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Biocompatible and Biodegradable Bioplastics Constructed from Chitin via a “Green” Pathway for Bone Repair Meng He, Xiaolan Wang, Zhenggang Wang, Lingyun Chen, Yao Lu, Xinjiang Zhang, Mei Li, Zhongming Liu, Yu Zhang, Hong Xia, and Lina Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02051 • Publication Date (Web): 14 Aug 2017 Downloaded from http://pubs.acs.org on August 16, 2017

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ACS Sustainable Chemistry & Engineering

Biocompatible and Biodegradable Bioplastics Constructed from Chitin via a “Green” Pathway for Bone Repair

Meng Hea,b#, Xiaolan Wangc#, Zhenggang Wanga, Lingyun Chend, Yao Luc, Xinjiang Zhangb, Mei Lic, Zhongming Liuc, Yu Zhangc, Hong Xiac*, and Lina Zhanga

a, College of Chemistry & Molecule Sciences, Wuhan University, No.16 Luojia Mountain Street, Wuhan, 430072, China b, School of Materials Science and Engineering, Yancheng Institute of Technology, No. 211 Jianjun East Road, Yancheng, 224051, China c, Guangdong Key Lab of Orthopaedic Technology and Implant materials, Key Laboratory of Trauma & Tissue Repair of Tropical Area of PLA, General Hospital of Guangzhou Military Command of PLA, No.111 Liuhua Road, Yuexiu District, Guangzhou, 510010, China d, Department of Agricultural, Food and Nutritional Science, University of Alberta, 9211 - 116 Street, Edmonton, Alberta T6G 2P5, Canada

* To whom correspondence should be addressed. Phone: +86-27-87219274. Fax: +86-27-68762005. E-mail: [email protected] (L. Zhang); [email protected]. 1

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ABSTRACT Biodegradable plastics are urgently needed in the biomedical field to avoid the secondary surgery for implants after completing the repair of non-load bearing bone defects. Herein, novel chitin-based plastics were successfully fabricated by changing the shape and aggregation state structure of chitin hydrogels through drying under a negative pressure, which led to a plastic deformation. The chitin hydrogels were prepared by using an environmentally friendly aqueous NaOH/urea solvent, and then radially oriented under the negative pressure to form chitin bioplastics (CP) on the basis of the removability of the chitin molecular bundles in the hydrogels. Moreover, hydroxyapatite (HAP) was in-situ synthesized to obtain the chitin/HAP composite plastics (CHP). Their structure and properties were characterized by SEM, FTIR, 13C NMR, X-ray diffraction and mechanical testing. The results indicated that the bioplastic preparation was a “green” physical process, and the incorporation of HAP reinforced significantly the tensile strength of CHP. The viability, biocompatibility, hemocompatibility and in vivo histocompatibility of the bioplastics were evaluated systematically. The introduction of HAP could improve the cell adhesion, proliferation and differentiation of the osteoblast cells. Moreover, CHP exhibited good histocompatibility, hemocompatibility and in vivo biodegradability, showing potential application in the bone tissue engineering field.

KEYWORDS:

chitin

bioplastic,

orientation

structure,

biocompatibility, in vivo biodegradability

2


hydroxyapatite,

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INTRODUCTION The repair of the damaged bone is a great challenge in the biomedical material field all over the world. When the bone couldn’t complete self-repair, orthopaedic implants are needed1. However, there is a need generally to undertake a secondary surgery or immune rejection and infection would occur after orthopaedic implants in fracture fixation and spinal fusion surgeries for non-load bearing bone defects 2, so exploring a suitable artificial bone material for repair of bone defects is an inevitable trend in the bone tissue engineering field. At present the commonly used bone repair materials are mainly divided into the following four categories. (1) Biomedical metal materials are the first generation of biomaterials for bone repair, among which the most widely used one is titanium alloy with high strength and excellent biocompatibility3. However, their mechanical mismatch with surrounding bones usually lead to the impairment of the normal bone repair and a secondary surgery is needed to remove the implanted metals when the repair of non-load bearing bone defects is completed4. (2) Biomedical polymers include both natural polymers and synthetic polymers5. Particularly, natural polymers derived from biomass are biocompatible and biodegradable,

which

are

beneficial

to

cell

adhesion,

proliferation

and

differentiation6-9. (3) Biological ceramic based materials includes bioactive and bioinert ceramic materials. Among these, hydroxyapatite (HAP) as one component of the animal bone, exhibits excellent biocompatibility, high strength and bone induction10, 11, thus is used widely for the repair of non-load bearing bone defects. However, pure hydroxyapatite is difficult to be processed into specific forms required

