Fabrication of nano-hydroxyapatite/chitosan membrane with asymmetric structure and its applications in guided bone regeneration

Abstract

The development of guided bone regeneration (GBR) technique brings a promising alternative for bone defects and fracture healing. In this study, an asymmetric nano-hydroxyapatite/chitosan (n-HA/CS) composite GBR membrane was fabricated by means of solution-blending and solvent-evaporating in vacuum. The membranes were characterized using SEM, XPS and contact angle. It was found that the composite membrane displayed an asymmetric structure, in which the upper surface was CS and the under surface was a complex of n-HA and CS, and some interactions between n-HA and CS were also confirmed to exist. The contact angle testing showed that the under surface was more hydrophilic than the upper surface. The in vivo experiments demonstrated that the asymmetric composite membrane had the ability to make osteoblasts mineralize and promote loose bone calcified, and then accelerate the bone regeneration. Compared with CS membrane, the asymmetric composite membrane displays a better bone regeneration ability and is suitable for GBR membrane.

Keywords

GBR technique nano-hydroxyapatite chitosan degradation asymmetric membrane

1. Introduction

Bone nonunion and bone defects are common clinical problems that are difficult to deal with. In clinic, bone grafting is considered to be the basic way to solve these problems, but a follow-up study showed that fibroblasts could migrate to the defect or the fracture part rapidly and formed a fibrous capsule, which could prevent blood vessels and osteoblasts from growing into the bone graft and ultimately led to bone nonunion [1].

Recently, the development of guided bone regeneration (GBR) technique brings hope to solve the above-mentioned problems. GBR technique is effective in halting bone destruction and promoting new bone formation, and is considered an important option for bone regeneration [2–4]. The basic principle of GBR technique is to create and maintain a secluded space by using a barrier membrane to prevent the invasion of fast growing fibrous tissue and other soft tissues from migrating into the bone defects, thereby allowing time for osteogenic cell populations originating from the host bone to inhabit the bone defect [5]. Membranes used in GBR technique must be biocompatible, have the proper degradation profile and should be equipped with satisfactory mechanical properties to accomplish the barrier functions, space maintenance and clinical manageability [6]. Consequently, completing the bone regeneration in the defect sites and achieving a better bone repair ability depend on the improvement and update of GBR membrane materials.

Currently, two sorts of GBR membranes are available: non-degradable and degradable membranes [7]. Although the non-degradable membranes have better space-maintaining properties than the degradable membranes, they still have two main disadvantages: one is the need for a second surgery to be removed off, which often creates additional surgical trauma to patients and raises their treatment costs; another is the increased risk of infection, which might lead to the necessity of early removal [4]. Biodegradable membranes can avoid secondary operation, reduce adverse reactions and sustain continuous neo-bone formation, thus draw more attention and be widely used in bone regeneration [8–10]. For instance, collagen has been wildly used in guiding bone tissue regeneration due to its excellent biodegradability, biocompatibility and cell affinity. Nevertheless, the poor mechanical performance and the mismatched degradation rate with the bone healing speed as well as the high cost limit its application widely [11]. Although many fabricating methods have been tried to improve the performance of degradable membranes, its mechanical strength and the ability to guide bone regeneration are still hard to meet the requirements of GBR technology up to now [12–14].

Chitosan (CS), a cationic natural biopolymer obtained from the deacetylation of chitin, has been extensively used for bone and skin tissue regeneration due to its excellent biocompatibility, non-toxicity, antibacterial ability, biodegradability and hemostatic properties. Several CS-based membranes have been reported to be used in GBR technique tentatively, but their barrier effects are not so satisfactory [10,15,16]. Nano-hydroxyapatite (n-HA), the main mineral component of bone and teeth, has been widely used in the field of hard tissue repair and regeneration because of its superior biocompatibility, high bioactivity and osteoconductivity [17–19]. We know that n-HA/CS composite membranes have also been investigated for hard tissue regeneration, but these membranes were often fabricated into uniform structure [20–22]. However, it is a pity that such a structure can guide both bone and fibrous tissue regeneration simultaneously. It is well known that fibrous tissues grow faster than bone tissue and always occupy the defect sites prior to bone tissue, thus restrain the bone regeneration [19]. Based on this, n-HA/CS composite membrane with an asymmetric structure was prepared in this study by solution-blending and solvent-volatilizing method in vacuum. The physic-chemical properties were investigated and the in vivo biological performances were also examined and analyzed comprehensively.

