Collagen-grafted porous HDPE/PEAA scaffolds for bone reconstruction
© The Author(s). 2016
Received: 29 April 2016
Accepted: 19 July 2016
Published: 27 July 2016
After tumor resection, bone reconstruction such as skull base reconstruction using interconnected porous structure is absolutely necessary. In this study, porous scaffolds for bone reconstruction were prepared using heat-pressing and salt-leaching methods. High-density polyethylene (HDPE) and poly(ethylene-co-acrylic acid) (PEAA) were chosen as the polymer composites for producing a porous scaffold of high mechanical strength and having high reactivity with biomaterials such as collagen, respectively. The porous structure was observed through surface images, and its intrusion volume and porosity were measured. Owing to the carboxylic acids on PEAA, collagen was successfully grafted onto the porous HDPE/PEAA scaffold, which was confirmed by FT-IR spectroscopy and electron spectroscopy for chemical analysis. Osteoblasts were cultured on the collagen-grafted porous scaffold, and their adhesion, proliferation, and differentiation were investigated. The high viability and growth of the osteoblasts suggest that the collagen-grafted porous HDPE/PEAA is a promising scaffold material for bone generation.
For bone reconstruction such as skull base reconstruction after tumor resection, an interconnected porous structure is critical to mimicking the bone extracellular matrix [1–8]. The pore size, porosity, and pore interconnectivity of porous bone scaffolds determine their performance in functions such as cell attachment and nutrient diffusion, which enhances soft tissue and bone ingrowth and eventually resistance to infection or deformation. Moreover, mechanical stability is mandatory for the mechanical support that is required during the repair and regeneration of damaged or degenerated bone [7, 9]. Porous scaffolds for biomedical applications have been successfully fabricated via the sol-gel process , salt-leaching method [8, 11–13], electrospinning [14–17], and microsphere-sintering technique [18, 19]. However, the lack of mechanical strength of the porous materials can cause instability of the pore structures and hence limit their biomedical applications, and thus the choice of scaffold material is crucial.
The performance of porous scaffolds can be optimized by controlling their surface chemistry, because the interface between the porous scaffolds and cells determines the cellular behavior, such as cell adhesion, spreading, and proliferation . Collagen is the main organic component of bones, and is hence a promising candidate material for the surface modification of porous scaffolds by promoting cell attachment and chemotactic responses .
High-density polyethylene (HDPE) shows excellent mechanical properties, and it has been widely used as an implant material for bone reconstruction [18, 21, 22]. Medpor® (Porex Technologies Co., USA) is one such porous HDPE scaffold for bone tissue engineering, used as an alloplastic material for craniofacial reconstruction [23, 24]. However, HDPE is inert and hydrophobic, and exhibits poor reactivity with biomaterials such as collagen. Several efforts have been made to improve the reactivity of PE for biomedical applications. The grafting of acrylic acid onto the PE film was conducted to improve protein immobilization and cell seeding . It was also reported that plasma treatment effectively provides HDPE with a hydrophilic surface, which results in better reactivity with bioactive molecules . The carboxylic acid groups of poly(ethylene-co-acrylic acid) (PEAA) make it an outstanding candidate to support the reactivity with collagen. Besides this, PEAA is mechanically stable, owing to the strong hydrogen bonds in its carboxylic acid groups, which can be effective crosslinkers between polymer chains.
In this study, the composite of HDPE and PEAA was chosen as scaffold material for cranial reconstruction owing to the high mechanical stability of HDPE and the high reactivity of PEAA with collagen. Before collagen grafting, the porous structure was prepared using a salt-leaching method, which can provide the proper pore size and high porosity. Osteoblast cells were then cultured on the collagen-grafted porous HDPE/PEAA scaffold, and the cell adhesion, proliferation, and differentiation were measured to investigate their bone tissue compatibility. Porous scaffolds of HDPE and HDPE/PEAA without collagen grafting were also fabricated and studied as controls.
