In Vivo Evaluation of S-Chitosan Enhanced Calcium Phosphate

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ABSTRACT: Calcium phosphate cements (CPC), made from dicalcium phosphate dihydrate and calcium hydroxide and reinforced with water soluble.

In Vivo Evaluation of S-Chitosan Enhanced Calcium Phosphate Cements XIAOHONG WANG,1,2,* JIANBIAO MA,2 Q. L. FENG1 AND F. Z. CUI1 1

Department of Materials Science & Engineering Tsinghua University, Beijing 100084 P.R. China

2

The State Key Laboratory of Functional Polymer Materials for Adsorption and Separation Institute of Polymer Chemistry, Nankai University Tianjin 300071, P.R. China ABSTRACT: Calcium phosphate cements (CPC), made from dicalcium phosphate dihydrate and calcium hydroxide and reinforced with water soluble S-chitosan, were investigated in vivo. Cylinders of these cements were prepared and prehardened before implantation into preformed radial defects in rabbits. Histological observations after 1, 4, 14 and 22 weeks, respectively, were performed on thin decalcified sections. No inflammation or other negative response was found in the S-chitosan containing cements (S-CPCs). After 4 weeks, newly formed trabeculae contacted with the implant directly in the lower S-chitosan sample, while a thin layer of fibers had formed between the newly formed bone and the implant in the higher S-chitosan samples. The degradation rates of the S-CPCs were significantly lower than the original CPC cement alone. Most of the S-chitosan cements were still present at the end of the 22 weeks. The implant material and the surrounding infiltrated fluid layer were examined by back scattered scanning electron microscopy and X-ray energy-dispersive spectrometry. KEY WORDS: bone substitutes, skeletal repair, S-chitosan, calcium phosphate cement, osteoconduction, trabecula, bone repair.

*Author to whom correspondence should be addressed. E-mail: [email protected] Journal of BIOACTIVE AND COMPATIBLE POLYMERS, Vol. 18—July 2003 0883-9115/03/04 0259–13 $10.00/0 DOI: 10.1177/088391103036042 ß 2003 Sage Publications Downloaded from jbc.sagepub.com at Tsinghua University on February 7, 2015

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T

he repair of large bones is always a difficult problem. The natural biological bone repair materials used by surgeons for autografts, allografts, vascularized grafts and other bone transport techniques, have many shortcomings such as high morbidity of autografts, high endemic risk of allografts, sophisticated infrastructure technique of vascularized grafts. Although some synthetic bone substitutes have been developed, there exists a lack of confidence in their biological performances, particularly in long term behavior, in vivo safety and efficacy [1]. For example, the most commonly used calcium phosphate ceramics are very different from hydroxy apatites developed in vivo and have limited application in orthopedics because of their low fatigue properties relative to bone [2]. Recently attention has been directed to self-setting calcium phosphate cements because they are highly osteoconductive, readily osteointegrated, easy to shape and gradually degrade [1–3]. The end products of calcium phosphate cements such as hydroxyapatite (HA), brushite, calcium-deficient hydroxyapatite or amorphous calcium phosphate [3] are similar to the mineral phase of natural bone. But the lack of organic phase may lead cements to decay and fatigue easily when in contact with blood or other body fluids. Our previous studies have shown that water-soluble phosphorylated chitin (P-chitin) [4] and phosphorylated chitosan (P-chitosan) [5] can enhance the mechanical strengths of two easily obtained and readily biodegradable calcium phosphate cements (CPCs) (i.e. monocalcium phosphate monohydrate (MCPM) with calcium oxide (CaO) in 1 M phosphate buffer (pH ¼ 7.4) and dicalcium phosphate dihydrate (DCPD) with calcium hydroxide [Ca(OH)2] in 1 M Na2HPO4 solution) as well as induce osteoconduction of the CPCs in vivo [6,7]. In vitro tests have shown that water soluble disodium (1,4)-2-deoxy-2-sulfoamino- -Dglucopyranuronan (S-chitosan, Mw 6.47  103, with 12.12% S) has the same properties as P-chitin and P-chitosan. Owing to the number of negative charges in these molecules, S-chitosan chelates Ca2þ and increases the dissolubility of the start materials in the cements. In a certain range of the liquid phase, the addition of S-chitosan greatly improve the mechanical strength of the CPCs [8], especially the mechanical strength of the dicalcium phosphate dihydrate (DCPD)/ calcium hydroxide [Ca(OH)2]/1M Na2HPO4 cement. The aim of this study is to examine the short-term effects of the S-chitosan containing cements in radial bone defects in rabbits.

