100 Vol.27 No.1 LI Hong et al: In vitro and in vivo Characterization of Homogeneous Chit...
In vitro and in vivo Characterization of Homogeneous Chitosan-based Composite Scaffolds
LI Hong1,2, ZHOU Changren1,2, ZHU Minying1,2, TIAN Jinhuan1,2, RONG Jianhua1,2
(1.Department of Materials Science and Engineering, Jinan University, Guangzhou 510630, China; 2.Education Ministry Research Centre of Artifi cial Organs & Materials, Jinan University, Guangzhou 510630, China)
Abstract: With a homogeneous distribution of hydroxyapatite (HAP) crystals in polymer matrix, composite scaffolds chitosan/ HAP and chitosan/collagen/HAP were fabricated in the study. XRD, SEM and EDX were used to characterize their components and structure, in vitro cell culture and in vivo animal tests were used to evaluate their biocompatibility. HAP crystals with rod-like shape embeded in chitosan scaffold, while HAP fine-granules bond with collagen/chitosan scaffold compactly. A homogenous distribution of Ca and P elements both in chitosan/HAP scaffold and chitosan/collagen/HAP scaffold was defined by EDX pattern. The presence of collagen brought a more homogenous distribution of HAP due to its higher ability to induce HAP precipitation. The results of in vitro cell culture showed that the composite’s biocompatibility was enhanced by the homogenous distribution of HAP. In vivo animal studies showed that the in vivo biodegradation was effectively improved by the addition of HAP and collagen, and was less infl uenced by the homogeneous distribution of HAP when compared with a concentrated distribution one. The composite scaffolds with a homogeneous HAP distribution would be excellent alternative scaffolds for bone tissue engineering.
LI Hong(李红): Assoc. Prof . ; E-mail:[email protected] by the National High Technology Development Program (No.
2007AA091603) and the National Natural Science Foundation of China (Nos. 30870612 and 20604010)
DOI 10.1007/s11595-012-0416-4
1 Introduction
Tissue engineering, which applies methods from engineering and life sciences to create artificial constructs to direct tissue regeneration, has attracted many scientists and surgeons with a hope to treat patients in a minimally invasive and less painful way. The important process of a tissue engineering paradigm is to isolate specific cells to grow them on a scaffold. A scaffold should be in combination with support for tissue architecture, bimolecular and selective transportation of ions in body fl uids. Chitosan (CS) is the partially deacetylated form of chitin that can be extracted from crustacean. Apart from being biocompatible, CS is easy to mould a 3-dimensional scaffold which can support tissue ingrowths, aid in the formation of tissue structure, promote growth and mineral rich matrix deposition by osteoblasts in culture for bone tissue engineer[1]. Even though CS shows many advantages that make it attractive biomaterials for bone
scaffolds, it is possible to improve its osteoconductivity by combining with calcium phosphate[2-5], collagen or gelatin[6,7]. CS in combination with hydroxyapatite (HAP), Ca10(PO4)6(OH)2, further enhance tissue regenerative efficacy and osteoconductivity, for HAP can accelerate the formation of bone-like apatite and can induce bone formation[2-5]. With the regarding, collagen is also biocompatible, biodegradable and osteoinductive, acting as an excellent delivery system for bone morphogenetic proteins (BMPs)[6]. For CS-based composite scaffold, both HAP and collagen endowed scaffolds with osteoconduction and osteoinduction. Many studies have focused on the composite scaffold based on CS/HAP materials for bone tissue engineering[8-11].The composite scaffolds had been prepared by different processing, including mixing of powdered HAP with CS and then lyophilizing the mixture[9,12], coating CS sponge with biomimetic calcium phosphate[13-15] and co-precipitating of HAP/CS composite[16,17]. In cell cultures, composites exhibited better biocompatibility than the individual materials alone[10, 18]. However, using powdered HAP and coating calcium phosphate, the fi nal materials with weak interface bonding between CS and inorganic particles were microscopically heterogeneous and often caused irritation or damage around the tissue when implanted[18,19]. Co-precipitation methods result
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in uniform distribution of calcium phosphate crystals in the CS matrix, together improving biocompatibility and osteoconductivity[16, 20, 21]. Although the ability of composite CS-based scaffold with HAP or collagen to support osteoblast attachment and proliferation in vitro has been well established[6,16, 21,22], the effect of a homogeneous structure of CS-based composite scaffold on properties, especially biocompatibility in vivo with respect to the plain CS, blended HAP/CS and HAP/CS/collagen scaffold, has not been systematically investigated. In the paper, the combination of the lyophilization method and in situ precipitation was applied to develop a homogeneous composite scaffold fabricated from biopolymer CS and (or) collagen, and the bioceramic HAP uniformly distributed in the CS matrix. The morphological and compositional properties of composites were investaged by X-ray diffraction(XRD), scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDX).The in vitro cell interaction (MC 3T3-E1) as well as in vivo tissue interactions (rabbit model) in term of plain CS, CS/HAP, CS/collagen/HAP and blended CS/HAP scaffolds was investigated.