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for bone repair. (4) Biocomposite materials may combine the advantages of all components such as the processibility from polymers and bone induction from HAP, showing a better role in the bone reconstruction12-18. Recently, the research on renewable resources has been listed as one of the 24 international frontiers due to the energy and sustainable development issues, and the utilizations of natural polymers related materials have attracted much attention19. Chitin is one of the most abundant biomasses, which widely exists in the skeleton of crustaceans and cell walls of algae and fungus as structural backbones1,

20, 21

.

Moreover, chitin is an important bioactive polysaccharide with biofunctions, intrinsic biocompatibility, biodegradability and low immunogenicity20, 22, thus has potential applications in the biomaterial field. However, low solubility of chitin has limited its research and applications. In our laboratory, NaOH/urea aqueous solution as an environmentally friendly solvent has been developed to dissolve chitin, and the regenerated chitin based materials such as hydrogels, aerogels and fibers have been fabricated23-28. These chitin materials, regenerated directly from its solution, have been proven to promote cell adhesion and proliferation. Only a small amount of chitin materials such as chitin whiskers, chitin based microgels and a calcium phosphate/-chitin nanofiber hydrogel have been fabricated for bone repair29-32. However, these chitin materials for bone repair were not bulk and entire, and chitin plastics with high toughness have never been reported in biomaterial area, especially for repair of non-load bearing bone defects. Plastics are materials that can be shaped or undergo plastic deformation when

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processed under certain conditions33. The bioplastics are biodegradable, and produced from renewable resources rather than fossil-fuel plastics34-36. In view of the above circumstances, a worthwhile endeavour would be to construct biocompatible chitin based bioplastics. It is well known that the close packing of chitin molecular chains leads to its difficulty in the removability, limiting its processability. On the basis of the removability of the chitin molecular bundles in the hydrogel state under negative pressure (Scheme 1), the conversion of the aggregated structure of the chitin hydrogels can be occurred under a pressure. In the present work, chitin solution was coagulated with ethanol to obtain hydrogel sheets, and then the chitin molecular bundles in the hydrogel could move in the drying process under the negative pressure to form the oriented structure, generating bioplastic. Due to the affinity of chitin towards inorganic compounds, the acetyl amino in the chitin chains could effectively fix Ca2+ to react with HPO42-, so HAP could be homogenously in-situ synthesized in the chitin hydrogel to construct the chitin/HAP composite bioplastics by drying under the negative pressure. The physical techniques in the processing and no toxic materials were introduced here, so chitin and HAP could retain their inherent bioactivities. The composites were demonstrated to have potential application as the repair of non-load bearing bone defects materials with good biocompatibility, histocompatibility and biodegradability. This work not only supplied a novel “green” pathway for the direct preparation of chitin based bioplastics, but also new materials for the repair of non-load bearing bone defects. We believe this would be beneficial to the sustainable development in the

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biomaterial field.

