J Mater Sci: Mater Med (2015) 26:152 DOI 10.1007/s10856-015-5490-7

BIOMATERIALS SYNTHESIS AND CHARACTERIZATION

Fabrication of gelatin–strontium substituted calcium phosphate scaffolds with unidirectional pores for bone tissue engineering Yu-Chun Wu1 • Wei-Yu Lin2 • Chyun-Yu Yang3 • Tzer-Min Lee2,4

Received: 1 September 2014 / Accepted: 8 February 2015 / Published online: 15 March 2015 Ó Springer Science+Business Media New York 2015

Abstract This study fabricated homogeneous gelatin– strontium substituted calcium phosphate composites via coprecipitation in a gelatin solution. Unidirectional porous scaffolds with an oriented microtubular structure were then manufactured using freeze–drying technology. The resulting structure and pore alignment were determined using scanning electron microscopy. The pore size were in the range of 200–400 lm, which is considered ideal for the engineering of bone tissue. The scaffolds were further characterized using energy dispersive spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. Hydroxyapatite was the main calcium phosphate compound in the scaffolds, with strontium incorporated into the crystal structure. The porosity of the scaffolds decreased with increasing concentration of calcium-phosphate. The compressive strength in the longitudinal direction was two to threefold higher than that observed in the transverse direction. Our results demonstrate that the composite scaffolds degraded by approximately 20 % after 5 weeks. Additionally, in vitro results reveal that the addition of strontium significantly

& Tzer-Min Lee [email protected] 1

National Laboratory Animal Center, National Applied Research Laboratories, Tainan, Taiwan

2

Institute of Oral Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan

3

Department of Orthopedic, National Cheng Kung University, Tainan, Taiwan

4

School of Dentistry, Kaohsiung Medical University, Kaohsiung, Taiwan

increased human osteoblastic cells proliferation. Scaffolds containing strontium with a Sr-CaP/(gelatin ? Sr-CaP) ratio of 50 % provided the most suitable environment for cell proliferation, particularly under dynamic culture conditions. This study demonstrates the considerable potential of composite scaffolds composed of gelatin–strontiumsubstituted calcium phosphate for applications in bone tissue engineering.

1 Introduction The demand for artificial materials suitable for biomedical applications has increased considerably due to the limited availability of autografts and the unfavorable immune responses associated with allografts. Tissue engineering is an interdisciplinary field with the aim of solving problems related to organ deficiency and tissue damage. The engineering of tissue involves three core elements: scaffolds, progenitor cells, and growth factors. Scaffolding is critical to the success of tissue engineering. Many parameters must be considered in scaffold design, including biocompatibility, pore size, porosity, interconnectivity, susceptibility to degradation, and mechanical strength [1]. In bone regeneration, effective scaffolding simulates the composition and structure of human bone, thereby enhancing integration with surrounding tissue. Bone defects can be caused by external forces, reaction to tumors, trauma, congenital abnormalities, non-union fractures, and osteoporosis. Therapies traditionally used to treat these conditions include autografts, allografts, and xenografts; however, the applicability of these methods are limited [2]. Thus, developing an artificial scaffold capable of providing both osteoconduction and osteoinduction as a support for

