Double-Network Interpenetrating Bone Cement via in situ Hybridization Protocol

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Double-Network Interpenetrating Bone Cement via in situ Hybridization Protocol

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By Jing Wang , Changsheng Liu , * Yufei Liu , and Shuo Zhang

A major obstacle to the development of a more effi cient calcium phosphate bone cement (CPC) is its relatively low mechanical strength and susceptibility to brittle fracture. Herein, a series of dual-setting hybrids have been devel-oped based on the in situ crosslinking polymerization of glycidyl methacrylate derivatized dextran (Dex-MA) and the synchronous hydration process of CPC. Such a strategy is highly desirable for bone regeneration and provides insight into hybrid cement designing. The structure and physical properties of the resulting hybrids are investigated. Compared with CPC, the initial setting time is shortened and may be modulated. As a result, the hybrid cement possesses characteristics of both of its components, and is tunable from stiff-but-not-brittle to ductile-but-not-soft depending on the composition of the double network. Introduction of the polymeric moiety does not obstruct the funda-mental hydrating process of CPC. Due to the synergistic effect produced by the double-network structure, the resulting hybrid has an optimal compres-sive strength of over 98.3 MPa. The mass ratio of the binary network and the size of apertures are shown to be key parameters for improving the compres-sive strength.

1. Introduction

There is a growing need for the ability to regenerate bone due to various clinical musculoskeletal defects such as trauma, tumor resection, congenital malformation reconstruction, and age-related diseases. [ 1–4 ] Though autogenous bone is usually the treatment of choice, unfortunately, the insuffi cient supply of bone and the risk of infection or pain at the donor site limits its applications. [ 5–7 ] Current artifi cial substitutes are far from ideal and each has its specifi c problems and limitations. Cal-cium phosphate cements (CPCs), fi rst developed by Brown and Chow in the early eighties, [ 8 ] have attracted much attention for

© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheiAdv. Funct. Mater. 2010, 20, 3997–4011

DOI: 10.1002/adfm.201000995

[∗] Prof. C. Liu State Key Laboratory of Bioreactor Engineering and Key Laboratory for Ultrafi ne Materials of Ministry of Education Shanghai, 200237 (P. R. China) E-mail: [email protected] Dr. J. Wang , Y. Liu , S. Zhang Key Laboratory for Ultrafi ne Materials of Ministry of Education, and Engineering Research Center for Biomedical Materials of Ministry of Education East China University of Science and Technology Shanghai 200237 (P. R. China)

their ability to combine the advantage of being freely moldable and osteoconduc-tive. This is a signifi cant step forward in the fi eld of bioceramics, since it provides a material which, in addition to being bio-active, is moldable and has the capacity of self-setting within the bone cavity at human body temperatures. [ 9–12 ] As a result, CPC was approved in 1996 by the FDA for repairing craniofacial defects in humans, thus becoming the fi rst CPC available for clinical use.

Although CPC is highly promising for use in a wide range of applications, its mechanical properties are non-ideal and it is known to degrade. [ 13 ] The relatively low strength and susceptibility to brittle fracture have limited its use to only non-stress-bearing applications. [ 14 , 15 ] One clin-ical study on periodontal repair demon-strated that the tooth mobility resulted in the early fracture and eventual exfoliation of the brittle CPC implants. [ 16 ] Besides low

other shortcomings with the conventional
strength, there areCPC regarding its practical application, including its prolonged setting time and the liklihood of washout upon early contact with physiological fl uids or when bleeding occurs, due to the diffi culty in some cases of achieving complete hemostasis.
Therefore, extensive studies have been performed to circumvent such problems, including the modifi cation of CPC granules, [ 17 ] adjustments to the solidifi cation process, [ 18 , 19 ] and introduction of polymers, [ 20–23 ] etc. Barralet and co-workers investigated the effect of citrate, lactate, glycolate, malate, and tartrate ions on the properties of CPC. They found these sodium salts of α -hydroxylated organic acids were involved in increasing the ζ potential that is responsible for the macroscopic liquefying effect, such that less water was required and higher strengths could be obtained. [ 24 , 25 ] Liu et al. [ 26 ] ] elucidated the effects of the granularity of CPC raw materials on the hydration process and compressive strength of the hardening body. Notably, the Xu research group [ 27 ] explored strong, bioactive composites by combining pre-hardened CPC particles and nano-silica-fused whiskers in a resin matrix of Bis-BMA. The whisker-CPC com-posites had a maximum fl exural strength of 164 MPa, which was 3 times higher than previous values. Furthermore, they incorpo-rated both chitosan and absorbable meshes for synergistic rein-forcement and obtained a desirable fl exural strength (43MPa) and a toughness higher than those cancellous bones. [ 28–30 ]

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Scheme 1 . Schematic procedure for the formation of CPC/Dex-MA double interpenetrating networks.

Figure 1 . Washout resistance test after 10 min in deionized water. Left: CPC/Dex-MA ( r = 5); right: CPC/Dex-MA ( r = 5, without APS/TEMED).