2. Materials and methods

2.1. Materials

Chitosan with more than 95% degree of deacetylation procured from Jinan Haidebei Marine Bioengineering Co. Ltd (Jinan, China) ( $Mw = 250000$ ); Nano-hydroxyapatite (n-HA) slurry was synthesized in our lab; Acetic acid, NaOH and ammonia were obtained from Chengdu jinshan chemical reagent Co. Ltd (Chengdu, China). All chemicals were analytical grade.

2.2. Methods

2.2.1. Preparation of asymmetric n-HA/CS membranes

According to the reference [23] we synthesized n-HA slurry at atmospheric pressure, then adjusted n-HA slurry to neutral. 4 wt% chitosan was prepared by dissolving 4 g chitosan powder in acetic acid aqueous (2 wt% in water). Next, the chitosan solution was slowly added into n-HA slurry, and the mass ratio of n-HA and CS was 4:6, then adjusted pH to 5 with aqueous ammonia and stirred for 5 h. Subsequently, the mixture was slowly poured onto a glass plate and dried at 60°C in vacuum oven. After being completely dried, the film was detached by immersing into 1 mol/L NaOH solution and then washed with distilled water to neutral, dried at room temperature. Finally, n-HA/CS membrane with asymmetric structure was obtained, in which the upper surface was mainly composed of CS and the under surface was made up of n-HA/CS complex. Besides, pure CS membrane was also fabricated using the same method to be used as the control in the following experiments.

2.2.2. Characterization of asymmetric n-HA/CS membrane

Both the pure CS and the asymmetric membrane were photographed digitally, and the cross-section of the n-HA/CS membrane was observed by scanning electron microscope (SEM, JEOL, JEM-100CX, Japan) after being sputter-coated with gold using Ion Sputter SBC-12. X-ray photoelectron spectroscopy (XPS, XSAM, 800SERIES-800SINS) was performed in order to determine the difference of components on the upper surface (non-attaching-glass surface) and the under surface (attaching-glass surface) of the asymmetric membrane. The wettability of the pure CS and the asymmetric membrane was determined by a contact angle testing meter (Shanghai Zhongchen Digital Technic Apparatus Co. Ltd, JC2000).

2.2.3. The in vivo implantation

The skull defect models of Sprague-Dawley rats were established to investigate the GBR behaviors of these membranes. The rats ( $200 g \sim 300 g$ , female) were randomly divided into the blank control (covered without membrane), the negative control (covered with CS membrane) and the positive control (covered with asymmetric n-HA/CS membrane), respectively. Firstly, the rats were generally anesthetized by gluteus injection of 0.5% Sumianxin with the dose of 0.8 ml/kg. After the parietal calvarium exposed, two full-thickness skull defects ( $Φ 6$ mm) for each rat were drilled with a trephine bur driven by a handpiece. For the experimental groups, the left defect was covered by the asymmetric n-HA/CS membrane with the size of $Φ 8$ mm (it is worth noting that the under surface must be adhered to the defect) and the right defect was covered by CS membrane; while for the blank control group, the defects were not covered. After implantation, the periosteum and the skin were sutured.

Ethical approval for this investigation was obtained from the Research Ethics Committee, Sichuan University of Medicine.

2.2.4. Histological evaluation

At each time point (1, 4, 8, 12 weeks), these rats underwent euthanasia by injecting overdose of pentobarbital sodium, then samples together with surrounding cranial tissues were cut and taken out. The specimens were fixed in 10 wt% neutral-buffered formalin, decalcified in 17 wt% EDTA solution for 7 days, dehydrated in an ascending graded series of alcohol, and embedded in paraffin. A series of 5 μm transverse sections in the center of the specimens were prepared and stained with hematoxylin-eosin (HE) and Masson Trichrome (MT) staining for the observation of light microscopy (BX51, Olympus, Japan). In order to observe the repair status of bone defect site more directly, X-ray radiographs for the defects of skulls were taken with a digital dental machine (0.03 mA).

3. Results and analysis

3.1. Morphology observation

Figure 1(a) and 1(b) showed the digital photos of CS and n-HA/CS composite membranes. We could see that the upper surface of the asymmetric membrane showed light yellow color similar to that of pure CS membrane, implying that they have the similar composition; the under surface of the asymmetric membrane was white, similar to the color of HA. Figure 1(c) displayed the cross-sectional SEM image of the composite membrane. It could be found that the membrane was about 170 μm in thickness and demonstrated an asymmetric structure, wherein the upper layer was denser and only 20 μm in thickness, and the under surface was looser and about 150 μm in thickness.

Fig. 1.

Photographs of (a) CS membrane and (b) asymmetric n-HA/CS membrane and (c) cross-sectional view of asymmetric n-HA/CS membrane.