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the growth and differentiation of osteo-progenitors could be highly beneficial to the treatment of bone damage. Natural bone is composed of calcium phosphate (mass 69–80 wt%, mainly hydroxyapatite (HA), collagen (mass. 17–20 wt%), and other substances, including water and proteins [3]. Previous investigators discovered that both collagen fibers and the c axis of HA have a preferred orientation, which is parallel to the direction of maximum load within the bone [3]. Optimizing integration into surrounding tissue requires scaffolds that mimic the components, structure, and function of natural bone and are capable of regenerating bone tissue. Gelatin is a derived polymer similar to collagen that has excellent biocompatibility, a large number of biological functional groups, and biodegradability in vivo [4, 5]. Gelatin also possesses other features that are advantageous for scaffolding, such as flexibility in shape and cost efficiency [6]. HA, a form of modified apatite, is a major constituent of bone and teeth and thus has excellent biocompatibility to bone [7]. However, HA alone is susceptible to brittleness, low viscoelasticity, and low resorbability, and is therefore not an ideal material for bone grafts [8]. Gelatin can be used for the biomimetic synthesis of nanocomposite scaffolds via the coprecipitation of HA. This process yields materials that are similar to natural bone [9, 10]. Substituting HA with various metal ions can alter the biological function according to the quantity of metal ions used in coprecipitation [11–14]. The use of strontium as a substitute for calcium in HA can significantly increase bone mineral content as well as bone mineral density [15]. The pharmacology of strontium has previously been determined. Moreover, the element has been demonstrated to be safe and biocompatible with bone [16]. In vitro and in vivo studies have further revealed that strontium has the ability to promote bone formation and reduce bone resorption, thereby leading to increased bone mass [17–20]. Strontium ranelate (SrR), a ranelic acid derived from strontium, is used in commercial injections for treatment of osteoporosis [21, 22]. SrR has been shown to have dual anabolic (stimulation of pre-osteoblast replication) and anti-catabolic (reduction in osteoclastic activity) effects [23–25]. Freeze–drying is a simple, clean method of controlling a network of ice crystals by introducing a unidirectional temperature gradient, which leads to the formation of axially-oriented pores [26]. To mimic anisotropic bone tissue, this study employed freeze–drying to fabricate unidirectional gelatin–strontium-substituted calcium phosphate (GSCP) scaffolds with the desired pore orientation [27]. The physical and chemical characteristics of these scaffolds were evaluated and the effect of strontium on the proliferation of osteoblast-like cells on scaffolds

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under both static and dynamic culture conditions was investigated.

2 Materials and methods 2.1 Co-precipitation in fabrication of gelatin– strontium-substituted-calcium-phosphate composite Gelatin–strontium-substituted-calcium-phosphate scaffolds with unidirectional pores were fabricated using coprecipitation and a modified version of the freeze–drying technique outlined by Kim [9]. Two types of gelatin solution were separately prepared in distilled water at 50 °C: gelatin ? Ca(NO3)24H2O ? Sr(NO3), (gelatin, Sigma; Ca(NO3)24H2O, Panreac; Sr(NO3), Panreac, and gelatin ? (NH3)2HPO4, ((NH3)2HPO4, Panreac). The gelatin ? (NH3)2HPO4 solution was titrated into gelatin ? Ca(NO3)24H2O ? Sr(NO3) solution at a pumping rate of 3.8 mL/min under stirring at 100 rpm, with the pH adjusted to 10 with NH4OH at 50 °C. The gelatin was fixed at 5 wt%, with the Sr/(Ca ? Sr) ratio fixed at 0.1 and the (Sr ? Ca)/P ratio fixed at 1.67. Three different mixtures were then prepared with Sr-CaP/(gelatin ? Sr-CaP) ratios set at 30, 50 and 66.6 wt%, respectively (referred to as S3CP, S5CP, and S6CP, respectively). After stirring for 24 h at 50 °C, 20 mL of each mixture was poured into a round polystyrene tube attached to a copper plate to ensure unidirectional freezing. The solutions were subsequently placed within a styrofoam container, quenched to -20 °C for 12 h, and freeze–dried for 24 h. A control group (5 wt% gelatin) was fabricated using conventional freeze– drying (without a copper plate) to form an isotropic scaffold, (referred to as GI). A schematic of the process used to prepare the unidirectional composite is presented in Fig. 1. The abbreviations used to designate the scaffolds fabricated in this study are listed in Table 1. The dried scaffolds were cross-linked in an acetone (Mallinckrodt) mixture containing 100 mM of 1-ethyl-3-3dimethylaminopropyl carbodimide hydrochloride (EDC, Aldrich) at 4 °C for 12 h. The samples were then thoroughly rinsed using double-distilled water to remove residual chemicals. This was followed by sonication in ethanol for 30 min, and drying under vacuum for 24 h for the preparation of porous scaffolds. In addition to the GI control group, three control groups of gelatin calcium phosphate (GCP) composites were also prepared. These were fabricated without additional Sr(NO3) and used a Ca/P ratio of 1.67. The GCP scaffolds were fabricated with CaP/(gelatin ? CaP) ratios of 30, 50, 66.6 wt%, respectively (referred to as 3CP, 5CP, and 6CP, respectively).

