Abstract
Objective
Highly porous apatite-based bioceramic scaffolds have been widely investigated as three-dimensional (3D) templates for cell adhesion, proliferation, and differentiation promoting the bone regeneration. Their fragility, however, limits their clinical application especially for a large bone defect.
Methods
To address the hypothesis that using a ZrO 2 /hydroxyapatite (HAp) composite might improve both the mechanical properties and cellular compatibility of the porous material, we fabricated ZrO 2 /HAp composite scaffolds with different ZrO 2 /HAp ratios, and evaluated their characteristics. In addition, porous ZrO 2 /HAp scaffolds containing bone marrow derived stromal cells (BMSCs) were implanted into critical-size bone defects for 6 weeks in order to evaluate the bone tissue reconstruction with this material.
Results
The porosity of a ZrO 2 /HAp scaffold can be adjusted from 72% to 91%, and the compressive strength of the scaffold increased from 2.5 to 13.8 MPa when the ZrO 2 content increased from 50 to 100 wt%. The cell adhesion and proliferation in the ZrO 2 /HAp scaffold was greatly improved when compared to the scaffold made with ZrO 2 alone. Moreover, in vivo study showed that a BMSCs-loaded ZrO 2 /HAp scaffold provided a suitable 3D environment for BMSC survival and enhanced bone regeneration around the implanted material.
Significance
We thus showed that a porous ZrO 2 /HAp composite scaffold has excellent mechanical properties, and cellular/tissue compatibility, and would be a promising substrate to achieve both bone reconstruction and regeneration needed in the treatment of large bone defects.
1
Introduction
Bone tissue has a high potential for self-repair and regeneration, however, when a fracture defect that needs to be bridged is too large, the result can be pseudarthrosis (nonunion of the fracture) and loss of function . Metal or ceramic materials are widely used for bone tissue repair, however, it is still hard to rebuild tissue with its original morphology. Although recent advances in scaffold based bone regeneration therapy have made it effective for bone tissue repair, there are still some limitations to its ability to repair large defects . Generally, biodegradation of the scaffold material would reduce the mechanical strength of the bone regeneration region during the healing period . We therefore considered that the combined achievement of both bone reconstruction and regeneration led by biomaterials would be an effective approach to the treatment of large bone defects.
Open pore structure of the scaffold enhances the new bone tissue formation, because the structure readily allows the nutrient supply, gas diffusion, and metabolic waste removal, which are important for cell survival and activity . Hydroxyapatite (HAp) is the first choice as a source material for bone reconstructive scaffolds because it is the main component of bone and is known for its excellent cellular and tissue affinity . Porous sintered HAp, however, does not have sufficient mechanical properties . The compressive strength of pure HAp porous blocks synthesized in previous works is only 0.3 MPa, whereas that of trabecular bone is 12 MPa and that of cortical bone is 200 MPa . Zirconia (ZrO 2 ) would be a good additive material because it is a stable inorganic material with high biocompatibility and good mechanical properties. ZrO 2 itself, however, does not have good cellular and tissue affinity . ZrO 2 and HAp mixtures having porous structure were therefore prepared in efforts to obtain a nondegradable and bone reconstructive substrate that can enhance the bone regeneration as well.
To evaluate the usefulness of the porous ZrO 2 /HAp composite material for bone tissue repair, in this study we investigated physical properties and cellular compatibility of the material. Moreover, we also implanted cell-loaded porous ZrO 2 /HAp scaffolds in critical-size bone defects to evaluate the effect of the material for bone tissue repair.