In this study, a simple, convenient, and effi cient strategy for directed hybridization based on an interpenetrating double net-work (DN) of calcium phosphate cement and dextran has been explored. Methacrylate groups were introduced onto dextran chains and were crosslinked to generate the fi rst hydrogel seg-ment, and the second cement network was formed by its sub-sequent hydration in the presence of the fi rst one ( Scheme 1 ). Though the similar dual-setting glass-ionomer system had been reported, the primary backbones used were polyacrylate or sodium alginate, the former was nonbiodegradable, while the unmodifi ed alginate gels would remain stable for over one year due to the absence of specifi c enzyme in vivo, thus caused incompatible degradation rate with tissue regeneration. [ 31,32 ]

The aim of this work is to improve the mechanical proper-ties of CPC-based cements through the synergy of polymer and CPC cement. We are interested in this system for several reasons: 1) the polymer selected in the system is dextran, a natural, biodegradable polysaccharide with good biocompat-ibility and low toxicity that has been widely used in biotech-nology applications and clinical treatments, studied as drug delivery vehicle, and more recently has been investigated as a biomaterial. Dextran hydrogels are particularly compelling as scaffolds for tissue engineering applications. [ 33 , 34 ] 2) Besides the hydration of CPC cement, dextran derivates polymerize under aqueous conditions to form a double-network system. The “hard” moiety of CPC cement and “soft” hydrogel segment incorporate into each other to make the hybrid cement stiff and ductile. 3) The interpenetrating network method is used to form novel composites consisting of three-dimensionally con-tinuous integrated phases. This in situ process is expected to facilitate a higher degree of interaction and bonding between the organic and inorganic components, translating into better mechanical performance.

2. Results

In the control group of CPC/Dex-MA, free radical poly-merization of methacrylate would not occur without an ini-tiator and an accelerator. Disintegration of the uncrosslinked

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linear Dex-MA was visible, and the uncon-verted cement also dissolved ( Figure 1 ). By contrast, all the crosslinked samples of CPC/Dex-MA with APS/TEMED showed good resistance against washout and main-tained their original shapes after immersion in deionized water. This apparent distinction caused by the initiator/accelerator might be direct evidence of crosslinking.

2.1. Structural Characterizations

The chemical structures of crosslinked dex-tran methacrylate and its composites with CPC are characterized by FTIR spectra, as shown in Figure 2 . The noticeable strong absorption at 3430 cm − 1 , indicating the sym-metric and asymmetric stretching vibration

modes of the hydroxyl groups, belongs to both the pristine dextran and its CPC-hybrid products. The absorbance peaks at 1720 cm − 1 and 2930 cm − 1 are assigned to the stretching modes of the carbonyl group and methylene on methylated dextran, respectively, confi rming the existence of the Dex-GMA moiety. The characteristic ν 3 PO 4 3 − stretching mode at about 1040 cm − 1 and ν 4 PO4 3 − bending mode at 562 cm − 1 and 604 cm − 1 of the apatite phase are attributed to CPC and its transformation process. The characteristic peak for glucop-yranose at about 700-800 cm − 1 is overlapped by the PO/P-O(H) stretching mode of dicalcium phosphate (DCPA). The absorption peak originating from the double bond on the methacrylate group at 990 cm − 1 , 918 cm − 1 , and 840 cm − 1 is not clearly visible in the series of CPC/Dex-MA composites, which is similar to results reported previously for polymerization of the methacrylate group. [ 35 ] However, since the characteristic peak for the phosphate group exists in the same region, confi r-mation of polymerization through IR spectra is insuffi cient.

Wide-angle X-ray diffraction (WAXD) patterns of crosslinked Dex-MA, CPC and CPC/Dex composites with different r values are displayed in Figure 3A . Crosslinked Dex-MA is an amor-phous polymer demonstrated by a broad diffraction peak centered at a 2 θ value of 18 ° . The series of CPC/Dex-MA com-posites show similar XRD patterns to CPC. The variations in

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Figure 2 . FT-IR spectra of CPC/Dex-MA hybrid materials with different mass ratios. a) CPC; b) CPC/Dex ( r = 10); c) CPC/Dex ( r = 5); d) CPC/Dex ( r = 1); e) CPC/Dex ( r = 0.33); f) Dex-MA. The insert shows the disap-pearance of C = C bending mode.

Figure 3 . A) WAXD patterns of the CPC, Dex-MA and CPC/Dex hybrid with different mass ratios. The period of time between fabrication and measurement was about 72 h. B) XRD patterns of the CPC/Dex com-posite ( r = 5) after different times of incubation in 100% humidity at 37 ° C. The main diffraction peaks of TECP are indicated with ( � ); the 002 and 211 refl ections of HA are indicated with ( ∗ ); and the peak of DCPA is indicated with ( ∗ ).

the distinct (002) peak (2 θ = 25.8 ° ) were used to evaluate the effects of polymeric additives on the conversion to hydroxyapa-tite. As evidenced in Figure 3 A, patterns of the series of com-posite samples exhibit clear peaks at about 2 θ = 25.8 ° and a broad peak centered at about 2 θ = 32 ° , which seemingly desig-nates low-crystalline HA, attributable to the hydration of CPC. This suggests that methacrylated dextran did not shelter the hydration of CPC and the main phase of the hydrated cement was hydroxyapatite. No other phases were found, except for the distinguishable peaks of hydroxyapatite and TECP. However, small but distinct peaks of TECP at about 2 θ = 29.2 ° and 29.8 ° are still present, suggesting the incomplete transformation of CPC.