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Fig. 1 Schematic diagram of process used in fabrication of GSCP scaffolds with unidirectional pores Table 1 Abbreviations of control and experimental groups Control group, 5 % gelatin

Experimental group I, (GCP) h i CaP ¼ 30; 50; 66:6 wt% CaP þ Gelatin

Experimental group II, (GSCP) ðSr þ CaÞ Sr P ¼ 1:67; ðSr þ CaÞ ¼ 0:1

Gelatin-unidirectional

Gelatin-30 %CaP

Gelatin-Sr30 %CaP

GU

3CP

S3CP

Gelatin-50 %CaP

Gelatin-Sr50 %CaP

5CP

S5CP

Gelatin-isotropic

Gelatin-66.6 %CaP

Gelatin-Sr66.6 %CaP

GI

6CP

S6CP

2.2 Characterization

where PT is the total porosity, qb is the bulk density, and qt is the true density.

2.2.1 Porosity of specimens The bulk density of specimens (cylinder: d = 13 mm, h = 5 mm) is defined as the ratio of total scaffold volume divided by scaffold weight. In this study, true density was measured using the buoyancy method. Specifically, after cutting and grinding the specimens, the volume of powder was measured by immersion and sonication in alcohol according to the Archimedes principle. Total porosity was then calculated as follows: PT ¼ 1 

qb qt

ð1Þ

2.2.2 Geometric characterization Geometric characterization of the sliced samples was performed using scanning electron microscopy (SEM; JSM6390LV, JEOL) and energy-dispersive X-ray spectroscopy (EDX) analysis. Micro-computed tomography (micro-CT; model 1076, Skyscan) was used to reconstruct the 3-dimensional structure of the specimens. Images were taken with the pixel size set to 9 lm. Additionally, the formation of Sr-CaP nanocrystals was using transmission electron microscopy (TEM; JEM-1400, JEOL). Scaffolds were sonicated in

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99.9 % ethanol to obtain supernatant (Sr-CaP nanocrystals), which was placed on a carbon-coated copper grid. 2.3 Pore size distribution Pore size distribution was measured using image analysis software (Image-Pro Plus, Media Cybernetics). Pores were measured from SEM micrographs in a perpendicular direction along the long and short axes. A total of 200 pores were measured for each specimen and the average values are reported as the mean pore size. The phase composition and chemical structure of the samples were examined using a high-resolution X-ray powder diffractometer (XRD; D/MAZ2500, Rigaku) and Fourier transform-infrared spectroscopy (FT-IR; System 2000, Jasco), respectively. 2.4 Compression testing Compression testing using a tensile/compression testing device (AG-I; Shimadzu) at a compression rate of 2 mm/min according to prior studies [28, 29]. Testing was performed in a vertical direction toward the smallest area of cubic specimens, which measured approximately 10 9 10 9 10 mm3. Compressive strength was calculated from the load-strain curve according to the ratio of the ultimate compressive load to the cross-sectional area of the specimen. 2.5 Degradation assay Degradation was evaluated at 3, 7, 14, 21, 28, and 35 days. The specimens were weighed prior to incubation (Wi) in phosphate buffered saline (PBS) within a humidified incubator at 37 °C (5 % CO2/balanced air). After 35 days, the specimens were removed from the PBS solution, gently blotted with filter paper to remove surface water, and immediately weighed again (Ws). The specimens were then dehydrated in an oven for 2 days to remove all remaining water, after which they were weighed (Wd) a third time. Scaffold degradation ratios were calculated using the following equation:

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2.7 Dynamic cell culture Scaffolds (d: 13 mm, h: 5 mm) were prepared and sterilized in 70 % ethanol for 5 min three times. Samples were then presoaked in culture medium for 10 min prior to placement in 6-well plates. Each plate had been sterilized prior to loading and contained 1 mL of culture medium seeded at 2 9 105 cells/mL. After 3 h of incubation, cell-containing scaffold were placed in orbital shakers and agitated at 20 rpm. 2.8 Cell attachment and proliferation After culturing, the scaffolds were removed and washed with PBS three times. The number of viable cells attached to the scaffolds was subsequently quantified using the Alamar blue assay at days 1, 3, 5, and 7. At each culturing period, the scaffolds were removed and transferred into new wells and 500 mL of Alamar blue (Life Technologies, US) was added to each sample. Following incubation at 37 °C for 60 min, absorbance was measured at 590 nm using a micro-plate reader. According to cell attachment tests, the seeding conditions were set at a concentration of 2 9 105 cells/scaffold. After incubation for 3 h, the morphology of cells on the scaffolds was evaluated. Cell proliferation was determined by holding scaffolds in 4 mL of the medium under static or dynamic culture conditions for up to 7 days in an incubator humidified with 5 % CO2/95 % air at 37 °C. Following proliferation, cells were fixed with 2.5 % glutaraldehyde, dehydrated in a series of graded ethanol concentrations (70, 90, and 100 %), and treated with hexamethyldisilazane solution (repeated twice for 10 min each). Cellular morphology was subsequently observed using SEM. 2.9 Statistical analysis

Degradation ratio ¼ ðWi  Wd Þ=Wi  100 %

The results of cell proliferation assays are presented as the mean ± standard deviation (SD) of eight replicates. Statistical differences were analyzed using one-way analysis of variance (ANOVA) with P values set to \0.05. All statistical analyses were conducted using SAS statistical software.