2 Experimental
2.1 MaterialsCS powder was supplied commercially with the
degree of deacetylation over 97% (Shanghai Boao Biotechnology Co., Shanghai, China; the viscosity-average relative molecular weight was 1.8×106 Da). HAP powder with average diameter of 15 μm was supplied by the Shanghai Boao Biotechnology, Shanghai, China. K2HPO4 (A R) and CaCl2 (A R) were acquired from Guoyao Co. (Shanghai, China). Collagen I (from calf skin) was supplied by Sigma Corporation.2.2 Preparation of the composite scaffolds
A CS aqueous solution was prepared by dissolving CS powder into acetic acid solution with magnetic stirring. The stoichiometric 2 M CaCl2 solution and 1.2 M K2HPO4 solution with a Ca/P atom ratio of 1.67 were also respectively added into CS aqueous solution with stirring. The dosage of chemicals was used as
Table 1 list. After stirring, the suspension was put into dishes (diameter in 30 mm, and depth in 5 mm), and then rapidly transferred into a freezer at presented temperature –40 ℃ to solidify the water and induced phase separation. The solidifying route was maintained at that temperature over night. In the next stage, frozen samples were lyophilized using a freeze-dryer (Uniequip, Germany) for 24 hrs. The obtained scaffolds were rinsed in a mixture of 0.1 N sodium hydroxide solution and pure ethanol with a 2:1 volumetric ratio for 24 hrs at 37 ℃. After immersion, the samples were washed with deionized water till the pH of washout water was about 7. Finally, the samples treated were freeze-dried again to obtain the porous scaffolds. The samples were denoted by H-CS.
The collagen I was added into the CS solution according to Table 1, and mixed into a homogenous solution. Then a stoichiometric 2 M CaCl2 solution and 1.2 M K2HPO4 solutions with a Ca/P atom ratio of 1.67 were also respectively added into CS/collagen I solution. The followed process was the same as described above. The samples were denoted by C-CS.
HAP/CS composite scaffolds were also prepared via blending method. Briefly, powdered HAP was added into a CS aqueous solution of acetic acid solution under magnetic stirring and followed by ultra sonic treatment. After the mixture was in a suspension state, the scaffolds were mould, frozen, lyophilized as described above. The samples were denoted by B-CS.
As a control, plain CS scaffolds were fabricated via lyophilizing the CS aqueous solution according to Table 1, while the plain CS scaffolds were denoted by CS.2.3 Characterization2.3.1 XRD
The phases of porous composite scaffolds were characterized by X-rays diffraction (XRD; MASL, Beijing, China, 40 kV, 20 mA, 1°/min). 2.3.2 SEM examination
The scaffolds were cut by a razor blade to expose the inner parts. After coated with gold in a sputtering device, all samples were observed under a scanning electron microscope (XL-30 ESEM, Philips Co., the
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Netherlands) with an accelerating voltage of 20 kV. 2.3.3 EDX
A energy-dispersive X-ray analysis (ISIS-300X, Oxford), which combines with SEM (S-520, Hitachi)was used for EDX map to show elemental composition and surface distribution.2.3.4 in vitro cell culture
Osteoblast cells line MC 3T3-E1 were cultured in DMEM supplemented with 10% fetal bovine serum (GIBCO Co., U.S.A.), 100 U/mL penicillin (Sigma, St. Louis, MO), and 100 μg/mL streptomycin (Sigma). Cells were incubated at 37 ℃ in a 5% CO2 incubator and the medium was changed every 2 days. When the cells reached the stage of confluence, they were harvested by trypsinization followed by the addition of fresh culture medium to create a cell suspension. A cell suspension with a concentration of 1 × 106 cells/mL was loaded into the 3-D propous scaffolds, with 300 μL of suspension for each scaffold. The scaffolds were put in a polystyrene 48-well fl at-bottom culture plate and incubated at 37 °C in a 5% CO2 incubator. After cells were attached, fresh culture medium was added until the total medium volume was 500 μL. Culture medium was changed every 2 days.