2
Materials and methods
2.1
Scaffold preparation
HAp was synthesized in the wet condition at 80 °C mentioned elsewhere . ZrO 2 /HAp slurries were prepared by dispersing 80 g of powder consisting of ZrO 2 (3% Y 2 O 3 –97% ZrO 2 , Tosoh, Japan) and HAp mixed at various wt% ratios (ZrO 2 /HAp: 0/100, 50/50, 60/40, 70/30, 80/20, and 100/0) in 100 ml of ethanol containing 6 ml of triethyl phosphate (TEP, Sigma–Aldrich, MO) and 6 g polyvinylbutyral (PVB, Sigma Aldrich) for 24 h. Porous scaffolds were fabricated by impregnating the struts of a polyurethane sponge (45 pores per inch, Customs Foam Systems Ltd, ON, Canada) with the slurry. The sponge block was dipped into the slurry and compressed slightly to remove the excess slurry on the foam. After the sponge was dried at 70 °C for 1 h, heated at 700 °C for 3 h to burn out the sponge block and binder, and then sintered at 1500 °C for 5 h.
2.2
Scaffold characterization
The size and shape of the crystals in the ZrO 2 and HAp starting powders were observed using a transmission electron microscope (TEM, H-800, Hitachi, Japan), and the morphology and pore size of the obtained scaffold were observed using a scanning electron microscope (SEM, JSM-6390BU, JEOL, Japan). X-ray diffraction analysis (XRD, 40 kV, 30 mA, RINT2000, Rigaku, Japan) was carried out to identify the phase composition of starting powders and the sintered scaffolds with different ZrO 2 /HAp ratios. The porosity of the porous scaffolds was measured based on Archimedes’ principle . The compressive strength of the porous scaffolds was measured by a mechanical tester (EZ-test, Shimazu, Japan) with a crosshead speed of 1 mm/min. Five samples were tested for each group, and the average and standard deviation were calculated.
2.3
Cellular affinity of ZrO 2 /HAp scaffold
MC3T3-E1 osteoblast-like cells (Riken, Japan) were cultured at 37 °C in Dulbecco Modified Eagle’s Medium (DMEM, Wako, Japan) containing 10% fetal bovine serum (FBS, Invitrogen, CA) with 1% penicillin/streptomycin (Nacalai Tesque, Japan) in a humidified atmosphere with 5% CO 2 . Cells were seeded (1 × 10 5 cells/ml) into the scaffolds, and after 3 and 5 days of culture the cells adhering to the scaffold surface were observed by using a SEM (JSM-6390BU, JEOL).
Cell proliferation in the scaffolds was investigated by seeding cells in the scaffolds at a density of 5 × 10 4 cells/ml and counting cells after culturing for 3, 7, 10, and 14 days. Cells were collected from the scaffolds by soaking the scaffolds in a diluted trypsin–EDTA solution for 20 min and using a hemocytometer to count the cells in aliquots of the resulting cell suspensions ( n = 4).
2.4
Dynamic array analysis
Nanofluidic real-time RT-PCR analysis using a BioMark™ 48 × 48 Dynamic Array (Fluidigm, CA) and TaqMan gene expression assay mixes (Applied Biosystems, CA) for osteopontin ( Opn ), osteocalcin ( Oc ), and Gapdh were carried out. Scaffolds containing osteoblasts were collected at days 10 and 20, cells in the scaffold were pulverized, and the total RNA was extracted by Trizol reagent (Invitrogen, CA) ( n = 4). RNA was isolated using an RNeazy mini kit according to the directions of the manufacturer (Qiagen, Germany). The first-strand synthesis of single-strand cDNA from RNA for use as a PCR template was carried out with a SuperScript First-Strand cDNA Synthesis Kit (Invitrogen). cDNA was preamplified before the real-time RT-PCR by using pooled 0.2 × TaqMan Gene expression Mixes and CellDirect™ One-Step qRT-PCR Kits (Invitrogen). The thermal cycling conditions for STA were 12 cycles of 95 °C for 15 s and 60 °C for 4 min. Preamplified cDNA was diluted with TE buffer (1:5) and was used for real-time RT-PCR. The PCR profile for Dynamic Array included a 10 min, 95 °C hot-start to activate the Taq polymerase, followed by 40 cycles of a two-step program: 15 s at 95 °C (denaturation) and 60 s at 60 °C (annealing and extension). Data were analyzed using BioMark™ Real-Time RCR Analysis Software v2.0 (Fluidigm). The results were normalized by dividing the amount of the target gene by the amount of Gapdh . The following TaqMan gene expression assay mixes were used in this study; Opn : Mm01204014_ml, Oc : Mm03413826_mH, Gapdh : Mm99999915_gl (Applied Biosystems, CA).
