Synthesis and Characterization of CSH/CS/n-HA Composite Scaffold for Bone Tissue Engineering


 In this paper, chitosan/hydroxyapatite (CS/n-HA) were synthesized by ultrasound-assisted precipitation combined with inverse crosslinking-emulsion method. In order to obtain a scaffold material with excellent properties, Calcium sulfate hemihydrate (CSH) were combined with CS-HA obtained CSH/CS/n-HA composite scaffold via setting citric acid as solidifying liquid, which possessed better biodegradability, bioactivity, mechanical properties. The physicochemical, morphological properties of scaffolds were characterized by FTIR, XRD and TFSEM. In addition, explored were the mechanical, degradable, biocompatibility and iron release properties. The mechanical strength study indicated that the compressive strength of the porous composite scaffold was influenced by adding an appropriate amount of CS/n-HA composite microspheres. It was proved that the composite scaffold with 6% CS/n-HA content obtained the highest mechanical strength (17.46±1.29 MPa). The results illustrated that the composite scaffold possessed biodegradability and can form hydroxyapatite by dynamic balance of Ca and P elements. The hemolysis tests demonstrate that materials are non-hemolytic and have good blood compatibility. Therefore, the developed composite scaffolds are safe medical inorganic materials, which can potentially be applied in bone tissue engineering.


Introduction
Skeleton is the hard organ of vertebrates with hierarchical structure, which mainly composed of inorganic phase (such as nano-hydroxyapatite) and organic phase (such as collagen). Skeleton has excellent mechanical properties, and also the source of hematopoietic cells and stem cells. It can protect important organs, store and release ions like calcium, exercise, support muscles and so on [1][2][3]. Therefore, it is important that the bone tissues are integrated and healthy. To date, there are as many as 10 million patients worldwide suffering from bone defects due to improper treatment of fractures and fractures, severe trauma, infection, bone tumors, etc. Bone defect is a common disease, mainly due to local bone loss caused by trauma and disease (such as infection, tumor, etc.) and require amount of medical resources [4,5]. For the repair of bone defects, due to the signi cant limitations of traditional treatments such as bone grafting, the search for ideal bone graft replacement materials has become a research hotspot. Ideal bone scaffolds for bone tissue engineering should exist interconnected porous structure, adequate mechanical properties, excellent biocompatibility, and osteoinductivity [6,7].
Hydroxyapatite (hydroxyapatite, Ca 10 (PO 4 )6(OH) 2 ) belongs to the apatite family and is one of the most common forms of CaP. Nano-hydroxyapatite, the main inorganic component of natural bone tissue, is often used in bone tissue engineering owing to its good biocompatibility, osteoconductivity and osteogenic capacity [8][9][10][11], low in immunogenicity and the capability to bind to hard tissue [12][13][14]. At present, the preparation methods of hydroxyapatite mainly include: (1) dry method, (2) wet method (mainly coprecipitation method, sol-gel method, emulsion method, hydrolysis method and hydrothermal method), (3) Alternating energy input methods (mainly microwave assisted, ball milling, sonochemistry and other methods) can be used to prepare hydroxyapatite particles of different shapes and sizes [15][16][17][18][19]. However, hydroxyapatite materials have di culties in forming and easy to agglomerate, and irregular or high-density particles usually cause in ammation, which limits their application in bone tissue engineering. It is well known that HA combined with organic polymer not only improve the mechanical properties of composite scaffold but also strengthen the biocompatibility of material.
Chitosan ((C 6 H 11 NO 4 )N) is a semi-synthetically derived polymer obtained by partial N-deacetylation of chitin, which is the second most abundant polymer in nature [20,21]. It is widely used in the treatment of water quality, agriculture, food, cosmetics and biomedical applications, and has a wide range of applications, due to its non-toxicity, biocompatibility, biodegradability, antibacterial and emulsifying properties [22][23][24][25]. Chitosan has become a new type of research-oriented biomedical material owing to its low toxicity, biocompatibility and biodegradability [26]. The previous literature has shown that CS and its derivatives accompanied by HA will enhance materials bioactivity [27]. Among the hydrophilic semisynthetic materials, chitosan has shown outstanding properties as tissue supporting materials [28][29][30].
Calcium sulfate hemihydrate (CaSO 4 0.5H 2 O), commonly known as "Paris stucco", is an surgical grade calcium sulphate cement that does not cause in ammatory reactions due to its good biocompatibility and has bone repairing ability. It promotes bone healing and can be used to ll areas of bone defects. At present, calcium sulfate hemihydrate has long been used as a substitute for bone grafting [31][32][33]. At the same time, the use of inorganic cement calcium sulfate hemihydrate can overcome the migration problems of granular materials [34].
In the present work, the precursor materials from nano-hydroxyapatite and chitosan/hydroxyapatite composite microspheres were prepared using ultrasonic assisted chemical precipitation and reversedemulsi cation cross-linking method, which were used to increase the compressive strength of composite scaffolds in subsequent experiments. A porous hemihydrate calcium CSH/CS/n-HA composite scaffold was prepared by combining chitosan/hydroxyapatite composite microspheres and calcium sulfate hemihydrate in different ratios. The content of doping CS/n-HA on the mechanical property were detected using a universal tensile testing machine. Additionally, the degradability of composite microspheres and property of iron release were investigated. Subsequently, the in vitro blood compatibility of n-HA, CS/n-HA, CSH/CS/n-HA and calcium sulfate hemihydrate/chitosan/hydroxyapatite composites were investigated by in vitro red blood cell hemolysis test. Through the above research, in order to obtain a new bone graft replacement material that is safe, non-toxic, and has osteogenic ability and anti-infective ability, which provides a new solution for the treatment of bone defects.