Fabrication of collagen-grafted porous scaffolds
Porous HDPE/PEAA scaffolds were fabricated by using a salt-leaching method10. HDPE (Mw 85,000, Mn 13,500; Korea Petrochemical Industrial Co., Korea) and PEAA (acrylic acid 20 wt%; Sigma-Aldrich Co., USA) beads (w/w = 3:1) were mixed with sodium chloride (HDPE/PEAA:NaCl = 1:9) with a particle size of 200–500 μm, using a melt mixing machine (Brabender, Plasti-Corder Co.) at 160 °C. Then, the mixture was cast in a circular mold (diameter 13 mm, thickness 1.3 mm) using a heat press machine (Yoochang Co., Korea). The resulting HDPE/PEAA/NaCl composite was immersed in distilled water to leach out the NaCl, leaving pores in the composite. The salt-free porous HDPE/PEAA was washed with distilled water and air dried.
For obtaining high reactivity between the scaffold and collagen, L-lysine was grafted onto the scaffold surface to improve the affinity of the carboxyl groups to the amine groups in collagen. Before the L-lysine grafting, the carboxylic groups on the HDPE/PEAA scaffold were activated by immersing the scaffold into a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (0.25 wt%; Sigma-Aldrich Co., USA) and N-hydroxysuccinimide (0.25 wt%; Sigma-Aldrich Co., USA) aqueous solution for 6 h at room temperature. Afterwards, it was immersed in 3 wt% L-lysine aqueous solution with gentle stirring. The carboxyl groups of L-lysine, attached to the scaffold surface, were also activated by this same method. Collagen-grafted HDPE/PEAA (HDPE/PEAA/Col) was produced by immersing the HDPE/PEAA scaffold in 3 wt% collagen solution (in distilled water containing acetic acid, pH 4.3) for 6 h with gentle stirring, and then it was washed with distilled water and dried.
Characterization of the scaffolds
The surface morphology of the porous HDPE, HDPE/PEAA, and HDPE/PEAA/Col scaffolds was observed under a field emission scanning electron microscope (FE-SEM S4300; Hitachi, Japan) after sputter-coating with platinum. The chemical bonds and elemental composition were characterized by Fourier transform infrared (FT-IR; Mattson, Galaxy 7020A) spectroscopy and electron spectroscopy for chemical analysis (ESCA; ESCA LAB VIG microtech, Mt 500/1, and so forth, East Grinstead, UK), respectively.
Tensile properties were measured via a universal testing machine (Instron, model 4465) with a Zwick Roell tensile tester equipped with a 1 kgf load cell, at 25 °C with an extension speed of 10 mm/min. The tensile strength and Young’s modulus measure of each sample were calculated from the averages of 10 specimens.
The porosity of the porous scaffolds was determined by using a mercury intrusion porosimeter (AutoPore IV 9520; Micromeritics Co., USA). The advancing and retreating contact angles of mercury were taken to be 140° and the surface tension was taken as 0.480 N/m (480 dynes/cm).
Cell behavior was observed by culturing osteoblast cells (5 × 104 cells/mL; MC3T3-E1, ATCC) on the scaffolds, at 37 °C in a humidified atmosphere with 5 % CO2, in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10 % fetal bovine serum (Gibco, USA) and 1 % penicillin G-streptomycin (Gibco, USA). After both 1 and 2 days of incubation, calcein-AM (1 mM in dimethyl sulfoxide) and propidium iodide (1.5 mM in distilled water) solutions were added and the scaffolds were left standing for 15 min. The fluorescence images were visualized with a confocal laser scanning microscope (CLSM, Carl Zeiss, LSM 700, Germany).
To evaluate the cytoskeletal organization of cells on the porous scaffolds, double staining was performed. After 3 days of incubating the cell solution with the scaffold samples, the cells were fixed with 4 % paraformaldehyde in PBS and permeabilized with 0.1 % Triton X-100 in PBS for 15 min. The samples were then incubated for 30 min in a PBS containing 1 % bovine serum albumin, followed by the addition of tetramethylrhodamine-5-isothiocyanate (TRITC)-conjugated phalloidin (Millipore, Cat. No. 90228). After 1 h, the samples were incubated with 4,6-diamidino-2-phenylindole (DAPI) (Millipore, Cat. No. 90229) for 5 min. The fluorescence images were taken with a confocal laser scanning microscope (CLSM 700).
