Article pubs.acs.org/journal/abseba
Fabrication and Osteogenesis of a Porous Nanohydroxyapatite/ Polyamide Scaffold with an Anisotropic Architecture Fu You,†,§ Yubao Li,† Qin Zou,† Yi Zuo,† Minpeng Lu,‡ Xiongbiao Chen,§ and Jidong Li*,† †
Research Center for Nano Biomaterials, Analytical & Testing Center, Sichuan University, No. 24 South Section 1, Yihuan Road, Chengdu 610064, P. R. China ‡ Department of Orthopaedic Surgery, Yongchuan Hospital, Chongqing Medical University, 439 Xuanhua Road, Chongqing 402160, P. R. China § Division of Biomedical Engineering, College of Engineering, University of Saskatchewan, 57 Campus Drive, Saskatoon, Saskatchewan S7N5A9, Canada ABSTRACT: Scaffolds are used in bone tissue engineering to provide a temporary structural template for cell seeding and extracellular matrix formation. However, tissue formation on scaffold outer edges after implantation due to insufficient interconnectivity may restrict cell infiltration and mass transfer to/from the scaffold center, leading to bone regeneration failure. To address this problem, we prepared nanohydroxyapatite/polyamide66 (n-HA/PA66) anisotropic scaffolds with axially aligned channels (300 μm) with the aim to enhance pore interconnectivity and subsequent cell and tissue infiltration throughout the scaffold. Anisotropic scaffolds with axially aligned channels had better mechanical properties and a higher porosity (86.37%) than isotropic scaffolds produced by thermally induced phase separation (TIPS). The channels in the anisotropic scaffolds provided cells with passageways to the scaffold center and thus facilitated cell attachment and proliferation inside the scaffolds. In vivo studies showed that the anisotropic scaffolds could better facilitate new bone ingrowth into the inner pores of the scaffold compared to the isotropic scaffolds. The anisotropic scaffolds also had improved vascular invasion into their inner parts, increasing the supply of oxygen and nutrients to the cells and thus facilitating revascularization and bone ingrowth. Enhanced cell and tissue penetration to the scaffold center was observed in the anisotropic scaffolds both in vitro and in vivo, indicating the axially aligned channels positively influenced cell and tissue infiltration. Thus, such scaffolds have great potential for applications in bone tissue engineering. KEYWORDS: bone, tissue engineering, anisotropic scaffolds, axially aligned channels designing scaffolds with a superior internal structure.5 The third criterion relates to two important characteristics of scaffolding materials: porosity6 and interconnectivity.7 Higher porosity enhances cell recruitment and vascularization, leading to improved osteogenesis.6,8 Pore interconnectivity affects the biological response of a biomaterial by influencing the diffusion of nutrients to and metabolites from the tissue-engineered constructs, uniform cell seeding and migration, and subsequent tissue ingrowth.9 Cellular adhesion and proliferation are affected by surface properties. Chemical surface properties concern the issue of cell adhesion to the material and the protein interactions with the latter. Topographical surface properties are related with the osteoconductivity,10 which is a process of osteogenic cells migrating to scaffold surface after scaffold implantation. Thus, selecting appropriate materials to produce scaffolds for bone
1. INTRODUCTION Requirements for the design and fabrication of scaffolds for bone tissue engineering are manyfold and challenging. Several specific criteria must be taken into consideration when designing bone tissue engineering scaffolds. They must be able to (1) define a 3D space that shapes the regenerating bone tissue; (2) provide temporary mechanical support in the defect as bone regenerates; (3) be highly porous with an interconnected pore network that facilitates bone ingrowth as well as the ingress of nutrients and the removal of metabolic waste; (4) exhibit excellent surface properties promoting cell adhesion, differentiation, and proliferation; (5) possess noncytotoxicity and osteoconductivity.1−3 Regenerating tissue to exactly match the original tissue shape can be achieved by medical imaging technologies in combination with computer-aided design (CAD) and computer-aided manufacturing (CAM), which allows for the production of scaffolds with irregular and complex geometries.4 Temporary mechanical support in the defect can be provided by using advanced biomaterials with excellent mechanical properties or © 2015 American Chemical Society