EXPERIMENT SECTION Materials Raw chitin powder was purchased from Jinke Chitin Co. Ltd (Zhejiang, China) and purified as we reported previously37, which was treated in 5 wt% NaOH, 7 wt% HCl, 5 wt% NaOH for 12 h and bleached in 4 wt% H2O2 at pH=8 and 75C step by step. The resultant chitin powder was washed repeatedly to neutral with distilled water after each step. The purified chitin powder was dried at 60 C and kept in a desiccator. The weight-average molecular weight (Mw), measured by dynamic light scattering (DLS, ALV/CGS-8F, ALV, Germany) in 5% LiCl/DMAc (w/w), were 3.0×105. The CaCl2, (NH4)2HPO4, NaOH, ethanol, NH3·H2O and urea (Shanghai Chemical Reagent Co. Ltd.,China) were used as received. All the chemical reagents were of analytical grade and used without further purification. Preparation of CP and CHP Chitin solution was prepared by dissolving the purified chitin powder in an 11 wt% NaOH/4 wt% urea solvent through a freezing/thawing method. The transparent 8 wt% chitin solution was centrifuged to degas at 6000 rpm for 15min, then the resultant chitin solution was kept at 5 C for 24 h. Subsequently, the chitin solution was poured into the mold and coagulated with ethanol to fabricate the chitin hydrogel sheets with the thickness of about 5.5 mm. The chitin hydrogel sheets were rinsed repeatedly with deionized water and soaked in 2 wt% glycerin aqueous solution for 12 h, whose

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periphery was then fixed tightly on the glass plate to let water evaporate only on the side away from the glass plate. Under negative pressure, the water molecules in the chitin hydrogels could evaporate completely, leading to a transition in the aggregated structure of the chitin from hydrogels to bioplastics. Thus, pure chitin bioplastics with the thickness of about 0.3 mm were obtained, coded as CP. Furthermore, the chitin hydrogel sheets was soaked in 0.25 M CaCl2 and 0.15 M (NH4)2HPO4 solutions for 6 h respectively, and this process was repeated for three times, and then the hydrogel sheets were put in the NH3·H2O atmosphere for 2 h to fabricate chitin/HAP composite gels, which were then rinsed with deionized water after each step. The composite gel sheets were soaked in the 2 wt% glycerin aqueous solution for 2 h, fixed on the glass plate to fabricate the composite plastic under vacuum at room temperature subsequently, denoted as CHP. The preparation process of CP and CHP is shown in Figure 1a. Characterization The morphologies of the surface and cross-section for CP and CHP were observed with a Field emission scanning electron microscopy (FESEM, Zeiss). The acceleration voltage for the FESEM observation was 5 kV. The chitin and chitin/HAP hydrogel sheets were frozen in liquid nitrogen, immediately snapped and then freeze-dried for SEM observation. The surface and cross-section of the plastics or hydrogels were sputtered with god, and then observed. FTIR spectra were carried out with a FTIR spectrometer (1600, Perkin–Elmer Co., MA) in the wavelength range from 4000 to 400 cm-1. The powdered and vacuum-dried CP and CHP were obtained,

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and the test specimens were prepared by using the KBr disk method. CP and CHP were also characterized by X-ray diffraction, two-dimensional X-ray diffraction and Solid-state

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C NMR, the details for these characterizations are provided in the

Supporting Information. The tensile strength (σb) and elongation at break (εb) of CP and CHP in dry state were measured on a universal tensile tester (CMT 6503, Shenzhen SANS Test machine Co. Ltd., Shenzhen, China) according to ISO527-3-1995 (E) at a speed of 2 mm/min. The light transmittance of CP and CHP was analyzed with UV spectrophotometer over a wavelength range 250−900 nm. The transmittance spectra were acquired using air as the background. In vitro cell assay and in vivo tests The cell viability and density of cells proliferation were characterized by the live/dead fluorescent staining and MTT assay of MC3T3-E1 cells, respectively. Cell morphology of CP and CHP was examined by scanning electron microscopy. The alkaline phosphatase (ALP) activities of CP and CHP were also evaluated. The details for these in vitro cell assays are provided in the Supporting Information. Fresh anticoagulant New Zealand White rabbit whole blood was diluted using physiological saline with the ratio of 1:1.25. The samples were put into the tubes and washed with distilled water thrice and rinsed with 0.9 wt% NaCl solution for 30 min, the soaking solution was poured out and 10 mL 0.9 wt % NaCl solution was added, then kept in 37 ºC constant temperature water bath for 30 min. 0.2 mL dilute whole blood was added and mixed homogenously, the resultant solution was kept in