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MATERIALS AND METHODS

Materials Chitin was obtained from Tianjin Zhengtiancheng Company, China. Perchloric acid was purchased from Tianjin Chemical Company Limited, China. Chlorosulfonic acid, pyridine, DCPD, MCPM, CaO, Ca(OH)2, chromium trioxide and other chemicals were purchased from Peking Chemical Company, China. All of the chemicals were of analytical grade. Commercial CaO was crushed in a pulverizer and heated at 900 C for 2 h to remove H2O and CO2 and then stored in a vacuum desiccator before use. Preparation of the S-Chitosan Reinforced CPCs (S-CPCs) Calcium phosphate cements and S-CPCs were prepared similar to the procedures reported by Takagi [9]. Briefly, cement powders were prepared by mixing calcium phosphate (DCPD) with basic calcium hydroxide [Ca(OH)2] in a stoichiometic ratio according to Equation (1) in an agate mortar. The liquid phase was an 1 M Na2HPO4 aqueous solution. The cement powder was mixed with the liquid phase using the solid-to-liquid (S/L) ratio (g/mL) of 2.29. Different amounts of S-chitosan (Mw was 6.47  103, and 12.12% S) using 0.03 g/mL (or 0.07 g/mL) dissolved into the liquid phase. After the two phases were mixed for 2 min, the paste was loaded into 3 mm (diameter)  8 mm (high) plastic molds that were stored in a 37 C, 80 RH box for 24 h. The cylinders were then sterilized under Co60 for 8 h before use. 6CaHPO4  2 H2 OðDCPDÞ þ 4CaðOHÞ2 ! Ca10 ðPO4 Þ6 ðOHÞ2 ðHAÞ þ 18H2 O

ð1Þ

Surgical Techniques After general anesthesia 12 adult white rabbits (2.0–3.0 kg) were shaved and disinfected in the areas over the left radii with a mixture of 15% ketamin hydrochloride and xylazine hydrochloride (1 mL/kg). The radii were exposed by a skin incision and bone defects (>8 mm in length) were surgically created with a saw. Eight of the twelve rabbits were implanted with cements with different amounts of S-chitosan. The other

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four were used as controls. The wounds were closed in layers with silk thread. Microscopic Observations and Histological Studies The rabbits were divided into three groups, four in each, and one from each group was sacrificed after 1 week, 4, 14 and 22 weeks after surgery, respectively. X-ray radiographs of the recovered rabbit radii with the implanted materials were taken with a Philips 1025 X-ray Radiograph system (Japan). The implant materials were harvested with the surrounding tissue and fixed in 10% neutral buffered formalin. For histological evaluation samples were decalcified with 8% nitric acid, dehydrated, embedded (in paraffin), sectioned (Leitz, Western Germany) to 7 mm thick and stained with Masson trichroism (M-T) or Hematoxylin-eosin (H-E). Isolated samples were also analyzed by back scattered scanning electron microscopy (BSE) and X-ray energy dispersive spectrometry (EDS) (Philips XL-30W/TMP, Japan). RESULTS

Macroscopic and Radiograph Observations Radiographic pictures of the experimental rabbit radii (Figure 1) indicate that S-CPC had integrated with the surrounding tissues. The implants appear stable in the defect areas and no osteolysis and hyperplasia was found in any of the implanted rabbits through out the study. Histological Observations After 1 week, the S-chitosan implants were still in their original shapes and some hematoma was observed between the implants and bone. In the higher S-chitosan (0.07 g/mL) sample (Figure 2), hematoma had changed into cartilage tissue with numerous chondroblasts. In the control group (cements without chitosan), a great deal of hematoma was found around the implant, however, bone formation with the CPC samples was not observed. After 4 weeks, the fibrous layer around the higher S-chitosan sample (0.07/mL) was a little thinner than that around the lower S-chitosan sample (S-chitosan: 0.03 g/mL) (Figure 3(a), 3(c)). Furthermore, the fibrous layer around the lower S-chitosan sample was even thinner than that of the control group. The S-chitosan samples, maintained their

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(a)

(b)

(d)

(e)

(c)

(f)

Figure 1. Radiographs of CPC samples after operation: (a) 1 week (S-chitosan: 0.07 g/mL); (b) 4 weeks (S-chitosan: 0.07 g/mL); (c) 14 weeks (S-chitosan: 0.07 g/mL); (d) 14 weeks (S-chitosan: 0.03 g/mL); (e) 14 weeks (S-chitosan: 0 g/mL); (f) 22 weeks (S-chitosan: 0.07 g /mL).