2.6 Cell culture

3 Results

Human osteoblastic MG63 cells were used to assess the cellular response to the scaffolds. The culture medium for the cells was Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10 % fetal bovine serum (FBS, Gibco). Following sub-culturing in a humidified atmosphere of 5 % CO2/95 % air at 37 °C, the cells were washed with PBS (Gibco), detached using trypsin–EDTA solution (0.25 % trypsin) at 37 °C for 5 min, and centrifuged and resuspended for further testing.

3.1 Characteristics of gelatin–strontium-substituted calcium phosphate scaffolds

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Strontium has been approved that promotes osteoblastic replication and helps the bone growth [22, 30]. This study employed co-precipitation techniques to synthesize GSCP composites. TEM (Fig. 2) was used to evaluate the morphology of strontium-substituted calcium phosphate crystals precipitated in gelatin solutions of various

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Fig. 2 TEM images of various crystals produced using coprecipitation method: a 3CP, b 5CP, c 6CP, d S3CP, e S5CP, and f S6CP

concentrations. The TEM images reveal crystals in the form of nano-needles measuring approximately 100 nm. SEM was used to evaluate the structure of the GSCP scaffolds produced by freeze–drying. The pore morphology results are presented in Fig. 3. The transverse pores of the scaffolds were round and evenly distributed, as shown in Fig. 3a (GU group, transverse). As shown in the longitudinal section of Fig. 3d, the pore direction was not strictly vertical; however, a single direction was maintained from bottom to top. As shown in Fig. 3b, e, the GI group (conventional freeze–drying without a copper plate) has no directional arrangement in its cross-section and longitudinal sections. The addition of strontium–substituted-calcium-phosphate nanocrystals within the scaffold increased the barriers between pores, but did not influence pore direction, as shown in Fig. 3f. Pore size distribution was determined using SEM images at various magnifications, as shown in Fig. 4. Pore sizes in groups with only gelatin (i.e., GU and GI) were approximately 200 nm. When the concentrations of

calcium and phosphorus were increased, pore size increased to approximately 400 nm; however, at the highest concentrations of Ca and P (i.e., 6CP and S6CP), pore aperture was reduced to 292 and 288 nm, respectively. The micro-CT scans in Fig. 5 show the cross-section surface and longitudinal reorganization. Porosity tends to lower in composites with higher concentrations of calcium phosphate or strontium-substituted calcium phosphate. EDX analysis was used to calculate the semi-quantitative elemental ratio. The concentrations of calcium and phosphorus were correlated with an increase in the Ca/P ratio. Specifically, the Ca/P ratios of groups 3CP, 5CP, and 6CP were 1.53, 1.57, and 1.70, respectively (Table 2). Through coprecipitation, the concentrations of strontium-substituted calcium and phosphorus groups increased from 30 to 66.6 wt%, with the (Sr ? Ca)/P ratios of groups S3CP, S5CP, and S6CP reaching 1.51, 1.62, and 1.71, respectively. The chemical structure of the GSCP scaffolds was characterized using FTIR spectroscopy, as shown in Fig. 6. Amide I and II bands appear in the spectra, corresponding

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Fig. 3 SEM cross-sectional images of scaffolds: a GU-transverse, b GI-transverse, c S5CP-transverse, d GU-longitudinal, e GI-longitudinal, and f S5CP-longitudinal. a–c Show image with 9100 magnification. d–f Show images with 930 magnification

Fig. 4 Average pore size of scaffolds: (Asterisk indicates statistical comparison with GU group, * and # indicate p \ 0.05, N = 10)

to the functional group of gelatin (at 1690, 1550, and 1250 cm-1). The absorption peaks at 1026 and 630 to 580 cm-1 represent the PO43- functional groups of hydroxyl apatite and the peaks at 3600–3200 cm-1 represent the OH functional groups of water molecules. The FTIR data didn’t showed the peak shift that indicated chemical bonding in our system. The XRD patterns in Fig. 7 are for various groups of scaffolds and HA. The angles of the diffraction peaks for calcium and phosphorus groups are similar to those of HA. However, when strontium substituted calcium, the main peak shifted to a lower angle by approximately 0.1°.