2.5
In vivo bone formation
Fibrin gel containing bone marrow derived stromal cells (BMSCs, 1 × 10 6 cells/ml) was injected into ZrO 2 /HAp scaffolds to obtain the cell-encapsulated scaffolds. Eight-week-old male SD rats (320–360 g) were used in this study (supplementary figure 1). All animal experiments were performed in accordance with Osaka University Animal Care and Use Committee guidelines (Approved number: 20-017-0).
After exposure of the parietal calvarial bone, a full-thickness skull defect 5 mm in diameter was formed using a slow-speed dental drill with continuous cooling with saline. ZrO 2 /HAp scaffolds (70/30 wt%) containing cells or without cells were implanted in the bone defects. Bone defects without implanted material were also prepared as controls. At 6 weeks after implantation, explanted samples were fixed in 10% formaldehyde solution, dehydrated in a graded series of alcohol, and embedded in methylmethacrylate ( n = 4). Sections 100 μm thick were made using a modified sawing microtome technique and stained with hematoxylin and eosin (HE) according to the routine histology protocol. For the detection of new bone formation, von Kossa staining was carried out. For immunofluorescent staining, sections were treated first with the primary antibody of osteopontin (OPN) or type I collagen (Col I) (1:500, Chemicon, MA) and then with AlexaFluoro488-conjugated secondary antibody (1:500, Invitrogen, CA). Image analysis software (Lumina Vision, Mitani Corporation, Japan) was used to quantify the expression ratio of protein in the sample.
At 3 and 6 weeks after implantation, harvested calvaria was scanned to evaluate the calvarial architecture by using a μCT scanner (SMX-100CT-SV, Shimazu, Japan) with a resolution of 57 μm (pixel size) and an exposure time of 200 s. The X-ray source was set at 42 kV and 33 μA to obtain the best contrast between bone tissues and soft tissues. Morphometrical analysis based on the μCT data image was also performed by using the 3D image analysis software VGStudio MAX 1.2.1 (Volume Graphics, Germany). A cylindrical region of interest (ROI, 3 mm × 3 mm × 1 mm) was co-centrically positioned over the defect site and was measured at both 3 and 6 weeks ( n = 4).
2.6
Statistical analysis
Statistical analysis was performed using one-way analysis of variance (ANOVA). Student’s t -test was used for comparison at a 95% confidence interval. All results are expressed here as the mean ± the standard deviation.
2
Materials and methods
2.1
Scaffold preparation
HAp was synthesized in the wet condition at 80 °C mentioned elsewhere . ZrO 2 /HAp slurries were prepared by dispersing 80 g of powder consisting of ZrO 2 (3% Y 2 O 3 –97% ZrO 2 , Tosoh, Japan) and HAp mixed at various wt% ratios (ZrO 2 /HAp: 0/100, 50/50, 60/40, 70/30, 80/20, and 100/0) in 100 ml of ethanol containing 6 ml of triethyl phosphate (TEP, Sigma–Aldrich, MO) and 6 g polyvinylbutyral (PVB, Sigma Aldrich) for 24 h. Porous scaffolds were fabricated by impregnating the struts of a polyurethane sponge (45 pores per inch, Customs Foam Systems Ltd, ON, Canada) with the slurry. The sponge block was dipped into the slurry and compressed slightly to remove the excess slurry on the foam. After the sponge was dried at 70 °C for 1 h, heated at 700 °C for 3 h to burn out the sponge block and binder, and then sintered at 1500 °C for 5 h.