FT-IR analysis
FT-IR analysis was carried out to verify the conversion between n-HA, CS and CSH in the reaction process, as shown in Fig. 1(a). For n-HA, the characteristic peaks of PO 4 3− are at 473cm − 1 , 567 cm − 1 , 602 cm − 1 , In addition, the peaks at 631 cm − 1 and 3571 cm − 1 were ascribed to the OH − of hydroxyapatite [42]. The spectrum of CS showed the characteristic peaks at 3444 cm − 1 (overlap of O-H and N-H stretch), 1654 cm − 1 (NH-CO stretch) and 1589 cm − 1 (N-H bend), respectively [43]. The absorption peaks at 894 cm − 1 , 1083 cm − 1 and 1153 cm − 1 originated from the glycosyl of chitosan. Figure 1a was the infrared spectra of CS/n-HA composite microspheres (c). The IR spectra of CSH/CS/n-HA was shown in Fig. 1b  that the crystallinity of n-HA was reduced after the two materials combined. Figure 2c is an XRD pattern of a CSH/CS/n-HA composite. The overall intensity of the diffraction peaks of the CSH/CS/n-HA composites was weakened.

Morphology analysis
SEM image of n-HA microsphere prepared by ultrasound-assisted chemical precipitation method showed that the synthesized n-HA exhibited a rod-like morphology with a uniform particle size distribution (Fig. 3a). As can be seen from Figs. 4a, b, more holes were observed on the surface of the CSH/CS/n-HA composite scaffold, but the distribution was uneven and irregular. According to the results of electron micrograph, Fig. 4c showed that calcium sulfate hemihydrate was a material of sheet structure. At the same time, the two materials were successfully combined according to the TFSEM image of the composite scaffold indicating that more CS/n-HA composite microspheres were embedded in the ake calcium sulfate hemihydrate and embedded in the scaffold (Figs. 4d, e, f). Therefore, an intuitive morphological characterization can verify the successful preparation of porous CSH/CS/n-HA composite scaffolds.

Mechanical strength study
The mechanical strength results indicated that the addition of an appropriate amount of CS/n-HA composite microspheres could effectively increase the maximum compressive strength of the porous composite stent. Brie y, the ve sets of samples were subjected to a compression test using a universal testing machine, and the results were shown in Table 1. Compared with CSH scaffolds (CS/n-HA content of 0%), the maximum compressive strength of the scaffolds increased with an increase of CS/n-HA content (2%, 4% and 6%). When the CS/n-HA content was 6%, the compressive strength of the composite scaffold reaches the highest, which is 17.46 ± 1.29 MPa. When the CS/n-HA content was increased to 8%, the compressive strength of the stent decreased, even lower than the pure CSH scaffold.