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The porosity values of the various scaffold groups are presented in Table 3. The control groups (GI and GU) have the highest porosity (approximately 90–91 %). An increase in the concentration of CaP tended to decrease porosity, with samples 6CP and 6SCP having the lowest porosity (approximately 76–77 %). The degradability of scaffolds was measured at 37 °C for 35 days. As shown in Fig. 8, the degradation rate of each group follows a first-order curve. The GU group showed the highest degradation (a weight loss of 21 % at day 35). When the concentrations of strontium and calcium phosphate were increased, the degradation ratio decreased from 19 % (S3CP group) to 14 % (S6CP group) at day 35. Compressive strength was tested using a material testing machine. A stress–strain diagram is presented in Fig. 9. In the GI control group, the vertical and horizontal compressive strength values were very close to 300 kPa. Additionally, the longitudinal compressive strength in all unidirectional scaffolds was 2–3 times greater than the transverse compressive strength. Scaffolds demonstrated improved compressive strength when the concentration of calcium and phosphorus was increased or when strontiumsubstituted calcium and phosphorus. 3.2 Effects of strontium concentration in scaffolds on osteoblasts To evaluate the effect of strontium concentration on osteoblastic cells, scaffolds S3CP, S5CP, and S6CP were

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Fig. 5 Micro CT cross-sectional scans of scaffolds: a S3CP-transverse, b S5CP- transverse, c S6CP-transverse, d S3CP-longitudinal, e S5CPlongitudinal, f S6CP-longitudinal. Scaffolds are cylinders with d = 13 mm, h = 5 mm

Table 2 Semi-quantitative elemental ratio of scaffolds based on EDX analysis Specimen

Elemental ratio

Specimen

Ca

Elemental ratio Sr

P

Sr þ Ca

Sr þ Ca

P

3CP

1.53

S3CP

0.096

1.51

5CP

1.57

S5CP

0.106

1.62

6CP

1.70

S6CP

0.112

1.71

seeded with approximately 2 9 105 cells/scaffold under static culture conditions. Figure 10a presents the morphology of cell attachment to the S5CP scaffold. The

MG63 cells began attaching to the surface of the S5CP scaffold at 3 h, as shown in Fig. 10a1. The high-magnification image shown in Fig. 10a2 reveals a single, round MG63 cell and filopodia that extend to adjacent surfaces at 3 h. After 7 days of proliferation on the S5CP scaffold, the cells appeared flat and secreted extra-cellular matrix, as shown in Fig. 10a3. The cell proliferation on scaffolds containing various concentrations of strontium was measured after 1, 3, 5, and 7 days using the Alamar blue assay, as shown in Fig. 10b. On day 1, the efficiency of cell seeding was approximately 50 % in all scaffolds and the concentration of strontium had no effect on cell attachment. All strontium-containing scaffolds presented

Fig. 6 FTIR analysis of 3CP and S3CP scaffolds

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Fig. 7 XRD patterns of scaffolds compared with that of HA internal standard. The star indicates that silicon was added in order to correct peak positions

Fig. 8 Degradation ratio of scaffolds (N = 3)

significant proliferation during the static culture period; however, proliferation ceased on day 7. The S5CP group showed significantly greater cell proliferation than those of S3CP and S6CP groups from day 3 to day 7 (P \ 0.05). Specifically, the number of cells on the S5CP scaffold increased from 9.86 9 104 cells on day 1 to 1.55 9 105 cells on day 7. 3.3 Cell proliferation on various scaffolds under static and dynamic culture conditions MG63 cells were cultured on three different scaffolds (GU, 5CP, and S5CP) under static and dynamic culture conditions, respectively, for 7 days in order to identify which scaffold material provided the best conditions for cell proliferation and tissue engineering. Half of the scaffolds were transferred to a dynamic culture system 3 h after cell seeding, while the others were held under static conditions. Cell proliferation was measured after 1, 3, 5, and 7 days using the Alamar blue assay, as shown in Fig. 11. No differences were observed between the three kinds of composite (GU, 5CP, and S5CP) in the initial attachment of MG63 cells in either of the culture systems. Throughout the culture period, the cells continued proliferating, suggesting that all the composites have good biocompatibility. The cell proliferation on the 5CP scaffold was slightly higher than that on the control group (GU). The S5CP