Received: May 11, 2015 Accepted: August 13, 2015 Published: August 13, 2015 825
DOI: 10.1021/acsbiomaterials.5b00199 ACS Biomater. Sci. Eng. 2015, 1, 825−833
Article
ACS Biomaterials Science & Engineering
slurry was prepared using the coprecipitation method in ethanol.5 Briefly, PA66 (Basf Co., Germany) with a viscosity-average molecular weight of 18 kDa was completely dissolved into ethanol solution at 70 °C for 4 h. Then, the nanohydroxyapatite (n-HA) slurry was gradually added into the PA66 solution with vigorously stirring at 70 °C for 4 h to obtain a homogeneous slurry system with 40 wt % n-HA. The viscosity of the slurry was carefully adjusted to a desirable state (apparent viscosity: 330 Pa·s, tested at room temperature, with rotor speed of 0.6 r/min, NDJ-8S digital viscometer, Shanghai, China) by evaporating ethanol solvent. 2.2. Scaffold Fabrication. Stainless steel wires of 300 μm diameter were coated with the n-HA/PA66 slurry. The wires were cut into lengths of 100 mm, with 40 wires then bundled in heat shrink tubing. These bundles were heated in an oven at 80 °C for 25 min to activate the heat shrink tubing and fuse the slurry-coated wires together through a melt pressing process. The oven temperature was then increased to 95 °C for 4 h to induce the evaporation of the ethanol and solidify the porous structure. The tubing and wires were then carefully removed to reveal the axially aligned channels, which were later cut as required into appropriate lengths for use of anisotropic scaffolds. Porous n-HA/PA66 isotropic scaffolds produced by the TIPS technique were used as a control. Briefly, the n-HA/PA66 slurry was cast into a glass container and heated in an oven at 95 °C until the solvent ethanol evaporated completely and a solidified porous structure was obtained. All samples were washed in deionized water. 2.3. Scaffold Characterization. The microarchitecture of the isotropic and anisotropic scaffolds was observed by scanning electron microscopy (SEM; Jeol 6500LV, Japan) at 20 kV. The anisotropic scaffolds were carefully sectioned with a razor blade in directions parallel and vertical to the oriented channels and mounted onto aluminum stubs. Each sample was coated with gold before examination. Porosity was evaluated by the method described by Zhang and Ma.32 Briefly, a scaffold with a known volume (V) and weight (M) was immersed in ethanol and ultrasonically vibrated to expel air from the pores, allowing them to be filled with ethanol. The porosity (p) of the scaffold was then expressed as p = (M1 − M)/(dV)100%, where M1 is the weight of the ethanol impregnated scaffold and d is the density of ethanol (0.79 g/cm3). Five specimens of each scaffold type (anisotropic scaffolds and isotropic scaffolds) were tested. The compressive strength and modulus of the scaffolds were measured using a mechanical testing machine (AGIC 50 KN, SHIMADZU, Japan). Specimens were prepared with diameters of 4 mm and lengths of 15 mm. Testing was conducted at room temperature and 65% relative humidity (RH). The crosshead speed was set at 0.5 mm/min and the load was applied until the specimen was compressed to 60% of its original length. To investigate the effect of anisotropic architecture on the mechanical behavior of the scaffolds, we applied the load along the direction parallel to the axially aligned channels. The compressive modulus was calculated as the slope of the initial linear portion of the stress−strain curve, and the compressive strengths were determined from the cross point of tangents on the stress−strain curve around the yield point. Five samples from each scaffold type (anisotropic scaffolds and isotropic scaffolds) were tested. 