A MTT assay was applied in this study to quantitatively assess the number of viable cells attached and grown on the tested scaffolds at 1, 3, 5, 7 day’s cell culture. Briefly, all the tested scaffolds were fetched to a new 48-well fl at-bottom culture plate with 500 μL DMEM. 40 μL MTT (Sigma) solution (5 mg/mL in PBS) were added to each sample, followed by incubation at 37 ℃ for 4 h for MTT formazan formation. The upper solvent was removed and 1 mL of 10% sodium dodecyl sulfate (Sigma) in 0.01 N HCl was added to dissolve the formazan crystals for 6 h at 37 ℃. During the dissolving period, the spongy scaffolds were squeezed every 30 min to ensure the complete extraction of the formazan crystals. 300 μL upper solvent was removed to 96-well flat-bottom to measure the optical density (OD) values. The OD value at 492 nm was determined against the sodium dodecyl sulfate solution blank. Five parallel replicates were read for each sample. 2.3.5 in vivo animal test
To evaluate the in vivo tissue interactions of these scaffolds, the rectangle scaffold with 5 mm×5 mm×5 mm were implanted into the under subcutaneous along the spine of rabbits. For this, healthy and skeletally mature male New Zealand white rabbits with a weight between 2.0 and 2.5 kg were selected
and cared according to Administration guidelines for the care and use of laboratory animals of Jinan University. The rabbits were operated on under general anesthesia (1 mL kg−1 body weight, 3% amobarbital, China). After their backs were shaved and disinfected with 70% ethyl alcohol, shallow incisions were made under subcutaneous tissue, and pockets were created. Specimens of each kind of the composite were inserted under the subcutaneous and the skin was closed with sutures (Silk, Ethicon, INC., USA). At 4, 8, and 12 weeks post-implantation, euthanasia was performed with an overdose of pentobarbital sodium. A total of 6 rabbits (2 rabbits per each period) were used for the implantation of scaffold sections. For histological study, the implants with surrounding tissue were removed, fi xed with 4% formaldehyde in PBS, and then decalcifi ed in 10% formic acid. After dehydration in a graded series of ethanol, the specimens were embedded in paraffin wax. Paraffin wax-embedded specimens were sectioned at 5 μm thickness with a microtome, and the sections were stained with hematoxylin and eosin (HE) for the observation by light microscopy (Axioskop 40, ZEISS).