Fig. 9 Compressive strength of scaffolds in longitudinal and transverse directions (asterisk indicates statistical comparison with GU group, * and # indicate P \ 0.05; N = 3)

scaffold had a significant proliferative effect from day 3 to day 7 (compared with the GU group, P \ 0.05). On day 7, S5CP presented the best cell proliferation of all scaffold groups under both static and dynamic culture conditions, with the number of cells reaching 1.45 9 105 cells/scaffold under static culture conditions and 1.72 9 105 cells/scaffold under dynamic culture conditions. Additionally, cells continued proliferating until day 7 under dynamic culture conditions; however, cells in the static culture ceased proliferation on day 5. Figure 12 presents a SEM

Table 3 Porosity of scaffolds (N = 3) Specimen

Porosity (mean ± SD) %

Specimen

Porosity (mean ± SD) %

Specimen

Porosity (mean ± SD) %

GI

89.58 ± 4.13

3CP

89.92 ± 0.62

S3CP

89.19 ± 0.98

GU

90.80 ± 2.11

5CP

84.85 ± 0.91

S5CP

84.70 ± 1.05

S6CP

77.34 ± 1.09

S6CP

76.31 ± 1.06

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Fig. 10 Cell attachment and proliferation on strontium-containing scaffolds: SEM images at a1 low and a2 high magnification showing morphology of MG63 cells attached to S5CP scaffold at 3 h (arrows indicate cells); a3 morphology of MG63 cells cultured on S5CP

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scaffold for 7 days; b proliferation of MG 63 cells cultured on scaffold produced using various concentrations of strontium (* indicates p \ 0.05 compared to other groups; N = 8)

Fig. 11 Cell proliferation on three types of scaffold under static and dynamic culture conditions (* indicates p \ 0.05 compared to GU group each time point; # indicates p \ 0.05, scaffold in static culture compared to dynamic culture; N = 8)

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Fig. 12 SEM images showing the morphology of MG63 cells produced on scaffolds under static and dynamic culture conditions for 7 days: a1 GU scaffold under static conditions; a2 GU scaffold under dynamic conditions. b1 5CP scaffold under static conditions.

b2 5CP scaffold under dynamic conditions. c1 S5CP scaffold under static conditions. c2 S5CP scaffold under dynamic conditions. Images were obtained at 91000 magnification

micrograph of the cellular morphology on GU, 5CP, and S5CP samples under static and dynamic culture conditions. As shown, the cells appear to have expanded and flattened over a large area and the scaffold surface was covered with an extracellular matrix that linked individual cells. These results demonstrate the ability of all scaffolds considered by this study to support cellular growth.

(GU group) presented smaller pore sizes of 200 lm due to the high degree of uniformity in the growth and distribution of ice crystals. Following the addition of calcium phosphate or strontium (groups 3CP, S3CP, 5CP, and S5CP), the pore size tended to increase. This may be explained by insoluble calcium phosphate or strontium crystals impeding the growth of ice crystals. When the growth of ice crystals was inhibited, larger crystals accumulated, which possibly resulted in the formation of larger pores. However, in groups 6CP and S6CP, which contained the highest concentrations of insoluble crystals, the growth space of ice crystals was limited, leading to a decrease in pore size. All scaffolds were cross-linked using EDC for 12 h, and then the degree of cross-linking was measured. The crosslinking reaction was used to prevent the deformation of the structures. All samples in this study presented cross-linking of approximately 80 % (data not shown), which falls within the category of a complete cross-linking reaction [33]. The GU group presented the highest weight loss resulting from hydrolytic degradation [34]. The weight loss in samples with added calcium phosphate or strontium was less pronounced. The difference in the degradation rate between GU and S6CP can be attributed to the presence of Sr-CaP crystals in the S6CP structure, which helped maintain the integrity of the structure. Although previous research reported that low porosity enhanced degradation [35], in our study, the concentration of Sr, Ca, and P had a