Figure 2. Tissue response of an S-CPC sample (S-chitosan: 0.07 g/mL) after 1 week (decalcified, magnification 40, M-T stain).

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Figure 3. Tissue response of S-CPCs after 4 weeks: (a) S-chitosan: 0.07 g/mL, decalcified, magnification 40, M-T stain; (b) magnification (400) of (a); (c) S-chitosan: 0.03 g/mL, decalcified, magnification 40, M-T stain.

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original shapes and the encapsulated fibers changed into trabeculae with active osteoblasts, monocytes and multinucleated giant cell formation. In the control group, part of the implants were infiltrated with body fluid and some large particles had detached. After 14 weeks, the S-chitosan implants were only slightly degraded compared to the CPC implants. Umbral materials observed by radiographs of the lower S-chitosan sample were less distinctive than those observed 4 weeks after the implantation (Figure 1(d)). The material was surrounded by newly formed dense trabeculae (Figure 4(a)). In the control group, about one fifth of the implants remained (Figures 1(e), 4(b)). The CPC implant had changed into trabeculae-like tissue after

Figure 4. Newly formed bone after 14 weeks: (a) S-chitosan: 0.07 g/mL, Decalcified, magnification 40, H-E stain; (b) BSE image of the pure CPC; (c) EDS analysis of the central part of the cement body in Figure (b); (d) EDS analysis of the body filtration cement in Figure (b).

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Figure 4. Continued.

infiltration by body fluid. The Ca/P(At) ratio in the center part of the implant was 1.28 (Figure 4(c)) and in the surrounding trabeculae region was 0.46 (Figure 4(d)). Sulphur (S), silicium (Si) and potassium (K) were found in the trabecula regions. After 22 weeks, three fourths of the higher S-chitosan (0.07 g/mL) (Figure 1(f)) and half of the lower S-chitosan (0.03 g/mL) had degraded. The more obvious degradation of the implants took place close to the heart side of the rabbits. The newly formed trabeculae was arranged in

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good order (Figure 5(a)) and there was significant evidence of remodeling during the final 8 weeks. EDS analysis revealed that the Ca/P ratio in the central part of the lower S-chitosan implant was 1.64 (Figure 5(c)), while in the infiltrated body fluid layer the ratio was 1.91 (Figure 5(d)). The remodeling of the defective control radius was complete after 22 weeks but the edges of the remodeled radius were not as smooth as those of the original radius.

Figure 5. (a) Remodeling of bone took place after 22 weeks (S-chitosan: 0.07 g/mL, magnification 40, H-E stain); (b) BSE image of the S-CPC (S-chitosan: 0.03 g/mL) after 22 weeks; (c) EDS analysis of the central part of the cement body in Figure b; (d) EDS analysis of the filtrated layer in Figure b.

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Figure 5. Continued.

DISCUSSION

Many implantation studies have confirmed that the conventional tetracalcium phosphate (TTCP) containing c-CPC shows excellent biocompatibility towards soft and hard tissue [10–12]. However, Miyamoto et al. reported that c-CPC induces a severe inflammatory response when the paste was implanted subcutaneously in rats immediately after mixing [13]. Takechi et al. [14] added chitosan to c-CPC and found that the chitosan containing CPC was surrounded by thin fibrous tissue with only a slight inflammatory response 1 week after a subcutaneous implantation. Our previous in vivo experiments on phosphorylated chitin and phosphorylated chitosan containing