Xu Yong et al. [24] had prepared asymmetric porous chitosan membrane [25,26] by immersion-precipitation phase inversion technique. Here we added n-HA into CS solution to prepare an asymmetric n-HA/CS membrane at a certain temperature and vacuum, and the formation process was schemed in Fig. 2. When the n-HA/CS mixture is cast onto a glass plate, the solvent evaporation rate will increase with the surrounding temperature, and the little amount of non-compounded CS molecules accompanied with the evaporation of solvents will move from the interior of the mixed solution to the surface and ultimately form a polymer layer on the surface (the yellow surface in Fig. 1(b)). In turn, the formation of the polymer layer with high viscosity will slow the solvent evaporation rate significantly. Also, the blocking effect caused by HA will decrease or even stagnate the migration speed of CS molecules too, so the polymer layer formed on the surface is thin and mainly composed of colloidal particles [27]. Nevertheless, phase separation for the composite solution under the polymer layer will occur with the solvent evaporating, and the separated water droplets form micropores, finally the under layer of the composite film appears cavernous loose structure. With the solvent evaporation, n-HA particles deposit at the bottom and form a n-HA-rich composite layer.

Fig. 2.

The formation process of asymmetric n-HA/CS composite membrane.

3.2. XPS analysis

The upper surface and the under surface of the asymmetric n-HA/CS composite membrane were recorded and compared using XPS (Fig. 3). Figure 3(a) exhibited the full-scan spectrum of the upper surface, and only C, O and N peaks showed up. Figure 3(b) was the full-scan spectrum of the under surface, except for C, O and N peaks, Ca and P peaks were also observed at 347.2 eV and 133.3 eV, illustrating that the upper surface was only composed of CS, and the under surface was the complex of HA and CS. The binding energy of the elements in the upper and the under surfaces were showed in Table 1. It could be seen that the binding energies of C1s, O1s and N1s centered at 284.8 eV, 532.0 eV and 399.4 eV in the upper surface respectively; while the binding energies of C1s, O1s and N1s at the under surface removed to 286.1 eV, 532.5 eV, 399.2 eV, respectively, indicating that the bonding interaction occurred between n-HA and CS. It may be ascribed to the easily-formed hydrogen bonds between the amino or/and hydroxyl groups in CS and the hydroxyl groups in n-HA, or attributed to the chelation between CS and n-HA through their amino and calcium ions.

Fig. 3.

XPS spectra of (a) upper surface and (b) under surface of the asymmetric n-HA/CS composite membrane.

Table 1

The binding energy of the elements in the upper and the under surfaces of the asymmetric n-HA/CS membrane

Peaks	Binding energy (BE), eV

	Upper surface	Under surface
C1s	284.8	286.1
O1s	532.0	532.5
N1s	399.4	399.2
Ca2p	–	347.2
P2p	–	133.3

3.3. Wettability

Table 2
The contact angles of the upper and the under surfaces of the CS membrane and the asymmetric n-HA/CS membrane

Type of membrane Contact angle (°)

Upper surface Under surface

CS membrane $83.0 \pm 1.1$ $80.0 \pm 1.5$

Asymmetric n-HA/CS membrane $81.0 \pm 1.0$ $67.5 \pm 2.8$

Type of membrane	Contact angle (°)
CS membrane	$83.0 \pm 1.1$	$80.0 \pm 1.5$
Asymmetric n-HA/CS membrane	$81.0 \pm 1.0$	$67.5 \pm 2.8$

From Table 2, we could see that contact angles for the upper and the under surfaces of CS membrane were $83.0 \pm 1.1$ ° and $80.0 \pm 1.5$ °, implying that the two surfaces demonstrated the almost same wettability. In contrast, the contact angle for the under surface of the asymmetric n-HA/CS membrane was $67.5 \pm 2.8$ °, suggesting that the incorporation of n-HA could improve the wettability, while the contact angle of the upper surface was $81.0 \pm 1.0$ °, similar to that of CS membrane, hinting that the upper surface was composed of CS, which was well agreed with XPS results.

3.4. Histological observation

Figures 4–6 showed histological sections of the blank control, CS membrane and asymmetric n-HA/CS membrane implanted for 1, 4, 8 and 12 weeks. We know that the CS membrane was smooth, so the connection between CS membrane and the surrounding tissues was not so firm and relative slide occurred easily after 1 week postoperatively, thus a large number of fibroblasts invaded into the bone defects and suppressed the formation of new bone (Figs 4(b1) and 5(b1)). In contrast, the under surface of the asymmetric composite membrane was rough and combined with the host tissues better, so we found that fibroblasts reduced significantly in quantity, which was favorable for the ingrowth of newly-formed bone tissues (Figs 4(c1) and 5(c1)). On the contrary, almost no fibroblasts or osteoblasts in the defect could be found for the blank control group after 1-week implantation, and we only found a large cavity around the bone stump, as shown in Figs 4(a1) and 5(a1).