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constant-temperature water bath at 37 ºC for 60 min and then centrifuged at 3000 rpm for 5 min. The absorbance of the supernatant was measured at 545 nm (Thermo, Multiskn Go, U.S.A.). 0.2 mL whole blood was diluted with 10 mL of distilled water or 0.9 wt% NaCl solution, which were treated by the same process with the sample group and used as the positive and negative control, respectively. Each group of samples was repeated three times, and the average value was used in this work. The hemolysis ratio (HR) was calculated by equation (1): HR (%) = (AS−AN) / (AP −AN) ×100

(1)

Where AS, AP and AN are the average absorbance of samples, positive controls and negative controls, respectively. Blood serum samples were taken from the rabbits, pre-operation and 1, 4, and 12 weeks post-operation. Tests for alanine aminotransferase (ALT), aspertate aminotransferase (AST), creatinine (CREA), and UREA were conducted on an Roche module P800 automatic biochemical analyzer. To evaluate the histocompatibility of CP and CHP in vivo, subcutaneously implanted plastics animal model was established by six to eight-month-old New Zealand White rabbits (2.5-3 Kg, Medical experimental animal center of Guangdong province, China). All experimental protocols were approved by the Animal Care and Use Committee of the General Hospital of the Guangzhou Military Command of PLA. Rabbits were first anesthetized using chloral hydrate, and then a defect (≈12 mm long) was incised under the skin. Then CP or CHP specimen was implanted into the defect site. Each type of sample was implanted in four animals. After 1, 4 and 12 weeks implantation, the animals were sacrificed to allow histological evaluation. The

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acquired skin specimens include materials were preserved in 4% (wt/v) paraformaldehyde solution overnight, dehydrated through a graded series of ethanol (75%, 85%, 90%, 95% and 100%), immersed in dimethyl benzene and then embedded in paraffin. Then, the frozen specimens were cut into cryo-sections, and one of the adjacent 5 μm thick sections was used for hematoxylin and eosin staining (HE, NanJing JianCheng Bioengineering Institute, China) to observe the morphological changes in the cells and tissues, and HE photographs were visualized with a fluorescence microscope (Olympus BX51, Japan).

RESULTS AND DISCUSSION Construction and structure of the CP and CHP bioplastics Figure 1 shows the brief preparation process of CP and CHP, and their photographs, as well as scheme to describe the interaction between chitin and HAP. The chitin and chitin/HAP hydrogels (Figure 1d1,2) transformed into the CP and CHP plastics (Figure 1e1,2), respectively, by drying under a negative pressure. The CP bioplastic with the thickness of about 0.30 mm exhibited high transparency and toughness (Figure1e1), as a result of the compact and uniform aggregation of chitin chains. The CHP bioplastic was also tough and displayed a little milky color (Figure 1e2), because the introduction of HAP could cause optical loss from light reflections and refractions, resulting in the transmittance decrease38. There was hydrogen bonding interaction between HAP and chitin (Figure 1b), so HAP could be fixed in the chitin matrix to disperse homogenously.

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Figure 2a shows WAXD spectra of the CP and CHP. The five characteristic peaks at 2θ=9.0°, 12.3°, 18.6°, 21.9°and 25.0°for the CP sheet were assigned to its five crystal planes of (020), (021), (110), (130) and (013) of -chitin, which were different from the raw chitin power37. This could be explained that the dissolution and regeneration processes broke the original hydrogen bonds, thus the chitin aggregates restructured in some extent during the drying process, leading to the change of crystal morphology and crystallinity. There was a critical peak at 31.2°, assigned to the (211) HAP (hkl) indice, appeared in CHP, indicating the successful in-situ synthesis of HAP in the chitin matrix. The intensity for the characteristic peaks of chitin became weaker, suggesting that the strong interaction existed between chitin and HAP, resulting in the decrease of the chitin crystallinity. FTIR was used to analyze the interactions between the organic and inorganic components. Figure 2b shows the FTIR spectra of the CP and CHP. Compared with the raw chitin powder (Figure S1), the peaks for -NH (asymmetric) and -NH (symmetric) stretching vibrations overlapped with the peak of -OH stretching vibration, indicating that the hydrogen bonds of chitin chains were wakened because the dissolution and regeneration process could broke their original hydrogen network24, 39. Moreover, compared with CP, the -OH stretching vibrations for CHP broadened and shifted to high wavenumbers, further confirming that the introduction of HAP broke the original hydrogen bonding interaction between chitin chains. These results supported the scheme in Figure 1 (Figure 1b). Figure 2c shows the cross polarization/magic-angle spinning (CP/MAS) 13C NMR