2.2
Scaffold characterization
The size and shape of the crystals in the ZrO 2 and HAp starting powders were observed using a transmission electron microscope (TEM, H-800, Hitachi, Japan), and the morphology and pore size of the obtained scaffold were observed using a scanning electron microscope (SEM, JSM-6390BU, JEOL, Japan). X-ray diffraction analysis (XRD, 40 kV, 30 mA, RINT2000, Rigaku, Japan) was carried out to identify the phase composition of starting powders and the sintered scaffolds with different ZrO 2 /HAp ratios. The porosity of the porous scaffolds was measured based on Archimedes’ principle . The compressive strength of the porous scaffolds was measured by a mechanical tester (EZ-test, Shimazu, Japan) with a crosshead speed of 1 mm/min. Five samples were tested for each group, and the average and standard deviation were calculated.
2.3
Cellular affinity of ZrO 2 /HAp scaffold
MC3T3-E1 osteoblast-like cells (Riken, Japan) were cultured at 37 °C in Dulbecco Modified Eagle’s Medium (DMEM, Wako, Japan) containing 10% fetal bovine serum (FBS, Invitrogen, CA) with 1% penicillin/streptomycin (Nacalai Tesque, Japan) in a humidified atmosphere with 5% CO 2 . Cells were seeded (1 × 10 5 cells/ml) into the scaffolds, and after 3 and 5 days of culture the cells adhering to the scaffold surface were observed by using a SEM (JSM-6390BU, JEOL).
Cell proliferation in the scaffolds was investigated by seeding cells in the scaffolds at a density of 5 × 10 4 cells/ml and counting cells after culturing for 3, 7, 10, and 14 days. Cells were collected from the scaffolds by soaking the scaffolds in a diluted trypsin–EDTA solution for 20 min and using a hemocytometer to count the cells in aliquots of the resulting cell suspensions ( n = 4).
2.4
Dynamic array analysis
Nanofluidic real-time RT-PCR analysis using a BioMark™ 48 × 48 Dynamic Array (Fluidigm, CA) and TaqMan gene expression assay mixes (Applied Biosystems, CA) for osteopontin ( Opn ), osteocalcin ( Oc ), and Gapdh were carried out. Scaffolds containing osteoblasts were collected at days 10 and 20, cells in the scaffold were pulverized, and the total RNA was extracted by Trizol reagent (Invitrogen, CA) ( n = 4). RNA was isolated using an RNeazy mini kit according to the directions of the manufacturer (Qiagen, Germany). The first-strand synthesis of single-strand cDNA from RNA for use as a PCR template was carried out with a SuperScript First-Strand cDNA Synthesis Kit (Invitrogen). cDNA was preamplified before the real-time RT-PCR by using pooled 0.2 × TaqMan Gene expression Mixes and CellDirect™ One-Step qRT-PCR Kits (Invitrogen). The thermal cycling conditions for STA were 12 cycles of 95 °C for 15 s and 60 °C for 4 min. Preamplified cDNA was diluted with TE buffer (1:5) and was used for real-time RT-PCR. The PCR profile for Dynamic Array included a 10 min, 95 °C hot-start to activate the Taq polymerase, followed by 40 cycles of a two-step program: 15 s at 95 °C (denaturation) and 60 s at 60 °C (annealing and extension). Data were analyzed using BioMark™ Real-Time RCR Analysis Software v2.0 (Fluidigm). The results were normalized by dividing the amount of the target gene by the amount of Gapdh . The following TaqMan gene expression assay mixes were used in this study; Opn : Mm01204014_ml, Oc : Mm03413826_mH, Gapdh : Mm99999915_gl (Applied Biosystems, CA).
2.5
In vivo bone formation
Fibrin gel containing bone marrow derived stromal cells (BMSCs, 1 × 10 6 cells/ml) was injected into ZrO 2 /HAp scaffolds to obtain the cell-encapsulated scaffolds. Eight-week-old male SD rats (320–360 g) were used in this study (supplementary figure 1). All animal experiments were performed in accordance with Osaka University Animal Care and Use Committee guidelines (Approved number: 20-017-0).