The XRD patterns at the various time intervals during set-ting are illustrated in Figure 3 B. It is clear that the character-istic crystalline phases of TECP (2 θ = 29.2 ° and 2 θ = 29.8 ° ) and DCPA (2 θ = 25.9 ° and 2 θ = 32.4 ° ) are dominant at the begin-ning of setting ( t = 0.5 h). The hydroxyapatite phase is subse-quently generated and the relative intensities of TECP and DCPA decrease with time. The spectrum of the sample, after incubating in 100% humidity at 37 ° C for 10 days, still shows small but distinct peaks of DCPA and TECP. [ 36 , 37 ]

Figure 4 shows TGA thermograms of CPC and CPC/Dex-MA composites with three different Dex-MA compositions. In the temperature range of up to 800 ° C, CPC was stable without sig-nifi cant loss of weight ( < 5%) while crosslinked Dex-MA dem-onstrated dehydration and a thermal degradation step. The onset degradation temperature, T d , for crosslinked Dex-MA was 268 ° C and it increased slightly as the amount of incorpo-rated CPC increased. It should be noted that the residue from the degradation of the crosslinked Dex-MA moiety also increased progressively with the amount of CPC. Given the weight fraction of residue for crosslinked Dex-MA and for pure CPC, which were 14.14% and 95.29%, respectively, the weight fractions of residue for the CPC/Dex-MA composites

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with different Dex contents can be predicted according to the principle of linear superposition. [ 38 ] For example, the res-idue of CPC/Dex-MA ( r = 1) can be calculated as 14.14% × 0.5 + 95.29% × 0.5 = 54.72%. The calculated values for the crosslinked CPC/Dex-MA composites with feed ratios of r = 10, r = 1, and r = 0.5 are 87.91% ± 0.97%, 54.72% ± 1.05%, and 41.16% ± 0.98%, respectively. These calculated values are not signifi cantly different from the experimental values of 90.57 ± 1.01, 57.75 ± 1.03, and 44.87 ± 0.95%. The results indicate that the composition of the fi nal crosslinked Dex-MA in the hybrid accords with that in the feed. Since the residual Dex-MA monomer dissolves during the immersion process, it confi rms that the polymerization of the methacrylate groups occurred successfully notwithstanding the presence of calcium phosphate.

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Figure 4 . TGA thermograms of CPC and crosslinked CPC/Dex-MA with different mass ratios.

Figure 5 . A) Effect of mass ratios on the setting time of hardened CPC (P/L = 1.85). B) Effect of P/L values on the setting time of hardened CPC ( r = 5). C) Effect of the TEMED concentration on the setting time, at a fi xed composition of r = 5 and P/L = 1.85.

2.2. Setting Time

There are two processes occurring in this mixture: transfor-mation of CPC into hydroxyapatite, and polymerization of methacrylate groups to form the crosslinked structure. As the polymerization is carried out in an aqueous liquid, a Dex-MA hydrogel is formed. Herein, setting time is investigated to explain whether the various networks are formed simultane-ously or sequentially.

Figure 5 A shows the infl uence of Dex-MA content on initial setting time at a fi xed powder-to-liquid (P/L) ratio of 1.85 g mL − 1 . Just after mixing the powder with the liquid phase, the paste is viscous and easily moldable for several minutes, after which the complex cements harden. The control group of CPC paste without Dex-MA set within 13.74 min when mixed with Na 2 HPO 4 . The setting time of Dex-MA-doped composites abruptly decreases to less than 6 min. Enhancing the concentra-tion of Dex-MA from 4.8 wt% ( r = 20) to 75 wt% ( r = 0.33), the setting time reduces to 3.9 min, which is not signifi cantly dif-ferent based on a Tukey’s family confi dence coeffi cient of 0.95. Moreover, these times are shorter than the gelling time of pure Dex-MA (data not shown): CPC particles act as a thickening fi ller in the process of polymeric crosslinking, making the paste rheologically dense.

The initial setting times of the cements prepared with dif-ferent P/L values. with the mass ratio r kept constant at 5, are compared in Figure 5 B. The setting time clearly decreases with the increase of P/L value. Nevertheless, setting time was greater for CPC samples without Dex-MA (13.74 min) even at the lowest P/L. As for CPC, setting was diffi cult for P/L values below 2.5 due to the excessive dilution of the slurry. On the contrary, all dex-tran-containing cements took a remarkably short time to harden regardless of the excessive liquid, demonstrating the synchronous effect of Dex-MA on the hardening reaction of CPC. The results indicate that the network of crosslinked Dex-MA is formed fi rst, and then the hydration occurs in the hydrogel sheltering.