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spectra of CP and CHP. The chemical shift of CP was similar to the raw chitin powder, supporting that the preparation of CP was a physical process, and no chemical reaction occurred here. Interestingly, there was a small peak appeared at 63 ppm, which was assigned to the crystal plane of C636, confirming that the new crystal area appeared, as a result of the orientation of the chitin aggregates during the drying process. Moreover, the intensity of the peaks for the C of chitin lost their resolution, further confirming that the introduction of HAP could weaken the hydrogen bonds among the chitin chains. Therefore, there was strong interaction existed between chitin and HAP in CHP. Figure 2d shows the X-ray diffractograms perpendicular to the surfaces of CP and CHP. Obviously, the CP bioplastic exhibited the oriented structure36 (Figure 2d), whereas which was not obvious in CHP. The strong interaction between HAP and chitin might weaken the rearrangement and orientation of chitin during the drying process. To clarify the transition in the aggregated structure and deformation of the chitin based hydrogels during the regeneration process, the morphologies of the freeze-dried hydrogels were observed by SEM. Figure 3 (a, b) shows the SEM images of the cross-section of the freeze-dried chitin and chitin/HAP composite gel sheets. There were homogenous submicron pores in the chitin hydrogel sheet (Figure 3a), which could provide ideal space for in situ synthesis of HAP. For the composite hydrogels, the HAP submicron particles distributed evenly in the chitin network (Figure 3b). It was not hard to imagine that chitin could effectively absorb and fix Ca2+ through complexation interaction with acetyl amino groups, and then the immobilized Ca2+ in

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the chitin matrix interacted with HPO42- to form HAP under the atmosphere of NH3·H2O. Figures 3 (c-e) and S2 (a, b) show the SEM images of the cross-section and surface for CP and CHP, respectively. Evenly multi-layered structure appeared on the CP and CHP bioplastics (Figure 3c, d), such multi-layered structure is beneficial to improve the material mechanical strength and flexibility, selective permeability, and high bioactivity40. HAP could be filled in the chitin pores to some extent, and was fixed in the chitin matrix, as a result of the strong interaction between HAP and chitin. It was noted that the oriented structure was weakened by the incorporation of HAP, which was consistent with the results of the X-ray diffractograms (Figure 2d). The surface of CP was relative smooth (Figure S2a), suggesting the homogenous structure formed on the single layer plane under the negative pressure at room temperature. The HAP submicron particles were distributed uniformly on the surface of CHP (Figure 3e, Figure S2b), which could increase the surface roughness. The HAP submicron particles could provide an ideal basis for the absorption of extracellular protein and improvement of cell adhesion41. Moreover, the EDS spectrum of CHP indicated that the HAP did exist in the chitin matrix, further confirming that HAP was in-situ synthesized successfully (Figure 3f). In view of the above results from SEM,

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C NMR and X-ray diffractogram, a

schematic diagram to describe the conversion process from the chitin hydrogel into plastic is proposed in Scheme 1. Due to the ordered aggregation of the extended chitin chains in solution42, the chitin as a molecular bundle, which was consisted of chitin