3 Results and discussion
3.1 Phase analysis
Fig.1 shows XRD patterns of CS-based composite scaffolds. The XRD patterns were verifi ed by the Power Diffraction File (HAP: Card No. 090432; CS: Card No. 391894). HAP is the main crystals phase in H-CS, C-CS and B-CS samples, but the difference still exists. When compared to the peaks of HAP in B-CS, a broader pattern in C-CS is indicative of the presence of smaller HAP crystals size. The lower intensity peaks occur in C-CS and H-CS are evidence of less crystallization degree. Generally, the reaction of the HAP formation in the CS matrix reaches a pseudo-stationary regime in 8-10 h and the transformation completed in about 24 h[22]. After 24 hrs precipitation, H-CS and C-CS show HAP formation with less crystallization degree
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from XRD pattern as shown in Fig.1. According to Rusu’s study[23], HAP in the CS composite exhibited a low degree of crystallinity even 24 hrs precipitation with a value of 58.74%. For biomedical application, samples containing HAP with low crystallinity are needed due to their high ‘in vivo’ resorbability rate. For bone tissue engineering, CS-based scaffolds with a low crystallinity HAP would improve their biocompatibility and biodegradation[24].3.2 Morphology analysis
The morphologies of the scaffolds were examined with SEM. All CS-based scaffolds showed a similar spongy appearance (Fig.2) in macroscopic morphology, which indicated that neither adding the HAP in the system nor percipitating the scaffolds change the porous structure (Fig.2 (c) and (e)), for their relative high strength can resist the distortion during the SEM sample preparation. However, the microscopic morphology on pore-wall surfaces was quite different. The surface of CS scaffold is smooth as seen in Fig.2 (b). Typical images of the surface morphology of the others prepared CS-based composite scaffold are shown Fig.2 (d), (f) and (h). For B-CS in Fig.2 (d), the HAP particles scattered at the surface with different size and shape. Some of them show less or no affi nity with the CS matrix. For H-CS in Fig. 2(f), the inorganic particles exhibit as rod-like shape with relatively
uniform particle size. These rod-like HAP particles with 5 μm in diameter were of random orientation. Most of them were of regular shape and clear contours without obvious agglomeration. No obvious delamination was observed between the inorganic phase and the polymer phase. For C-CS in Fig.2(h), the inorganic fi ne granules, which are about less than 5 μm in diameter, densely occurred at the surface with relatively uniform size and compactly bonded C-CS scaffold.
By the way, the presence of collagen in C-CS sample resulted to a more homogeneous structure formed-HAP particles than those in H-CS, which was further certified by EDX element maps as shown in Fig.3. In Fig.3, the concentrated Ca and P elements distribution (one site identifies as square frames in Fig.3) occurred at B-CS composite, which means HAP particles just mix into the polymer matrix. In Fig.3, a homogeneous distribution of Ca and P elements was observed at the surface of B-CS and C-CS, which implied the formation of HAP precipitation was affected by CS or ( and) our study as shown in Figs.2 and 3. Further, when the HAP nucleus grow, the particles will grow in a rod-like shape attributed to its crystallization feature if there are enough ionic concentration[30], just like the HAP particles in H-CS scaffold. However, there were a lot of nucleation sites in C-CS since collagen also carried much functional group, together with CS, which is responsible for mineralization. The more the nucleation sites, the
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more the HAP nucleus formed; and then much more Ca2+ and PO4
3- were consumed for crystal nucleation. At this moment, nucleus can not grow in their typical crystallization way due to lower ionic concentration, so that densely tiny HAP granules other than rod-like HAP occurred in C-CS sample with more homogeneous distribution and more tightly compact with matrix as shown in Figs.2 (g) and Fig.3.3.3 in vitro cell test
The biocompatibility of the scaffolds was assessed on cells' proliferation. Cell proliferation was examined with MTT assay (Fig.4). The same amount of MC 3T3-E1 cells were seeded on the scaffolds. The cell density gradually increased with prolonged culture time, but the difference was still observed. At early time (3 days) points, an MTT value of CS is the highest level than others, and a significant difference
against others exists. At 5 and 7 days’ culture, H-CS and C-CS with homogeneous dispersion of HAP show higher OD value levels, and also show significant differences against plain CS. No signifi cant difference occurred between plain CS and B-CS with relatively concentrated HAP dispersion at 5 and 7 days’ culture. When compared with B-CS, H-CS and C-CS have a significantly higher OD level respectively. It is accord with other’s reports [18, 20], that a homogeneous dispersion of HAP enhanced in better cell proliferation. However, the amount of cells on C-CS was obviously higher at 7 days' culture than on others, which indicated that the MC 3T3-E1 cells showed much better viability properties on C-CS.
Furthermore, although the incorporation of HAP in scaffold via precipitation shows better cell viability properties , the presence of collagen improved the cell proliferation more effectivelly as the study’s result. The reason why the diffrence vialiaility between H-CS and C-CS exist not only in the prense of collagen but also the morphology of fi ne formed-HAP on polymer matrix for nanosize HAP always show a capacity of enhancing the preosteoblasts proliferation[10].3.4 in vivo evoluation
A rabbit model was used to evaluate the composite scaffolds’ tissue interactions. During the experiment, all rabbits remained in good health and did not show any wound complications. At explantation, no infl ammatory signs or adverse tissue reaction were
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seen. Macroscopically all the scaffolds were enveloped in thin tissue capsules.