Previous studies on polymerization of glycidyl-methacrylated dextran showed that TEMED acted as an accelerator by promoting the homolytic scission of APS and generating free radicals.

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Similarly, it could be seen that the setting process started imme-diately after the addition of TEMED. Figure 5 C shows the infl u-ence of the TEMED concentration on the setting time at a fi xed composition ( r = 5) and P/L value (1.85). In the hybrid system, it appears that an increase in TEMED concentration results in a faster setting. At higher TEMED concentrations the conversion is almost instantaneous.

2.3. Morphology

Scanning electron micrographs (SEMs) of typical microstruc-tured fracture surfaces of distinctive, aggregated, particle-like CPC samples of different compositions are shown in Figure 6a . All hybrid samples exhibit rough hydroxyapatite crystals and an amorphous crosslinked dextran matrix: the latter penetrates the HA crystals and an interconnected interface is formed with visible micropores. The resulting hybrids are microscopically phase-separated, but macroscopically uniform.

The crystals are irregular in shape and size, and individual crystals have different shapes depending on the Dex-MA con-tent. In the lower Dex-MA content ( r = 10), SEM examinations show formation of tiny, fl uffy, entangled hydroxyapatite clusters (Figure 6 b).

The morphologies of the fracture surface change with increasing polymer content. The morphologies of r = 5 sam-ples are fl ake-shaped or needle-like crystals. The fl ake-shaped crystals are about 1-2 μ m in width, arrayed together and spread outward, forming fl ower clusters (Figure 6 c). Furthermore, the platelets possess a subtle net-like texture consisting of radialized slender needles located on the surface, which have a length of


Figure 6 . Scanning electron micrographs of CPC (a) and CPC/Dex-MA compocontents (b–d). b) r = 10. c) r = 5; the inset bottom right shows that the con the surface of the fl ask. d) r = 0.33; the image in the inset shows the arrcrystals (arrow).

approximately 1 μ m as shown in the insert of Figure 6 c. These results indicate that instead of sheltering the hydration of CPC, the introduction of crosslinked Dex-MA could induce the depo-sition of hydroxyapatite.

Groups of Dex-dominant composites show a different micro-structure compared to those previously discussed. Figure 6 d illustrates the fracture surface of specimen CPC/Dex-MA ( r = 0.33). It is evident that the inorganic particles embed in the Dex-MA matrix. At higher magnifi cation, the agglomerations locally exhibit closely-arrayed, blade-like crystals.

2.4. Swelling Studies

Figure 7 depicts the relationship between Dex-MA content and water uptake. All composites show a signifi cantly higher capa-bility of water uptake. The hybrid materials swell quickly within the fi rst 3 h of incubation, and reach equilibrium weight within about 48h. The degree of swelling increases with time in a non-linear manner with Dex-MA content.

It was originally thought that differences in swelling might be controlled by the extent of the double bond in the methacry-late groups that crosslink the system, i.e., a higher content of Dex-MA would lead to greater formation of a gelled polymer network, thereby restricting the degree of swelling. However, the data in Figure 7 A suggest that this parameter only has a small infl uence on the systems studied. The implication is that the hydrophilicity is stronger, and may be the dominant factor regarding the water uptake capacity of the hybrids.

The volume-shrinkage behavior of CPC/Dex-MA compos-ites was investigated, and the results are shown in Figure 7 B.

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site with different rystals had grown ayed of blade-like

The swelling process is reversed during gradual drying from the surface to inte-rior, thereby resulting in vaporization and volume contraction of the hydrogel. [ 39 ] In accordance with the dependence of water uptake on the Dex-MA content, specimens with higher Dex-MA content dis-played an increased volume contractibility due to the higher equilibrium swelling ratio.

2.5. Distribution of Pore Size

The pore size distribution in the range 3–100 000 μ m was measured by mercury intrusion, and with N 2 adsorption for pore sizes ranging from 300 to 1 nm. The pore size distribution function was calculated as the differential mercury intrusion volume multiplied by the density, and it was plotted against the entrance pore size. [ 40 ] Different CPC systems had their own specifi c pore size distributions. [ 41 , 42 ] For example, Bar-ralet reported the majority of pores in their brushite cement were of diameters between 10 nm and 10 μ m. [ 43 ] Compared with tradi-tional pure CPC, which shows a bimodal dis-tribution at 2–100 nm and 0.1–5 μ m, [ 41 , 43 , 44 ]

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Figure 7 . A) Water uptake of CPC/Dex hybrid with different Dex content as a function of time. B) Volume contractibility of CPC/Dex rod with dif-ferent mass ratios.