4 Discussion Biomimetic GSCP scaffolds with unidirectional pores were synthesized at the nanoscale using strontium-substituted calcium phosphate (*100 nm) through precipitation in a gelatin solution. Bone is a complex biomineralized system with a hierarchical structure, comprising nano-apatite minerals deposited in a type I collagen matrix [31]. Artificial bio-mineralization processes are able to create nanobiological structures with good biocompatibility and biological function. Moreover, biomimetic fabrication can be used to produce nanostructures with specific biological functions that mimick those found in natural bio-mineralization processes [32]. In this study, scaffolds fabricated via freeze–drying presented high porosity and interconnectivity with pore sizes in the range of 200–400 lm. This size range is considered to be well suited to the growth of bone tissue [1]. The material produced with pure gelatin

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greater influence than that of porosity on the degradation rate. Unidirectional scaffolds were fabricated with an anisotropic pore structure that mimics that of natural bone [26, 36]. With an anisotropic pore structure, compressive strength parallel to the pore axis is much higher than compressive strength perpendicular to this axis. In this study, the compressive strength of the longitudinal section of unidirectional scaffolds was on averaged 2.5 times higher than that of the transverse section. Higher concentrations of CaP or Sr-CaP increased the mechanical strength longitudinally due to a reduction in porosity and the additional mechanical support provided by CaP or SrCaP crystals [37, 38]. Previous studies have reported that strontium induces osteoblast proliferation via calcium-sensing receptors [39, 40]. This study evaluated scaffolds produced with various concentrations of Sr-CaP. The S5CP group presented the highest cell proliferation, as compared with those of the GU scaffold, the 5CP scaffold (Fig. 11), and other Sr-CaPcontaining scaffolds (Fig. 10b). Boanini et al. [41] found the rat osteoblasts derived from osteoporotic bone cultured on SrHA exhibited higher proliferation and increased the level of alkaline phosphatase and collagen type I than that of HA. Aina et al. [42] found Sr substituted in HA increased the proliferation of MG63 osteoblastic cells and enhanced cell differentiation. They proposed that the positive effects of Sr2? in Sr-HA materials are probably due to the co-action of other ions, Ca2? or PO43-. Chattopadhyay [39] reported that Sr2? could potentially modulate the activity of CaR in cooperation with Ca2? and increased the expressions of c-fos and egr-1, which are involved in rat calvarial osteoblasts proliferation. In our system, the S5CP scaffold had a higher content of Sr2? and a synergic effect with Ca2?, which may be responsible for the enhance osteoblastic cells proliferation; In addition, the large pores of the S5CP scaffolds promoted nutrient transmission. The S6CP samples had smaller pores than those of S3CP and S5CP. Lower porosity tends to limit cell growth. Joseph [28] reported that Sr-ranelate induced a dose-dependent increase in the proliferation of MC3T3-E1 (pre-osteoblastic cell line), with 5 mM Sr-ranelate having the strongest effect and 10 mM Sr-ranelate producing a slightly reduced response. In this study, S6CP produced fewer cells than did S5CP, perhaps due to the same mechanism. Under static culture conditions, cell proliferation in all Sr-containing scaffolds ceased on day 7 due to restricted nutrient transport following the growth of cells over the surface of the scaffolds. Dynamic culture conditions enhanced the transport of nutrients, thereby enhancing the proliferation of cells beyond what was achieved in a static culture. These results are consistent with those of Araujo [39]. In terms of promoting cell proliferation, the

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scaffold materials can be ranked as follows: GSCP [ GCP [ gelatin. In addition, dynamic culturing is preferable to static culturing.

5 Conclusion This study demonstrated the advantages of combining coprecipitation with freeze–drying in the fabrication of unidirectional porous GSCP scaffolds. The resulting materials provided greater longitudinal compressive strength, increased proliferation of osteoblastic cells, and formed anisotropic structure similar to that found in natural bone. Of all materials considered in this study, S5CP showed the greatest cell proliferation rate due to its high porosity, suitable pore size, and superior mechanical strength. Thus, the S5CP scaffold shows the greatest potential for bone tissue engineering. In addition, dynamic culturing is recommended in order to provide cells with optimal nutrient transport conditions required for the engineering of bone tissue. The findings of this preliminary study suggest that GSCP scaffolds with unidirectional pores provide osteoconductive and osteoinductive support for the growth of osteoblastic cells. Future experiments will investigate how various dynamic culture parameters influence cellular differentiation and bio-mineralization in strontium-containing scaffolds. Acknowledgments This work was supported in part by Grant NSC 100-2221-E-006-263 from the National Science Council of Taiwan.

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