2.4. Cell Infiltration Studies. Cell infiltration into both isotropic and anisotropic n-HA/PA66 scaffolds was evaluated by culturing with MG63 cells. MG63 cells are derived from human osteosarcoma and express the characteristic features of osteoblasts. MG63 cells were cultured in F12 medium (cell-culture grade, Biowhittaker, Walkersville, MD) supplemented with 10% volume fraction of calf serum (cell-culture grade, Gibco, Rockville, MD), 1% penicillin/streptomycin, and 1% Lglutamine. Prior to seeding, both anisotropic and isotropic scaffolds were cut into discs of 4 mm (diameter) × 5 mm (height), autoclavesterilized and immersed in the culture medium for 24 h. MG63 cells were seeded onto the top of prewetted scaffolds (2 × 104 cells per well). The seeded scaffolds were then cultured in a humidified incubator (37 °C, 5% CO2) for 11 days with the medium changed every 2 days. Cell viability and infiltration were assessed using the Live/Dead viability/ cytotoxicity assay (Molecular Probes, USA), and cells were observed and imaged using fluorescence microscope (TE2000-U, Nikon, Japan). Samples of both groups were carefully sectioned with a razor blade along
tissue engineering is of vital importance since the materials properties will determine the properties of the scaffold to a large extent. A variety of processing technologies have been developed to prepare porous 3D scaffolds for bone regeneration, including solvent casting and particle leaching,11,12 gas foaming,13 thermally induced phase separation (TIPS),5 and freezedrying.14 However, scaffolds prepared by these conventional techniques share one disadvantage-limited interconnectivity, which has been shown to limit cell infiltration and new tissue formation to the outer edge or superficial pore layer, leading to a necrotic core.7,15 Emerging rapid prototyping (RP) techniques16,17 enable researchers to design and produce scaffolds with 100% interconnected pores, high porosities, and precisely controlled architecture. However, the general low resolution of RP techniques only allows the fabrication of scaffolds with pore sizes larger than the dimensions of a cell, which is not optimal for cell attachment, proliferation, and migration.18 This may result in slow tissue formation,19 nonuniform tissue,20 and different cell differentiation behaviors.21 Thus, conventional scaffold fabrication techniques are still indispensable as they can produce scaffolds with pore sizes comparable to cell dimensions, thus facilitating cell homing within the scaffolds. Therefore, a simple process based on a conventional TIPS technique was utilized in this study to produce scaffolds with axially aligned channels as well as a microporous structure with the aim to improve both porosity and interconnectivity. Cortical bone exhibits a honeycomb-like structure with axially aligned pores, with diameters in the micrometer range, filled with blood capillaries (Haversian canals, located in the center of the osteons).22 This type of structure contributes to the outstanding longitudinal mechanical properties of bone tissue and efficient mass transport within the bone. Thus, we hypothesize that scaffolds with axially aligned channels can better mimic the osteon structure of bone. Moreover, axially aligned channels within scaffolds build an anisotropic architecture, which would improve interconnectivity of the scaffold23,24 and help the infiltration of cells and tissue into the scaffolds.25 Previous studies from our laboratory demonstrated the biocompatibility and osteogenesis of biomimetic nanohydroxyapatite (n-HA)/polyamide66 (PA66) composite scaffolds for bone tissue engineering, in which hydroxyapatite and polyamide simulate the mineral crystals and the collagen matrix of natural bone.5,26,27 The n-HA/ PA66 composite has been applied in clinic.28,29 Therefore, this study continued to use n-HA/PA66 as composite scaffolding materials. Moreover, previous work is extended in this paper by incorporating axially aligned channels within the porous n-HA/ PA scaffolds and assessing performance both in vitro and in vivo. Notably, increased porosity and interconnectivity is usually accompanied by decreased mechanical properties; however, we report here an increase of porosity and interconnectivity without such a sacrifice.