Figure 8 . A) Pore size distribution of CPC/Dex-MA with different mass ratios measured by mercury intrusion. B) The N 2 adsorption isotherm for the hydrated cements. C) Pore size distribution curve with different mass ratios determined by BET.

mercury intrusion porosimetry indicated that introduction of the Dex-MA network results in a decrease in the distribu-tion of pore diameters: the majority of pores are between 2–20 nm ( Figure 8A ).The porosity data of the different pore size intervals as well as the total porosity of various mass ratios are described in Table 1 . It is rational that the P/L value is depressed with increasing Dex-MA content, resulting in higher total porosity and pore area. In curve b ( r = 1.25) and curve c ( r = 0.5), a weak distribution at 1–10 μ m is also detected, formed via evaporation of the occupied liquid. On the other hand, higher polymeric content leads to further volumetric shrinkage: a compacted pore structure was obtained. Therefore, the Dex-based group was dominated by meso-pores, despite few large pores.

Figure 8 B presents the N 2 adsorption-desorption isotherm for the CPC/Dex-MA ( r = 0.5, r = 5). The curve can be identifi ed as a type III isotherm, [ 45 ] corresponding to a nearly continuous distribution of pores. The small pore size distribution meas-ured by BET is detailed in Figure 8 C. Compared with CPC-based cement ( r = 5), Dex-based cement ( r = 0.5) had a much

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higher percentage of pores of smaller volume. There is an obvious majority in the 2–10 nm range that corresponds with the incompletely defi ned maximum around 10 nm in the mer-cury intrusion curves. In addition, the presence of larger pores

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Table 1. Effect of CPC/Dex-MA ratio on total porosity and pore area .

Mass ratio, r [w CPC /w Dex-MA ]

Dex-MA content [%]

Average Pore Radius [nm]

Total porosity [%]

Total pore area [m 2 g − 1 ]

5 16.7 6.8 27.97 46.81

1.25 44.4 8.4 28.22 44.57

0.5 66.7 8.1 8.15 12.20

of diameters 10–100 nm was also detected, which might belong to the clearance between hydration products and both moieties.

2.6. pH Variation During and After Setting

Figure 9 compares the variation in pH of CPC and CPC/Dex-MA ( r = 5) slurries during setting. The initial pH values of both were 6.1. In general, no obvious difference in the pattern of pH changes is observed between CPC and the CPC/Dex-MA composite. Both curves steeply increase during the initial process and reach the crest value of 8.9 for CPC and 9.1 for CPC/Dex-MA within 20 min. The CPC slurry maintained a pH value of 8.89–8.92 up to 120 min, and the variation in pH for the CPC/Dex-MA group is very gentle and remained at 9.0–9.1 up to 45 min. Then the pH of both groups declines, but at dif-ferent rates, such that their trends intersect. After 24 h, as the hydrating proceeds to the stage of basically set, the pH values of the two groups were vicinal to 6.8. After that, both pH values gradually reached equilibrium at a constant pH of 6.5.

2.7. Mechanical Properties

Figure 10 depicts the representative stress-strain behavior of CPC and CPC/Dex-MA composites with series mass ratios under uniaxial compression. All of the specimens were kept at a saturated humidity for 24 h. Pure CPC without polymeric additives was brittle with a lower compressive strength at about 24 MPa. At lower Dex-MA contents ( r = 10 and r = 5), shown in


Figure 9 . Variation of pH in the slurry of CPC and CPC/Dex-MA ( r = 5).

Figure 10 A, the yield on stress-stain curves did not show very clearly and the stress after the yield was not a constant value but increased slightly with increasing strain.

As indicated in Figure 10 B, when the CPC/Dex-MA mass ratio decreased to 1.25, an obvious yield appeared. The typical yielding fl ats reveal ductile-soft properties of the specimens, suggesting that the brittle deformation had converted to toughness. Considering the fragility of CPC, we postulate that the ductility is triggered by incorporation of the elastomeric crosslinked Dex-MA moiety.

Compressive strength and compressive modulus of CPC/Dex composites are plotted as a function of Dex-MA content ( Figure 11A ). All CPC/Dex-MA specimens were 2–3 times stronger than that of pure CPC cement under the same solidifying conditions, indicating synergistic reinforcement via incorporation with Dex-MA. Furthermore, the results illustrate that the mechan-ical properties are dependent on the Dex content. The variation of modulus with composition was similar to that of the yield stress. In Figure 11 A, for the CPC-based composites, the com-pressive strength signifi cantly increased from 24.13 ± 3.9 MPa


Figure 10 . Typical compressive stress-strain curves of CPC and CPC/Dex with different Dex content. A) CPC; CPC/Dex r = 10; r = 5; B) CPC/Dex r = 2.5; r = 1.25; r = 1; r = 0.5; r = 0.33.