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macromolecules and their aggregates (nanofibers) in parallel form, existed in the hydrogel. On the basis of the removability of macromolecules in the hydrogel state, the chitin molecular bundles could move continuously to aggregate as the water evaporation. Because the periphery of the hydrogels was fixed tightly to prevent the shrinkage, thus the orientation of the chitin molecular bundles in the hydrogels occurred rapidly under negative pressure, which was driven by the stretch force in the drying process to obtain pure chitin bioplastics. As shown in Scheme 1, the orientation occurred in the parallel direction, resulting in a radial orientation, forming a homogenous layer plane supported by Figure S2a. After complete vacuum drying, the chitin could assemble along the out-of-plane direction, forming a united multi-layered structure on the plane, supported by Figures 3c. The mechanical properties and optical transmittance of CP and CHP Figure 4a shows the mechanical properties of the CP and CHP at the dry status. The tensile strength (σb) and elongation at break (εb) of CP were 51.5 MPa and 34.1% respectively, displaying high mechanical properties and excellent flexibility. Importantly, the tensile strength of CHP could reach 99.2 MPa, indicating that the introduction of HAP significantly improved the mechanical strength of CP. This could be explained that the HAP submicron particles formed a “network” structure, and combined with chitin tightly to disperse the stress effectively. The tensile strength values of CP and CHP were much higher than that of PCL@MS, PCL, PCL/OA and PCL/OA/Hap (13.1-26.7 MPa) as well as the collagen network in the bone (15.4-23.8 MPa), which could satisfy the strength requirement of the cortical bone (cross, 40-60

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MPa) and trabecular bone (2 MPa)43-47. The modulus values of CP and CHP were 827 and 2017 MPa respectively, which were much higher that of porous SLM316L, PCL@MS, PCL, PCL/OA and PCL/OA/Hap (120-244 MPa) for bone repair43-46. Moreover, the elongation at break for CHP was 13.9%, indicating that the CHP was still flexible. The area under the stress-strain curves usually reflects the toughness37, the toughness values for CP and CHP were 14.3 and 11.3, respectively, which were much higher than that of calcium phosphate cement (CPC) scaffolds with different chitosan contents (0.18-0.23), and could satisfy the high toughness requirement (above 8.8) for bone repair such as that of a ceria stabilized zirconia–alumina nanocomposite46-50. Figure 4b shows the light transmittance of CP and CHP. As expected, the CP exhibited light transmittance of above 70% in the range of 400-800 nm, and the light transmittance could reach 86% at 800 nm, confirming its high optical transmittance (Figure 1b). CHP still exhibited relative high optical transmittance of about 70% at 800 nm, which was in accordance with Figure 1c, further confirming that there was certain miscibility between chitin and HAP. Biocompatibility and in vivo biodegradability evaluations Previous research suggests that chitin has excellent histocompatibility29. The cell attachment and proliferation on the CP and CHP bioplastics were then studied by fluorescent images, SEM images and MTT assay using the MC3T3-E1 cells. Figure 5 (a, b) shows the fluorescent images of MC3T3-E1 cells cultured on CP and CHP for 48 h. Most of the MC3T3-E1 cells on CP exhibited both spherical shape and shuttle-shape with the existence of some dead cells (red), only a small amount of

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MC3T3-E1cells had pseudopodia (Figure 5a). However, all the MC3T3-E1 cells on CHP exhibited a shuttle-shape and had obviously extended pseudopodia and no dead cells were observed (Figure 5b), indicating the information exchange among the cells. Therefore, the cells could adhere and grow well on CHP, namely the embodiment of the good biological activity. It has been reported that the introduction of HAP with high affinity to protein could improve the absorbability of protein on chitin in the nutrient solution51. It was not hard to imagine that CHP containing HAP was beneficial for cell adhesion. Furthermore, the biocompatibility of CP and CHP was studied by the DAPI method (Figure S3). The DAPI stain images of MC3T3-E1 cells on CP and CHP for 60 and 120 min revealed that the introduction of HAP could effectively improve the cell adhesion ability. Figure 5 (c-f) shows the SEM micrographs of the MC3T3-E1 cells on CP and CHP after 24 h of culture. The MC3T3-E1 cells could adhere and spread uniformly on CP (Figure 5c). The cells on CP exhibited shuttle-like shape and discrete filopodia (Figure 5e), which enabled the cells to be anchored on the surface of CP, indicating good cell adhesive ability. Interestingly, as shown in Figure 5(c, d), the number of cells adhered on the surface of the CHP was much larger than those on CP, which was consistent with the fluorescent images results. The cells on CHP also exhibited shuttle-like shape with more discrete filopodia (Figure 5f), further confirming that CHP had better MC3T3-E1cell adhesion performance. The results further demonstrated that the introduction of HAP significantly improved the osteoblast cell affinity.