2. MATERIALS AND METHODS 2.1. Preparation of n-HA/PA66 Slurry. All chemical reagents used in this study were analytical reagent (AR) grade. The n-HA slurry was prepared by our group.30 Ca(NO3)2·4H2O and (NH4)3PO4·3H2O were separately dissolved in aqueous solution. Ca(NO3)2·4H2O solution was added drop by drop into the (NH4)3PO4·3H2O solution with stirring and heating at 70 °C. The pH value was kept above 10 by ammonium hydroxide. The apatite precipitation was treated at 100 °C under normal atmospheric pressure for 3 h. The apatite precipitation turned to needlelike n-HA crystals after treatment.31 The obtained n-HA slurry was then washed to neutrality by deionized water. The n-HA/PA66 826
DOI: 10.1021/acsbiomaterials.5b00199 ACS Biomater. Sci. Eng. 2015, 1, 825−833
Article
ACS Biomaterials Science & Engineering
Figure 1. Surgery process: (a) radius of New Zealand rabbit, (b) bone defect model of rabbit radius, and (c) repair of the defect of the rabbit radius with implant. the direction parallel to the axially oriented channels for anisotropic scaffolds or along the central axis for isotropic scaffolds after Live/Dead staining, fluorescent photos were taken to observe the cell infiltration into the inner pores of two structural scaffolds. Cell proliferation on scaffolds were evaluated by MTT assay. The absorbance at 570 nm was measured with a microplate reader (PerkinElmer, USA) after 1, 4, 7, and 11 day (s). For visualization of the growth and distribution of the MG63 cells within the scaffolds, cells-seeded scaffolds were rinsed using phosphate buffered saline (PBS), fixed with 3% glutaraldehyde, dehydrated in graded ethanol concentrations (25, 50, 75, 90, 95, and 100% v/v in distilled H2O), rinsed with isoamyl acetate, then dried in supercritical CO2. Thereafter, the seeded scaffolds of both groups were carefully sectioned with a razor blade along the direction of the central axis of the cylindrical scaffolds. SEM was used to evaluate cell infiltration. 2.5. Tissue Infiltration Studies. Twenty-three adolescent New Zealand white rabbits from both sexes and each weighing 2.0−2.5 kg were used for in vivo studies. Surgery was carried out under sterile conditions, starting with anesthetizing the rabbits with an intraperitoneal injection of pentobarbital sodium. The forelimb was clipped and scrubbed with povidone-iodine and a 75% ethanol solution. A longitudinal incision was made over the radius and the underlying muscles retracted, exposing the mid-diaphysis of the radius. Critical 15 mm segmental bone defects were created in the radius using a burr drill under irrigation with sterile saline. All bone debris and interosseous membrane at the defect site were washed away with physiological saline. Scaffolds were autoclave-sterilized prior to implantation. The anisotropic or isotropic n-HA/PA66 scaffold was fitted into the defect of right or left radius of a rabbit (Figure 1). For the control group, the same defects were created without any treatment. Normal bone segments removed were retained for comparative testing as normal group. Finally, muscles and skin were sutured layer by layer without fixation on the operated limbs. All rabbits were housed and fed separately to reduce the risk of infection. The study was approved by the Animal Care and Use Committee of Sichuan University, and all operation procedures and animal care were performed with in compliance with the Guide for the Care and Use of Laboratory Animals. Rabbits were sacrificed at 2, 4, and 12 weeks after implantation. The scaffolds together with the surrounding tissue were excised (Figure 2). The harvested specimens were placed in 4% paraformaldehyde, embedded in paraffin after decalcification, sectioned parallel to the diaphysis at a thickness of 5 μm, examined after hematoxylin-eosin (HE)