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Figure 11 . A) Infl uence of Dex-MA content on compressive strength and modulus of CPC/Dex hybrid. B) Infl uence of Dex-MA content on fracture energy of CPC/Dex hybrid. C) Cross section morphology at fracture inter-face of CPC/Dex ( r = 0.5) hybrid.

without Dex-MA to 82.82 ± 7.5 MPa with a Dex-MA mass fraction of 16.67% ( r = 5) ( p < 0.05). The corresponding mod-ulus signifi cantly improved from 3.93 ± 0.4 GPa to 7.5 ± 0.6 GPa, superior to the strength of cancellous bone. However, further increasing the Dex-MA content resulted in both the compressive strength and modulus dropping off to 59.05 ± 5.1 MPa, 3.98 ± 0.04 GPa, respectively, with a Dex-MA mass fraction of 44.4% ( r = 1.25). As the mass fraction of Dex-MA increased and exceeded 0.5 ( r = 1), the hybrid may be consid-ered a Dex-based composite. It should be mentioned that the addition of Dex-MA was an effi cient approach to enhance the compressive strength as well as the modulus. A second peak was obtained at about 80.24 ± 0.7 MPa and 6.34 ± 0.6 GPa with a mass ratio of 66.7% ( r = 0.5). Then, with a higher content of Dex-MA, both compressive strength and modulus declined again. This might be due to the shrinkage discrepancy between the two moieties which resulted in obvious defects of cracks.

Work-of-fracture can represent the fracture toughness of a material and its ability to resist crack propagation.The infl u-ence of Dex-MA content on the work-of-fracture of CPC/Dex composites is shown in Figure 11 B. As expected, the results conform to the stress-strain behavior. The work-of-fracture was signifi cantly increased along with the increase in Dex-MA content, distinctly demonstrating the toughness achieved. The work-to-fracture at 66.67% Dex-MA mass ratio ( r = 0.5) in particular was 8.35 ± 0.45 kJ m − 2 , an increase of two orders of magnitude over the 0.084 ± 0.33 kJ m − 2 of the non-Dex control.

In addition, under high compression, CPC/Dex-MA did not fracture into separate smaller pieces but remained intact, although numerous microcracks appeared in the matrix, as depicted in Figure 11 C. Despite the cracks formed, the propaga-tion was retarded and the crack opening was displaced, changing direction into the Dex-MA moiety where the crack tip could be absorbed owing to the toughness. [ 46 ] From stress–strain curves of compression strength measurements the same phenomenon was observed, especially with the 66.67 wt% Dex-MA ( r = 0.5) sample, where compressive strength remained at a constant level after reaching yield strength.

Apparently, the Dex-MA content of 16.7 wt% ( r = 5) was optimal for achieving the highest mechanical strength for the CPC/Dex-MA formulations at degree of substitution 8.8. The infl uence of the powder-to-liquid ratio on the mechanical properties was examined while maintaining the Dex-MA content at 16.7 wt% ( r = 5), and the results are plotted in Figure 12A . The curve shows that the P/L ratio has a signifi cant effect on compressive strength. At each P/L ratio, the strength of the CPC/Dex-MA composite is signifi cantly higher than that of the CPC control without polymer, and enhancing the P/L ratio signifi cantly increases the compressive strength for both materials (p < 0.05). The compressive strength of CPC/Dex-MA is (78.58 ± 1.8) MPa at P/L ratio = 2.27; signifi cantly higher than the (19.1 ± 0.6) MPa for the CPC control (Tukey’s multiple comparison test; family confi dence coeffi cient = 0.95). At P/L ratio = 1.85, CPC/Dex-MA has a strength of (46.37 ± 1.2) MPa, signifi cantly higher than the (8.5 ± 1.7) MPa for the CPC control at the same P/L, and so much higher than the (24.13 ± 3.9) MPa for the CPC control at a P/L ratio of 3.3 (Tukey’s at 0.95). If the P/L ratio were higher than 2.56, Dex-MA would not dissolve

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completely; and at a P/L ratio lower than 1.85, CPC would not solidify.

Figure 12 B shows the effect of the degree of substitution (DS) of the methylacrylate group on the compressive strength. Increasing the DS of the MA group from 0 to 8.8 resulted in a dramatic enhancement of compressive strength from 22.56 MPa to a maximum of 98.33 MPa. This is reasonable, because neither the crosslinked structure nor the interpen-etrating network can be formed without the double bond on the MA groups, thus leading to a loss of strength. At odds is

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Figure 12 . A) Infl uence of P/L ratio on compressive strength. B) Depend-ence of compressive strength on the degree of substitution. C) Compres-sive strength of CPC/Dex-MA and CPC as a function of time.

the fact that further increases in DS lead to a gradual decline in compressive strength, which is distinctly different from the common speculation of dense crosslinking leading to higher strength. The implication is that the close-knit reactive site may


involve crosslinking between chains as well as the hydrated hydroxyapatite and unconverted CPC particle acting as fi llers, which leads to the irregular tendency.

Figure 12 C displays the time-dependant evolution of com-pressive strength of the CPC/Dex-MA composite ( r = 5) and of the pure CPC. Each datum was acquired without further drying. The strength of the CPC was enhanced gradually over 24 hours, ending with the conversion of hydroxyapatite. A similar trend was seen in the typical CPC/Dex-MA hybrid. It should be noted that the introduction of the polymeric network gave the sample about twice the initial strength (about 7.13 MPa) of the pure CPC (3.64 MPa) at preliminary setting for 15 min. We suspect that the polymeric network contributes the original strength; after-wards the conversion of inorganic Ca-P cement and the poly-meric network act in concert to supply the succedent strength.