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Figure 6a shows the results of MTT assay for the CP and CHP bioplastics. For the culture time of 1 d and 3 d, the optical density (OD) values of CHP were similar to that of CP. It has been stated that chitin is biocompatible and suitable for osteoblasts culture29, 51-53. From the MTT results for 5 d and 7 d, the cell viability increased obviously with the incorporation of HAP. HAP is one of the components of natural bone, so it is suitable for osteoblasts attachment and proliferation. These results of MTT indicated that the OD values of CHP were equal to or higher than that of CP, suggesting that the CHP was more appropriate for the culture of MC3T3-E1 cells. Figure 6b shows the osteogenic differentiation results of ALP assay. A minimal increasing expression of alkaline phosphatase activity on CHP could be observed after culturing for 7d and 14d. The ALP activity in CHP was higher than that in CP at day 21d, as shown in Figure 6b. The osteogenic differentiation on the CHP sheet was attributed to the signaling initiated by the introduction of HAP. Materials with high hemcompatibility should not induce the thrombosis and denaturation of blood components54,

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, and it is generally believed that if the

hemolysis rate of biomaterials is below 5%, the biomaterials can satisfy the requirements for hemolysis testing of medical equipments. The hemolysis rates of negative control group, positive control and the samples are summarized in Table 1. The destruction to red blood cells for biomaterials reduced with the decrease of the material hemolysis rate. The results indicated that the hemolysis rates of CP and CHP of blood cells were both below 5%, showing no hemolysis and high hemcompatibility to red blood cells.

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Furthermore, body temperatures of New Zealand White rabbits after implanting CP and CHP were tested after 0, 1, 2 and 3 h (Figure S4). Postoperative 1 and 2 h, body temperatures of New Zealand White rabbits decreased but the temperature rebounded to normal body temperature after 3 h. The results showed that both CP and CHP had no thermogenic action. Moreover, there were no statistical differences in the ALT, AST, Cr and UREA between the CP and CHP bioplastics (Table S1), suggesting that CHP had noliver and kidney blood toxicity. The hematoxylin and eosin staining (HE) photographs of CP and CHP implanted for 1, 4 and 12 weeks are shown in Figure 7. After being implanted in the rabbits for 1 week, CP and CHP were wrapped with granulation tissue (Figure 7, 1 w). The collagen generated around CP, whereas the inflammatory cells decreased. Importantly, the CP fragments appeared in the tissue after 4 weeks (Figure 7, CP-4 w), indicating the biodegradability and histocompatibility of CP and. The rabbit tissue could grow tightly around CHP, leading to the appearance of collagen and decrease of inflammatory cells (Figure 6, CHP-4 w), indicating good biocompatibility of CHP. Most of CP and CHP disappeared and only small fragments were found in the tissue, further confirming that both CP and CHP were biodegradable (Figure 7, 12 w). Newly generated tissue occupied the original site of implantation and integration of the implants with the tissue increased obviously especially after being implanted for 12 weeks. Therefore, CP and CHP both exhibited good biocompatibility, histocompatibility and in vivo biodegradability, showing great potential as the repair materials of non-load bearing bone defects.