staining, and observed using a light microscope (Nikon TE2000-U, Japan). 2.5.1. Quantification of Newly Formed Bone. To quantitatively determine the amount of newly formed bone in the inner pores or channels of the two scaffold types, histological sections from 4 and 12 weeks postimplantation were statistically analyzed. Three histological sections were chosen from both the anisotropic and isotropic n-HA/PA groups. After HE staining, each section was observed under light microscope with 100× magnification, and at least 10 images were obtained from the inner parts of the implanted scaffolds, especially from the center of the scaffolds. Using Image-ProPlus analytical software (Media Cybernetics, USA), new bone volume (NBV) was expressed as the percentage of newly formed bone area in the available pore space (bone area/pore area ×100%). 2.5.2. Immunohistochemistry. Histological sections were stained using antibodies for BMP-2 or CD31 (Abcam, England).33,34 Briefly, sections were first stained with antimouse BMP-2 (ab6285, Abcam, England) or antirabbit CD31 (ab28364, Abcam, England) primary antibody (Concentrations BMP-2:1:100; CD31:1:50), and then stained with labeled streptavidin−biotin reagents (EnvisionTM Detection Kit, Peroxidase/DAB, rabbit/mouse; Dako). 2.5.3. Biomechanical Testing. To estimate the mechanical stability of the regenerated bones, the radius and ulna excised at 12 weeks (including the control and normal groups) were wrapped in gauze, hydrated with PBS, and preserved (frozen) until mechanical testing. Specimens were subjected to a three-point bending test using a mechanical testing machine (AGIC 50KN, SHIMADZU, Japan) at room temperature and 65% RH. Briefly, the specimens were positioned on two supports spaced 8 mm apart, and the bending load was applied at the midpoint of the defect at a constant displacement rate of 5 mm/min until breakage. The data generated were automatically recorded (n = 5 parallel samples/group). 2.6. Statistics. For quantitative analysis, Student’s t tests were used to assess differences between two groups and multiple comparisons were performed via one-way ANOVA test using SPSS (v. 11.0). P values 60%) than 829
DOI: 10.1021/acsbiomaterials.5b00199 ACS Biomater. Sci. Eng. 2015, 1, 825−833
Article
ACS Biomaterials Science & Engineering
the anisotropic scaffolds (Figure 11a). In contrast, positive staining for BMP-2 was only observed at the edges of the
Figure 9. Hematoxylin-eosin (HE) stained sections of bone ingrowth (inside of scaffolds) for (a, c) anisotropic n-HA/PA scaffolds and (b, d) isotropic n-HA/PA scaffolds harvested at (a, b) 4 and (c, d) 12 weeks postimplantation. Scale bars represent 100 μm. OB denotes the host bone tissue, NB denotes newly formed bone tissue, CF denotes the collagen fibers and S denotes scaffold materials.
Figure 11. Immumohistochemical staining of BMP-2 for (a, c) anisotropic n-HA/PA scaffolds and (b, d) isotropic n-HA/PA scaffolds harvested at (a, b) 2 and (c, d) 12 weeks postimplantation. Scale bars represent 100 μm. S denotes the scaffold materials and brown indicates positive staining for BMP-2.
isotropic scaffolds (Figure 11b). At 12 weeks postsurgery, large amounts of newly formed bone tissue grew into the anisotropic scaffolds, and were surrounded by BMP-2 expression areas (Figure 11c). BMP-2 was also expressed in the isotropic scaffolds (Figure 11d), but this was generally weaker than for the anisotropic group. The platelet endothelial cell adhesion molecule-1 (CD31, an angiogenesis marker), triggers blood vessel growth and indicates the presence of blood vessels. The tissue sections were stained for CD31 to evaluate the degree of angiogenesis in the bone defect regenerated by anisotropic or isotropic scaffolds (Figure 12). At 2
Figure 10. Quantification of newly formed bone at the bone defect area repaired using isotropic and anisotropic n-HA/PA scaffolds at 4 and 12 weeks postimplantation. New bone volume (NBV) is expressed as bone area/pore area ×100%. * showed the statistical difference between 4 weeks and 12 weeks in the isotropic group. Δ showed statistical difference between 4 weeks and 12 weeks in the anisotropic group. ◇ showed the statistical differences between isotropic group and anisotropic group for 4 weeks and 12 weeks. Error bars represent mean ± SD for n = 6.
Figure 12. Immumohistochemical staining against CD31 antibody in (a) anisotropic n-HA/PA scaffolds and (b) isotropic n-HA/PA scaffolds harvested at 12 weeks postimplantation. Scale bars represent 100 μm. S denotes the scaffold materials and asterisks denote newly formed blood vessel in the pores of scaffolds.
in the isotropic scaffolds (