When implanted for 4 weeks, for the CS membrane group, there was still a lot of fibrous tissue invading into the center of the defect accompanied by a few of inflammatory cells. In addition, we also found that small amount of bone-like tissues (denoted as arrow) formed around the bone stump and some osteoblasts were active at the edge of the defect, as shown in Figs 4(b2) and 5(b2). As for the asymmetric composite membrane group, it could be found that its under surface was covered by newly-formed bone tissues, and only a little of fibrous tissue showed in the defect (Fig. 4(c2)). It was clearer from Masson stained section (Fig. 5(c2)) that the asymmetric membrane prevented fibrous tissues from growing into the defect and accelerated more new bone formation (denoted as arrow). Besides, osteoblasts were exciting in the proliferation and differentiation stage and the inflammation was almost disappeared (Fig. 5(c2)). Such phenomena were better than those reported elsewhere [12]. In comparison, there were only a lot of fibrous tissues around the defect and neither new bone nor osteoblasts were observed for the blank control group, just shown in Figs 4(a2) and 5(a2).

At the end of the 8th week, the newly-formed bone tissues increased significantly for both CS membrane group (Figs 4(b3) and 5(b3)) and asymmetric membrane group (Figs 4(c3) and 5(c3)), and also extended into the center of the bone defect. However, for the blank control group, the amount of newly-formed bone was very little, as shown in Figs 4(a3) and 5(a3). But at higher magnification, we could see that the mineralization of neo-bone tissue was not so mature for the CS group (Fig. 6(b)), and the new tissues were mainly composed of woven bone; while for the asymmetric membrane group, the mineralization degree of neo-bone was higher, and part of woven bone had calcified into lamellar bone, which could be clearly observed in Fig. 6(c) (because the lamellar bone could be stained into red in the Masson-stained sections). From Fig. 4(c3) we can also see that the asymmetric membrane was attenuated and peeled off from the newly-formed bone tissues, indicating that it was degraded and became loose, which implied that the asymmetric membrane could maintain its morphological integrity for almost 2 months. Nevertheless, the reported GBR membrane [28] began to degrade after 1-week implantation. We all know that it is necessary for GBR membrane to maintain its barrier function for 4–6 weeks in clinic [29].

After 12 weeks implantation, for CS membrane group, the newly-formed bone tissues was in the stage of remodeling and became more mature, and more lamellar bone formed, but the remodeling was not so complete that gaps still existed (denoted by arrow), and the degradation of the CS membrane was not so obvious, as shown in Figs 4(b4) and 5(b4); however, for the asymmetric membrane, the newly-formed bone tissues had occupied the whole defect and the mineralization of new bone was more active, and more lamellar bone had formed and also became thicker. At the same time, we could find that the membrane degraded more obvious, just shown in Figs 4(c4) and 5(c4). But for the blank control shown in Figs 4(a4) and 5(a4), we found that plenty of connective tissues filled the defect, and only minute neo-bone tissue could be observed.

Fig. 4.

Histological micrographs of the specimens with H&E staining: (a) not covered, (b) covered with CS membrane and (c) covered with the asymmetric n-HA/CS membrane for different periods. (M: Membrane; B: Bone; nB: new bone. Magnification $\times 100$ .)

Fig. 5.

Histological micrographs of specimens with Masson’s trichrome staining: (a) not covered, (b) covered with CS membrane and (c) covered with the asymmetric n-HA/CS membrane for different periods. (M: Membrane; B: Bone; nB: new bone. Magnification $\times 100$ .)

Fig. 6.

Histological micrographs of the specimens implanted for 8 weeks. (b) covered with CS membrane, (c) covered with asymmetric n-HA/CS membrane. (nB: new bone; Magnification $\times 200$ .)