3. Discussion

3.1. Formation of the Hybrid Interpenetrating Network

Previous studies have shown the advantages of introducing polymers into calcium phosphate-based cements due to their extraordinary properties based on the combination of different building blocks. [ 47 ] A critical challenge is the mixing and inter-facial bonding between the two dissimilar phases. Physical mix-tures of organic polymers and preformed inorganic particles can lead to separation in discrete phases, resulting in poor mechan-ical properties. In this study, an interpenetrating network strategy is adopted to incorporate a polysaccharide hydrogel into inorganic calcium phosphate cement via an in situ process, which facilitates the mixing of modulated compositions.

The apatite phase was produced in the presence of the free radical polymerization of methylacrylated dextran as evidenced by the XRD results (Figure 3 ). No other new chemical conjugations between the moieties are displayed except for the respective ori-gins of dextran derivates and calcium phosphate-based cement. The data observed here suggest that the apatite does not form at the very beginning, but grows up gradually during the process of hydration. Close examination of the broad nature of the XRD pattern indicates that such an apatite phase has a low-crystalline nature, and segments of amorphous calcium phosphate coexist. This was coincident with the persistence of residual TECP peaks in XRD patterns even after 10 days, inferring the incomplete and delayed transformation to hydroxyapatite, which might be ascribed to the grain refi nement and lattice distortion resulting from the introduction of polysaccharide as the liquid and interac-tions between the Ca-P moiety and polymeric phase. Other poly-mers and macromolecules have also been observed to delay such transformations. [ 48 ] Though the conversion of CPC is imperfect, the calcium phosphate remnants are covered or included in the polymeric matrix, acting as fi llers between coexisting phases.

On the other hand, although the variation of uptake as shown in the IR is not enough evidence to prove the occurrence of polymerization owing to the overlap of other phosphate groups, it distinctly demonstrates that the typical CPC/Dex-MA sample remains in its original shape while uncrosslinked CPC/Dex-MA (without initiator/accelerator) collapsed rapidly during


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the fi rst 10 minutes (Figure 1 ). Since the hydration of calcium phosphates was far from fi nished at that moment according to the XRD patterns, good resistance against washout confi rms the maintenance of an insoluble block, which comes from the crosslinking of the methacrylated dextran network, otherwise the water-soluble linear Dex-MA would inevitably give rise to disentanglement. Moreover, as conversion of HA appears to be a stepwise process, the faster-setting feature of the CPC/Dex-MA hybrid is mainly ascribed to the crosslinking of the methacry-lated dextran. Further thermogravimetric analysis also sup-ports the idea that the chain polymerization of methylacrylated dextran is carried out inviolately, since the compositions of the fi nal crosslinked CPC/Dex-MA are consistent with the feed ratio before crosslinking. It is inferred that free radical poly-merization might survive when encountered with either calcium phosphate particles or cement, and the unreacted dextran will dissolve and be eliminated during the soaking process, together with the remnants of the plethora of initiator and accelerator.

3.2. In situ Growth of Calcium Phosphate Cement in the Polymer Matrix

Thus, both crosslinked dextran hydrogel and solidifi ed calcium phosphate cement are concomitant in the CPC/Dex hybrid. The uptake data reported in Figure 3 b points to a gradual rather than instant transformation of hydroxyapatite. But, surprisingly, the gradual process does not seem to affect the setting time. Com-pared to conventional CPC, not only does almost every hybrid sample solidify rapidly, but also setting time decreases obviously with the addition of the Dex-MA (Figure 5 A), indicating the polymeric hydrogel is mainly acting as a setting accelerator in the system. Distinct from the fast-setting times of CPCs [ 49 ] due to the precipitation of chitosan, dextran derivates are soluble in aqueous solutions unless crosslinked into a hydrogel network. The implication is that polymerization occurring antecedent to the hydration, forming the hydrogel shelter, may involve short-ening the setting time. More evidence can be gleaned from temporal fl uctuations of mechanical properties from the begin-ning of setting (Figure 12 C). The initial compressive strength generally originates from the polymeric network, which sup-plies higher strength during the earlier stage.

Thus it can be seen that the formation of double networks is carried out by a sequential two-step process wherein a poly-meric hydrogel is formed in the primary step, instead of by the simultaneous formation of the binary networks. [ 50 ] Rapid forma-tion of a sol-gel network avoids the macroscopic phase separa-tion [ 51 ] and disentanglement. As expected, the resulting hybrids are microscopically phase-separated, but macroscopically uni-form, as demonstrated in SEM. Therefore, the two-step process includes the formation of a crosslinked hydrogel moiety by free radical polymerization of methylacrylated dextran, and penetra-tion into inorganic particles. The hydration of calcium phos-phates occurs subsequently and hydroxyapatite crystalline grows in situ in the hydrogel matrix. Thus the hybrid interpenetration network comes into being (Scheme 1 ). In fact, the data for set-ting time does not represent the actual setting of CPC, but an apparent gelation time of Dex-MA. Most of the calcium phos-phate particles go through the hydration process and convert


into an apatite phase gradually, serving as the inorganic skel-eton. The remainder disperse therein, acting as the thickening fi llers that cause the elevated viscosity of the slurry and the faster gelation behavior as compared with the pure hydrogel.