In-vitro degradation
Biodegradation is a vital factor to assess whether the scaffold can be implanted in human body which also should possess a controllable speed, biocompatible and non-toxic property. In this experiment, ve groups of CSH/CS/n-HA composite scaffolds (cylinder, Φ10 mm×10 mm) with CS/n-HA content of 0%, 2%, 4%, 6% and 8% were immersed in SBF solution to initially evaluate its degradation performance. The weight was taken every 24 h and the fresh SBF solution was replaced. The daily weight variable rate of the stent samples with increasing soaking time is shown in Fig. 5a. During the experiment, the ve groups of stents were immersed until the 15th day, and the remaining weight had dropped to 1-4% of the initial weight until the simulated body uid immersion experiment was stopped. As shown in Fig. 5a, the trend line of the daily weight loss rate of the CSH stent decreases day by day, with no obvious uctuations. In general, there was no signi cant difference in the trend between the other four groups of stent samples, and the uctuations in the rst 5 days were larger. After the sixth day, the daily weight loss rate tended to change smoothly and decreased as time ows. It was speculated that this was a tightly structured scaffold formed by the exquisite CSH powder attributed to the addition of CS/n-HA composite microspheres so that the gap of the CSH/CS/n-HA composite scaffold was increased, which was more conducive to the entry of the SBF solution into the scaffold promoting the degradation of the stent.

Concentration change of Ca in soaking liquid
The variation of Ca 2+ concentration with the immersion time was observed as shown in Fig. 5c. The EDTA complexometric titration method was used to determine the concentration of calcium ions in SBF soaking solution of the ve groups of samples, and the fresh SBF solution was set as the control (x = 0, y = 0.0026). During the immersion experiment, Ca 2+ was released from the 5 sample holders, and the Ca 2+ concentration was always higher than that of the SBF stock solution. However, the Ca 2+ concentration of the soaking solution decreased day by day compared with the sample before immersion.

Concentration change of phosphorus in soaking liquid
The reagent blank was set as the control, and the PO 4

In-vitro hemolysis study
The hemolysis rates of the different concentrations of n-HA, CS/n-HA and CSH/CS/n-HA suspensions were less than 5% in accordance with the Standard Practice of American Society for Testing and Materials Designation (ASTM:F 756-00), when the hemolysis rate is below 2% and 2% between 5%, those materials were viewed as non-hemolytic and slightly hemolytic, respectively. The results of in-vitro hemolysis experiments are shown in Fig. 8. Overall, the hemolysis rate of the three materials increased slightly with the increase of concentration of the suspension, and the maximum concentration of material hemolysis rate is less than 5%, indicating that no hemolysis occurs in the three concentrations of the material in the experimental concentration range, which meets the hemolysis experiment requirements and national standards of medical materials.

Discussion
FTIR spectra of CS/n-HA composite microspheres (Fig. 1a), which compared with the spectra of n-HA and pure CS powder. Some characteristic peaks of n-HA and CS overlap on spectra of CS/n-HA. The peak at XRD pattern further con rmed the existence of CSH/CS/n-HA composites. The characteristic peak shape of n-HA was sharper, indicating that the crystallinity of n-HA synthesized by ultrasonic-assisted chemical precipitation was high. The above XRD pattern analysis showed that the CS/n-HA composite microspheres were further con rmed to contain both CS and n-HA components, and no other impurity peaks appeared. The phase results of CS/n-HA showed that the diffraction peak intensity of the XRD pattern was weak. Therefore, the overall intensity of the diffraction peak of the CSH/CS/n-HA composite is weakened attributed to the addition of the CS/n-HA composite.
From mechanical strength results, it is know that when the content of CS/n-HA increases to 8%,, the adhesion between the materials decreased, and the stent was more easily loosened, resulting in a decrease in the compressive strength of the sample. The in-vitro degradation illustrated that the cumulative trend of the weight loss rate of the ve groups of samples showed no signi cant difference with time, increasing day by day, and almost completely degraded by soaking until the 15th day. Therefore, the results revealed that the CSH/CS/n-HA composite scaffolds with different CS/n-HA content are biodegradable, and the addition of CS/n-HA does not affect the degradation rate.
The results of in vitro hemolysis experiments illustrated that the suspensions of n-HA, CS/n-HA and CSH/CS/n-HA were in the range of 0.025 ~ 0.8 mg/mL, and the hemolysis rate was less than 5%. There is no hemolysis, and both meet the requirements of hemolysis experiments and national standards for medical materials. Through the above experimental research, it can be preliminarily believed that these three self-made materials will not cause acute hemolysis and have good biosafety.