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CONCLUSIONS The highly biocompatible chitin-based bioplastics were successfully fabricated by changing the shape and aggregation state structure of chitin hydrogels via a “green” pathway. On the basis of the removability of the chitin molecular bundles consisted of macromolecules and their aggregates in the hydrogels, ordered orientation and rearrangement of the chitin structure in hydrogel occurred during drying under negative pressure, leading to the formation of the transparent plastic. Through in situ synthesis, HAP was incorporated into the chitin matrix to form composite bioplastics. The HAP submicron particles were uniformly distributed in the chitin matrix, resulting in the improvement of the mechanical strength. The bioplastics preparation was mainly physical processes without any involvement of toxic materials. The introduction of HAP could significantly improve cell adhesion, proliferation and differentiation of the osteoblast cells. Moreover, the CHP bioplastics had good in vivo histocompatibility, hemocompatibility and biodegradability. Therefore, the CHP bioplastics derived from biomass would be important for the sustainable development in the biomaterial field.

ASSOCIATED CONTENT Supporting Information The details for X-ray diffraction, two-dimensional X-ray diffraction and Solid-state 13

C NMR measurements and in vitro cell assay. The FTIR spectrum of raw chitin

19



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powder, FESEM images of the surface for CP (a) and CHP (b), Fluorescent images of MC3T3-E1 cells on CP and CHP for 60 and 120 min by DAPI method, Body temperatures of New Zealand White rabbits after implanting CP and CHP for 0, 1, 2 and 3 h, ALT, AST, Cr and UREA of the CP and CHP at baseline to 12 weeks. This information is available free of charge via the Internet at http://pubs.acs.org/. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51503177, 81501859), the Major Program of National Natural Science Foundation of China (21334005), the Major International (Regional) Joint Research Project (21620102004),

the

Natural

Science

Foundation

of

Guangdong

province

(2015A030312004) and the Science and Technology Planning Project of Guangzhou city (201604020110). We thank Prof. Shuguang Yang for kindly providing two-dimensional X-ray diffraction measurements. Author Contributions # These authors contributed equally to this work.

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Table 1. Hemolysis rates of CP, CHP and the controls. Sample

HR (%)

Normal saline (Negative control)

100

Distilled water (Positive control)

0

CP

0.31±0.20

CHP

0.15±0.08

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Figure 1. Preparation process of CP and CHP by in situ synthesis of HAP in the chitin gel sheet (a), the scheme of the interaction between chitin and HAP in CHP (b), photographs of chitin solution (c), the chitin(d1) and chitin/HAP (d2) hydrogels, and the CP (e1) and CHP (e2) bioplastics.

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Figure 2. The WAXD patterns (a), FTIR (b) and Solid-state 13C NMR (c) spectra of CP and CHP, and X-ray diffractograms perpendicular to the surfaces of CP and CHP (d).

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Figure 3. The FESEM images of cross-section for chitin gel (a) and chitin/HAP gel (b) prepared by the freezing dried process (The blue arrows indicate HAP micro-plates), the cross-section for the CP (c) and CHP (d) and the surface of CHP (e), and EDS spectrum (f) of CHP.

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Scheme 1.The conversion of a chitin hydrogel to a chitin bioplastic by vacuum drying.

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Figure 4. Stress-Strain (σ-ε) (a) and UV transmittance curves (b) of the CP and CHP.

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Figure 5. Fluorescent images of MC3T3-E1 osteoblasts seeding on CP (a) and CHP (b) for 48 h, SEM images of MC3T3-E1 cells cultured on the surfaces of CP (c, e) and CHP (b, f) for 24 h.

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Figure 6. MTT assay results of MC3T3-E1 cultured on the CP and CHP sheets for 1, 3, 5 and 7 days at 490 nm (a). ALP activity analysis of MC3T3-E1 cells cultured on the CP and CHP sheets for 7, 14 and 21 days at 405 nm (b). Data are presented as the mean ±standard deviation.

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Figure 7. Hematoxylineosin staining (HE) photographs of the CP and CHP implanted in rabbits for 1, 4 and 12 W, respectively. C represents CP or CHP，S represents skin tissue.

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For Table of Content Only

Brief synopsis “Green”

pathway

for

construction

of

histocompatible

and

biodegradable

chitin/hydroxyapaptite bioplastics with enhanced mechanical strength, osteoblast cells adhesion for bone repair.

39


Pathway for Bone Repair - ACS Publications

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