffold indicated that the percent porosity decreased as the fibrinogen concentration increased. These results are consistent with a previous report (24), where it was suggested that the porosity reduction might have been due to the filling in and subsequent closure of porous structures. Therefore, porosity was likely reduced because the spaces between each granule were closed by fibrin layer formation due to the high fibrinogen concentration. In contrast, compressive strength increased as fibrinogen concentration increased. Compressive strength is closely related to porosity (25). The SEM images (Fig. 1C) showed several fibrin networks and layers bridging between the bone granules. Furthermore, a more extensive fibrin network and thicker fibrin layers were formed as the fibrinogen concentration increased. Therefore, the degree of fibrin fiber formation and the thickness and branches of the fibrin layer may have affected the compressive strength of the scaffolds. In the proliferation assay, after 1 day of culture on the scaffold fabricated with 80 mg/mL fibrinogen, the OD was significantly higher than that of cultured cells on the scaffold fabricated with 10 mg/mL fibrinogen. These results suggest that cells might have been more adherent initially to the scaffold fabricated with 80 mg/ mL fibrinogen than to the other scaffolds. Scaffold surface area has a major influence on cell attachment, as higher surface area enhances cell attachment, and the initial cell attachment influences the proliferation rate (26). As shown by SEM (Fig. 1C), many more fibrin layers were observed as fibrinogen concentration increased. This may be because the fibrin network and fibrin layers eventually increased the surface area. However, the growth rate of cells cultured on the scaffold fabricated with 80 mg/mL fibrinogen slowed at 15 days. This may be due to the reduction in scaffold porosity, resulting in a reduction in the surface area available for cell adhesion and growth (27). Some studies have reported that cell proliferation decreases with increasing fibrinogen concentration when the cells are embedded in three-dimensional fibrinogen gels (23,28). Bensaid et al. reported that, at a fibrinogen concentration of 18 mg/mL, cells could not spread or proliferate well (28). They demonstrated that the fibrinogen concentration could affect cell behavior when cells were embedded into FG as the cell carrier. In contrast, in our study, we used FG as the cross-linking agent to fabricate a porous scaffold. Therefore, the higher fibrinogen concentration may not have directly inhibited cell spreading and proliferation because the cells were not directly embedded in the fibrin clot. Previous studies may explain our proliferation results. Natural fibrinogen has a b 15e42
epitope region (29) that promotes cell spreading and proliferation. This epitope is normally hidden, but it becomes exposed after thrombin converts fibrinogen to fibrin (30,31). Exposure of the b 15e42 epitope has been shown to promote cell spreading and proliferation on a fibrin matrix (32). In the present study, FG/bone powder scaffolds were fabricated through fibrinogen cross-linking by thrombin. Therefore, the b 15e42 epitope might be exposed during scaffold fabrication, and this region could then aid in cell proliferation and spreading. Our cell adhesion and proliferation results suggest that osteoblast differentiation is dependent on fibrinogen concentration. To address this issue, the expression of ALP and OC mRNA was evaluated in hMSCs cultured on each FG/bone powder scaffold type. ALP is one of the most commonly used intermediate markers of osteogenesis because it reflects the proportion of cells undergoing osteogenic differentiation (33). OC is frequently used as a late marker of osteoblast differentiation (34). When cells were cultured on the scaffolds in OS-containing medium, the expression of both ALP and OC mRNA increased as fibrinogen concentration increased. A previous study demonstrated that the effect of fibrinogen on osteogenic differentiation is concentration-dependent. In their study, higher fibrinogen concentrations induced higher ALP activity. However, in their experiments, a commercial fibrin glue product (Tisseel) was used, and they thought that the ALP induction was due to the growth factors, such as TGF-b1 and b-FGF, that were contained in the fibrinogen complex of Tisseel (35). In contrast, in the present study, we used a Greenplast fibrin glue product that does not contain any growth factors in the fibrinogen component. This finding suggests that osteoblast differentiation is not influenced by the growth factors because ALP and OC mRNA expression increased as the concentration of fibrinogen, without growth factors, increased. Li et al. (36) reported that cell confluence affects intracellular signaling and cell differentiation. Our results showed that cells rapidly adhered, proliferated, and reached confluence when the fibrinogen concentration used to fabricate the scaffold was increased. In the in vivo experiment, we did not observe any remaining fibrin glue in the defects (data not shown). New bone formation typically occurred in the margins of the defect. Furthermore, the FG/bone powder scaffold implanted groups exhibited significantly more bone regeneration as compared with the empty group after 8 weeks of implantation. This suggests that the fibrin polymers could affect fibroblast and vascular cell migration into mature collagenous connective tissue (37,38), and the bone powder could affect osteoinductivity. These results suggest that in the stage of bone healing, the FG/bone powder scaffold supported an environment favorable for cell adhesion, which leads to the growth of bone tissue. In summary, the FG/bone powder three-dimensional scaffolds were fabricated using various fibrinogen concentrations. The fabricated FG/bone powder scaffolds had numerous fibrous fibrils and fibrin layers on the scaffolds. Higher concentrations of fibrinogen provided increased compressive strength. In contrast, porosity decreased with increasing fibrinogen concentrations. Furthermore, the FG/bone powder scaffold provides a suitable environment for the adherence, proliferation, and differentiation of hMSCs into osteoblasts and stimulates bone formation in vivo. Our findings demonstrate that the properties of FG/bone powder scaffolds can be optimized for applications in bone tissue regeneration. ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korean Ministry of Education, Science and Technology (2013-0731).
Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
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Please cite this article in press as: Kim, B.-S., et al., Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.014