Analysis of the scaffold sections via light microscopy revealed tissue reaction and materials’ degradation levels at 4, 8, and 12 weeks post-implantation as shown in Fig.5. Throughout the observation periods, fibrous tissue was observed around the implanted zones. The most significant difference in the scaffold sections is the degradation of the materials. At 4 weeks implantation, it was observed that the composite scaffolds of H-CS and C-CS showed obvious breaks of materials’ sections and B-CS had a little break; however, the plain CS scaffold kept its shape very well. At 8 weeks, most of the composite scaffolds broke into segments while no obvious change can be seen in the plain CS scaffold. At 12 weeks, also as shown in Fig.6, scaffold segments in C-CS became chips, while extensive segments remain in H-CS and B-CS. However, the plain CS scaffold still kept no obvious change. As we know, CS scaffold degradation can be modulated by varying the DDA, crystallinity, or molecular weight of the CS, or the amount of calcium phosphate included in the scaffold. The study by H Kashiwazaki thought that the homogeneous HAP/CS composite showed improved biocompatibility and biodegradation[21], the same result as Kashiwazaki’s was obtained at the initial 4 weeks. However, our study shows the presence of HAP, more than a homogeneous structure, was beneficial for composite scaffold biodegradation at the next periods. Also, crystalline calcium phosphate degrades slowly in vivo, so the presence of calcium phosphate in the scaffolds, may have contributed to the slow degradation rate[20], but no obvious difference in the study could be observed between H-CS with lower crystallinity
degree HAP and B-CS with higher crystallinity degree HAP from Fig.6. However, C-CS with HAP granules enhanced degradation of CS composite scaffold in vivo effectively; even a chemical binding was formed in blending of collagen with CS[32]. Okada T implanted the collagen suture in the subdemal tissue of rabbit, the degradation product of collagen was detected at 4 wk post-implantation[33]. Cui also reported that collagen got a higher weight loss in vivo when compared with nano-HAP in nano-HAP/collagen/poly(L-lactide) composite[34]. Basically, the faster biodegradation of C-CS composite is mostly attributed to collagen’s in vivo degradation.
To our present knowledge, the duration when the scaffold should maintain its shape while new bone is forming is not known. However, scaffold degradation should occur to allow for bone regeneration into the defect site. A slower degradation would limit tissue ingrowths while a faster degradation can not maintenance of mechanical strength for functional bone to regenerative. In the next studies, techniques to better control the degradation rate, such as incorporation of lysozyme[35], modifi cation of CS[36], or modulation size of calcium phosphate included in the scaffold will be investigated in an effort to balance the requirements for time to develop functional bone tissue and time to resorb or degrade the scaffold.
4 Conclusions
The CS-based composite scaffolds using a lyophilization method and in situ precipitation were prepared. The morphological and compositional properties of composites were investigated by XRD, SEM and EDX. HAP particles in rod-like shape were distributed homogeneously within the H-CS matrix; while fine HAP granules densely occurred in C-CS scaffold due to the presence of collagen. The in vitro cell culture (MC 3T3-E1) as well as in vivo tissue interaction (rabbit model) for 4, 8 and 12 weeks in term of plain CS, H-CS, C-CS and B-CS was investigated. A homogeneous HAP distribution in the composite scaffolds enhanced cell proliferation, together with the presence of collagen. All the scaffolds were enveloped by fibrous tissue in vivo. C-CS showed a higher degradation rate than H-CS and B-CS scaffolds in vivo after implantation; and plain CS kept no obvious change in shape even after 12 weeks implantation. However, the homogeneous distribution of HAP in of H-CS and C-CS have no obvious difference in in vivo
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degradation than the concentrated distribution in B-CS. It is argued that apart from the incorporation of HAP in the CS matrix, collagen plays a major role in the scaffold degradation in vivo.
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