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two calcium phosphate cements have shown that the two cement systems, MCPM/1 CaO/1M phosphate buffer (pH: 7.4) and DCPD/ Ca(OH)2/1M Na2HPO4, were safe and biocompatible when used in rabbits [6,7]. The P-chitosan containing CPCs (P-CPCs) and lower P-chitin concentration P-CPCs had an excellent tissue response without any fibers appearing during the bone repair processes. In this study, the DCPC/Ca(OH)2/1 M Na2HPO4 system was chosen to test the S-chitosan containing cements in the rabbits. It was expected that the order of Ca2þ binding ability of the three kinds of chitin derivatives should be S-chitosan > P-chitosan > P-chitin. This was based on the in vitro degradation rates of the three chitin derivative cements at the same polymer proportions that are in the order: P-chitin cements>P-chitosan cements>S-chitosan cements. Similar to P-chitin and P-chitosan, S-chitosan has a strong Ca-bonding ability and also increases the solubility of the cement starting materials [4,5]. Consequently, it was expected that along with the increased mechanical strengths that the degradation rates of the S-chitosan containing cements should be decreased. Like the former two chitin derivative reinforced cements, the degradation rates of the S-chitosan cements were proportional to the added S-chitosan. Histological examinations revealed that hematoma, which appeared in the rabbit implants after 1 week and the connective tissue in the implants after 4 weeks, were related to the S-chitosan proportions. This might be due to the blood-anticoagulation properties of S-chitosan [15]. After 4 weeks, fibers changed into trabeculae along with the broken side of the host bone. The repair processes are different from the self-healing process for fractured bone; fibrocartilaginous callus, cartilage and bone callus form before fibers were changed into trabeculae. Since the degradation rates of the S-chitosan implants were slow, especially for the higher S-chitosan sample, the newly formed bone was rather dense. After 22 weeks, about three fourths of the higher S-chitosan implants and half of the lower S-chitosan implants were still intact. One protruding edge of the higher S-chitosan implant was clearly reflected by the radiograph (Figure 1(d)). Compared to the EDS analysis results for the lower S-chitosan implants, the Ca/P ratio in the central part of the implants were 1.64 but 1.91 in the infiltrated body fluid layer. This suggested that some regenerated biomaterials were present in the latter. The similar amounts of S-chitosan in these two parts indicated that the basic components had not changed very much. In the control group, the implants changed into trabeculae-like tissue after infiltration by body fluid while a typical osteoconductive phenomenon seems to be induced by the S-chitosan implants.

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A thin layer of fibers around the specimen after about 1 week was observed by histopathological examinations. No inflammatory response to the S-chitosan was observed. Cell-mediated bone remodeling had taken place. The newly formed trabeculae were the continuation of the broken bone and the amount increased with time. To meet different bone repair requirements, different degradation rates are needed; in this study we have found that S-chitosan can be used to control this variable. Based on the outcome of our histological evaluations, which are in accord with the radiographic evidence, S-CPCs are biocompatible. Consequently, S-chitosan containing calcium phosphate cements (S-CPCs) are expected to have special use in bone tumor and leukemia treatments. ACKNOWLEDGMENT

The project is supported by the funds of the National Natural Science Foundation of China and the Education Ministry of China. REFERENCES 1. Langstaff, S., Sayer, M., Smith, T.J.N. and Pugh, S.M. (2001). Resorbable Bioceramics Based on Stabilized Calcium Phosphates. Part II: Evaluation of Biological Response, Biomaterials, 22: 135–150. 2. Constantz, B.R., Ison, I.C., Fulmer, M.T., Poser, R.D., Smith, S.T., Vanwagoner, M., Ross, J., Goldstein, S.A., Jupiter J.B. and Rosenthal, D.I. (1995). Skeletal Repair by in Situ Formation of the Mineral Phase of Bone, Science, 1267: 1796–1799. 3. Yoshimine, Y., Sumi, M., Isobe, R., Aan, H. and Maeda, K. (1996). In vitro Interaction Between Tetracalcium Phosphate-based Cement and Calvarial Osteogenic Cells, Biomaterials, 23: 2241–2243. 4. Wang, X., Ma, J., Wang, Y. and He, B. (2002). Reinforcement of Calcium Phosphate Cements with Phosphorylated Chitin, Chinese J. Polym. Sci., 4: 325–332. 5. Wang, X., Ma, J., Wang, Y. and He, B. (2001). Structural Characterization of Phosphorylated Chitosan and their Applications as Effective Additives of Calcium Phosphate Cements, Biomaterials, 22: 2247–2255. 6. Wang, X., Ma, J., Wang, Y. and He, B. (2002). Bone Repairs of Rabbits with Calcium Phosphate Cements, Biomaterials, 23: 4167–4176. 7. Wang, X., Ma, J., Feng, Q. and Cui, F. (2002). Skeletal Repair in Rabbits with Calcium Phosphate Cements Incorporated Phosphorylated Chitin, Biomaterials, 23: 4591–4600.

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