The cell viability and proliferation on the porous scaffolds were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and enzyme-linked immunosorbent assay (ELISA). For the MTT assay, the scaffold samples were immersed in 50 μL of MTT solution (5 mg/mL in PBS) for 4 h. After removing the solution, the water-insoluble formazan product was dissolved in 0.04 N HCl-isopropanol in the dark. ELISA was performed using 5-bromo-2-deoxyuridine (BrdU), which is incorporated during DNA synthesis in the cells. The BrdU ELISA was conducted according to the manufacturer’s instructions (Roche Molecular Biochemicals, Germany). The absorbance was measured at 570 nm, using a kinetic microplate reader (EL × 800; Bio-T Instruments, Inc., Highland Park, USA).
Cell differentiation was tested by several cell staining methods, using alizarin red S, von Kossa, and alkaline phosphatase (ALP) staining. The osteoblast cells (5 × 104 cells/mL) were cultured for 15 days on the three porous scaffolds and then fixed using 10 % formaldehyde. For alizarin red S staining, the samples were treated with an alizarin red S solution and incubated for 20 min. For the von Kossa assay, the fixed samples were treated with 5 % AgNO3 solution for 20 min under ultraviolet radiation, followed by the addition of 5 % Na2S2O3 solution for 5 min. ALP staining was done by a standard procedure according to the manufacturer’s instructions (Alkaline phosphatase, Leukocyte, Procedure No. 86; Sigma-Aldrich, USA), using an alkaline dye mixture (1 mL of sodium nitrate, 1 mL of FBB-alkaline solution, 1 mL of naphthol AS-BI alkaline solution, and 1 mL of deionized water) and a neutral red buffered solution for counterstaining . The digital images of the stained cultures were obtained with a digital camera (Canon A2000 IS, Japan) and an optical microscope (Carl Zeiss, Germany).
The results are displayed as the mean ± standard deviation. The statistical significance of differences between the scaffolds was determined by a Student’s two-tailed t test. Scheffe’s method was used for multiple comparison tests at a level of 95 %.
Results and discussion
Intrusion volume and porosity of the porous HDPE, HDPE/PEAA and HDPE/PEAA/Col scaffolds
Intrusion volume (mL/g)
The pore characteristics are also key factors that affect the performance of porous scaffolds in bone reconstruction because the pore size and porosity of scaffolds affect the diffusion of nutrients and osteoblast cell attachment, migration, proliferation, and differentiation, which are vital for bone formation. Additionally, a porous surface is known to drive mechanical stability at the interface between the implant materials and the surrounding tissue . Even though there is disagreement about the optimum pore size of porous scaffolds, it is generally agreed upon that the pore size and porosity play essential roles in their compatibility to cells such as osteoblasts, and pores of a few hundred microns are highly required [3–5, 8]. Therefore, on the basis of the results of Fig. 1 and Table 1, it can be concluded that the pore size of the HDPE-based scaffolds prepared by the salt-leaching method is appropriate for porous bone scaffolds.
Chemical composition of porous scaffolds calculated from their survey scan spectra
C 1 s
O 1 s
N 1 s
Na 1 s
Cell viability and proliferation
For bone reconstruction, porous scaffolds were fabricated using HDPE/PEAA composites via a salt-leaching method. The surface of the porous HDPE/PEAA scaffold was modified using collagen to enhance bone tissue compatibility. The surface modification was confirmed via FT-IR spectroscopy and ESCA by detecting the nitrogen component in collagen. It was shown that the pore size and porosity are suitable for osteoblast attachment, as confirmed by the surface images and porosity results. The cell viability and proliferation were measured by MTT and BrdU assays, with results showing that the collagen-grafted HDPE/PEAA surface is favorable for the adhesion and proliferation of osteoblast cells. Furthermore, cell differentiation was studied using several staining methods, where it was seen that osteoblasts on the collagen-grafted scaffold have outstanding differentiation. It is concluded that collagen grafting on the porous HDPE/PEAA scaffold effectively improves its biocompatibility and potential use as a bone scaffold.
This research was supported by Kyungpook National University Research Fund for the year 2013.
This research was supported by Kyungpook National University Research Fund for the year 2013.
Availability of data and material
All data are available on Journal portals in submitted manuscript. No other supporting files/data are needed along with this submission.
CSK and IKK designed the experiments. KHJ helped in writing the manuscript. HK and CBK have conceived the ideas of this study, and participated in its design. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The manuscript has been submitted with the consent of all authors and data of any other person not included.
Ethics approval and consent to participate
Manuscript does not include human ethics value. Hence no consent is needed.
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