4. Discussion

In this study, we developed a novel method to fabricate an asymmetric n-HA/CS membrane to be used as the GBR membrane, which showed excellent biocompatibility and biodegradability. Currently, the development of asymmetric membranes is a new trend in the biomedical field. Fwu-Long Mi et al. prepared a sponge-like asymmetric chitosan membrane by immersion-precipitation and phase-inversion method to be used as wound covering [30]. Zhi-Kang Xu et al. developed an asymmetric membrane with poly(acrylonitrile-co-maleic acid) to improve the surface biocompatibility [31]. Generally, the asymmetric membrane has two sides of which one is dense and another is loose; wherein, the dense layer is designed to prevent the fibrous connective tissue from invading into the defect space, and the loose layer directly contacts the bone defect space and can promote the adhesion, proliferation and differentiation of osteoblasts, then guide the bone regeneration.

The mechanical properties of GBR membranes need to be enhanced to satisfy the clinical requirements. In this study, we chose n-HA to improve the mechanical property of CS membrane. According to our previous work [32] the tensile strength and the elongation at break of the n-HA/CS asymmetric membrane were higher than those of pure CS membrane under wet condition. Wettability is one of the most important factors to determine the quantity and the quality of adsorbed proteins on biomaterials, while the contact angle to water could just reflect directly the wettability of a material [33,34]. The contact angle to water has been proven to be the most significant determinant for the adhesion strength of proteins and, in turn, cell growth rates depend on the adhesion strength of proteins [35]. In the present study, the contact angle of the under surface for n-HA/CS asymmetric membrane was significantly lower than that of the upper surface, illustrating the upper and the under surfaces was asymmetrical, agreement well with the results of XPS and SEM. Besides, the under surface was more hydrophilic than the upper surface, and so was more conducive to the adhesion and the growth of osteoblasts, thereby could accelerate the regeneration of bone defects.

The rat skull defect model was established to investigate the bone regeneration behavior of the GBR membranes. In this study, critical size defects with 8 mm in diameter were made. The newly-formed bone was observed in n-HA/CS asymmetric membrane group at 4 weeks, and with the time elongation, the defect was full of neo-bone, which transformed into lamellar bone ultimately. The degradation rate of membranes directly affects the maintenance of barrier function and tissue regeneration space. In clinic, the GBR membrane should maintain their barrier function for 4–6 weeks [36]. The n-HA/CS asymmetric membrane prepared here maintained its structure integrity until 8 weeks, and the obvious degradation happened at 12 weeks implantation, suggesting that the asymmetric membrane could meet the requirements of GBR membrane in clinic.

5. Conclusions

We fabricated a novel asymmetric n-HA/CS membrane using the method of solution-blending and solvent-volatilizing in vacuum. The resulting n-HA/CS membrane has an asymmetric structure coupled with a loose layer and a dense layer; the loose layer is composed of n-HA/CS composite which can promote osteoblast adhesion, and the dense layer is mainly composed of CS, which can prevent fibrous tissue infiltrating into the bone defects. Additionally, the in vivo biocompatibility and biodegradation of CS membrane and the asymmetric n-HA/CS membrane was evaluated by animal experiments. Compared with CS membrane, the asymmetric composite membrane shows more excellent biocompatibility and biodegradability, and is hopeful to fulfill the requirements to guide bone regeneration.

Supporting information

Supporting information is available from the Wiley Online Library or from the author.

Footnotes

Acknowledgements

This work was supported by the National Basic Research Program of China (973 Program) (Grant No. 2012CB933902), the National Natural Science Foundation of China (Grant No. 51273028), and the Key Technologies Research Program of Sichuan Province (Grant No. 2014SZ0019-6).

Conflict of interest

The authors have no conflict of interest to report.

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Fabrication of nano-hydroxyapatite/chitosan membrane with asymmetric structure and its applications in guided bone regeneration

Abstract

Keywords

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Methods

2.2.1. Preparation of asymmetric n-HA/CS membranes

2.2.2. Characterization of asymmetric n-HA/CS membrane

2.2.3. The in vivo implantation

2.2.4. Histological evaluation

3. Results and analysis

3.1. Morphology observation

Table 2 The contact angles of the upper and the under surfaces of the CS membrane and the asymmetric n-HA/CS membrane Type of membrane Contact angle (°) Upper surface Under surface CS membrane 83.0 ± 1.1 80.0 ± 1.5 Asymmetric n-HA/CS membrane 81.0 ± 1.0 67.5 ± 2.8

5. Conclusions

Supporting information

Footnotes

Acknowledgements

Conflict of interest

References

Table 2
The contact angles of the upper and the under surfaces of the CS membrane and the asymmetric n-HA/CS membrane

Type of membrane Contact angle (°)

Upper surface Under surface

CS membrane $83.0 \pm 1.1$ $80.0 \pm 1.5$

Asymmetric n-HA/CS membrane $81.0 \pm 1.0$ $67.5 \pm 2.8$