Conclusion
The hydroxyapatite which exhibits a rod-like morphology with uniform particle size distribution and high crystallinity was successfully fabricated by ultrasound-assisted chemical precipitation method. CS/n-HA composite microspheres with rules, uniform particle size, high regularity and good dispersion were synthesized under the optional conditions (the volume ratio of water to oil phase was 1:5, the content of n-HA was 40%, the reaction temperature was 50°C, the amount of emulsi er was 0.75 mL, and the amount of crosslinking agent was 0.6 mL). The mechanical strength of CSH/CS/n-HA was higher than CS/n-HA composite microspheres, which revealed that adding the appropriate amount of CS/n-HA composite microspheres can effectively improve the maximum compressive strength of porous composite stent. When the CS/n-HA content was 6%, the compressive strength of the composite scaffold reached the highest ( There is no hemolysis phenomenon, and all meet the hemolysis experiment requirements and national standards of medical materials. In this paper, porous hemihydrate calcium sulfate/chitosan/hydroxyapatite composite scaffold with good biosafety was successfully prepared. Porosity is an important property of tissue engineering, which is conducive to cell adhesion and proliferation. It was implied that the CSH/CS/n-HA scaffold have potential as candidate for application in bone tissue engineering.

Synthesis of CSH/CS/n-HA composite microspheres
In this work, n-HA were synthesized by ultrasonic-assisted chemical precipitation method. Ca(NO 3  The CS/n-HA composite microspheres with different properties (weight ratio, 0%, 2%, 4%, 6%, 8%) and 1% sodium bicarbonate as foaming agent were well blended with calcium sulfate hemihydrate and appropriate amount of calcium sulfate dihydrate as coagulant. The citric acid solution was used as solidifying solution, with the ratio of liquid to solid was 0.4 mL/g, and then stirred. Porous CSH/CS/n-HA composite scaffolds were prepared by pouring the mixture slurry into the self-made silica gel tubes and curing.

Characterization of materials
The structural compositions of samples were examined by FTIR (Perkin Elmer Spectrometer 100 device) analysis in the range of 4000 − 400 cm − 1 . Scanning electron microscopy (TFSEM) were used to observe the morphology of CSH/CS/n-HA composite scaffolds. The phase and crystallinity of samples were detected using X-ray diffraction analysis (XRD, D8 Advance Diffractometer, Bruker, Germany) with 40 KV and a scanning range of 10°C to 70°C. After preparing porous CSH/CS/n-HA composite scaffolds with different CS/n-HA content, the ve sets of samples were subjected to compression test using a universal tensile testing machine.

In-vitro degradation of scaffolds
Five groups of samples (pure CSH, CSH/2wt%CS/n-HA,CSH/4wt%CS/n-HA, CSH/6wt%CS/n-HA, CSH/8wt%CS/n-HA) were dried to constant weight and their initial weight M 0 were weighed. The in vitro red blood cell hemolysis test was carried out according to the method described in the related literature [37], and the "Standard Practice for Assessment of Hemolytic Properties of Materials" from Chinese National Standards (GB/T 16886.  to evaluate the blood compatibility of the n-HA, CS/n-HA and CSH/CS/n-HA microspheres [38]. Above all, the blood of New Zealand male rabbit (Purchased from Southern Medical University Experimental Animal Center, China, SCXK2016-0041) was obtained by ear edge incision. Accurately measuring 10 mL of red blood cells into a 500 mL volumetric ask and dilute with saline to obtain 2% red cell suspension (RCS). After grinding the CSH/CS/n-HA composite scaffold into powder, the n-HA, CS/n-HA and CSH/CS/n-HA microspheres were con gured as a series of sample suspensions (0.025 ~ 0.8 mg/mL ) with saline. Then, 2.5 mL of the sample suspension of different concentration and 2% RCS with gently shaking and mixing were placed in a 37 o C water bath for one hour. The mixture was centrifuged at low speed, and the supernatant was collected and the absorbance was measured at 538 nm by ultraviolet-visible spectrophotometer, and parallel experiments was performed 3 times. At the same time, a negative control and a positive control group were set up. The negative control group was added with 2.5 mL of physiological saline, and the positive control group was added with 2.5 mL of Watson's distilled water. The hemolysis rate is calculated according to the following formula: Where A sample is the absorbance of the testing sample and A negative control and A      The result of hemolysis test