In agreement with previous studies, an increased P/L value results in a shorter setting time (Figure 5 B). Furthermore, even below the critical P/L value of pure CPC, the hybrid still loses its fl uidity, as if independent of the hydration, confi rming the dominant contribution of Dex-MA on the primary setting time.

Another parameter that infl uences the setting of CPC/Dex-MA is the condition of polymerization, such as Dex-MA concentration and amount of initiator/accelerator. Given atten-tion to both networks, a too-high Dex-MA concentration would lead to inhomogeneous crosslinking and hamper the entangle-ment between the hydrogel and calcium phosphate crystals. Investigation of the effect of TEMED (Figure 5 C) exhibited the speciality of free radical polymerization, consistent with the hypothesis of the sequential formation of the polymer frame-work. As a result, it is feasible to modulate setting time through the amount of TEMED as well as the P/L value.

It is evident in SEM images that the crystalline HA and amorphous polymer phases coexist. As a rule, interpenetrating polymer networks (IPNs) have a micro-heterogeneous structure caused by the thermodynamic incompatibility of the constituent network. As deduced above, the polymerization of methacrylated dextran occurrs followed by gradual hydration of calcium phos-phate, and sequential formation of the dual organic and inor-ganic network, entangling each other. The incompatibility arises in the course of polymerization and hydration and leads to the microphase separation. After the onset of microphase separation the reactions proceed sequentially in two evolved phases whose composition also changes with time. The phase separation is not complete due to the formation of the crosslinked structure.

The incorporated ratio of the inorganic and polymeric moie-ties can be varied over a wide range, from a calcium phosphate-based hybrid to a polymeric-based hybrid. It is important to mention that the mass ratio employed for the hybrids has a cru-cial infl uence on the microstructures in the resulting material, directly infl uencing its properties. As shown in the morphology observations (Figure 6 ), different micro-architectures appeared, such as fl uffy agglomerates, needle crystals, fl ower clusters, and entrapped blade-shapes, etc. Moreover, the dimensions of the crystals correlate with the composition, changing from microm-eter to nanometer. In this sense, the polysaccharide hydrogel not only contributes the soft-moiety and retards the macro-phase segregation via interpenetration, but also serves as the matrix used for the in situ growth of crystalline HA. The apatite crystals nucleate on its surface, forming a linear growth of apa-tite crystals (surface-controlled process) and a potential growth of apatite crystals (diffusion-control process). As a result, both the polymerization and hydration processes form an entangled network that is responsible for the mechanical properties.

3.3. Effects of the Polymeric Network on the Hybrid Cement

Besides setting time, key parameters concerned with hydration process have been investigated to elucidate the effect of con-comitant polymerization on the CPC transformation.


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Since the CPC hydration and pH value are relatively inter-dependent, the pH variations may allow deduction of the mechanisms of the hydration process in aqueous Dex-MA. The essential pH trend of the composite was similar to that of the CPC paste (Figure 9 ). The pH value in the slurry moves to the basic range quickly at the initial stage as the TECP dissolves. Fortunately, the basic environment does not obstruct free-radical polymerization, and the crosslinking of methacry-lated dextran occurs rapidly. The calcium phosphate particles are embedded in the hydrogel network and carried further through by dissolution and diffusion through the liquid. It is deduced that such a process continuously takes place until Ca 2 + and PO 4 3 − gradually reach supersaturation levels and convert into HA. At a range of high pH values, the solubility of the acidic DCPA increases while the solubility of the basic TECP decreases, which leads to a drop in pH. Steady-state is fi nally achieved after 30 hours and the calcium phosphates are trans-formed into HA gradually. By this it may be inferred that the coexistence of the polymer moiety does not affect the principal hydration process of CPC slurry, irrespective of the difference between the liquids.

Compared with pure CPC, CPC/Dex hybrids show distinct features of swelling, attributing to the crosslinked hydrogel moiety. As summarized in Figure 7 A, the water absorption of the CPC/Dex-MA composite reduces along with incorporation of CPC. After hydration, the solidifi ed calcium phosphate-based cement serves as a temporary inorganic barrier, preventing water from permeating into the Dex-MA hydrogel, which would postpone the attenuation of mechanical properties of CPC/Dex composite under moist conditions.

The noticeable volumetric shrinkage during the deswelling process aroused our attentions. The external layer contracts prior to the contraction of the inside layers when drying is started from the outside. The shrinkage force generated outside is applied on the inside of the hybrid. Therefore the CPC/Dex can be self-reinforced by behavior of shrinkage in the process of drying. This accidental behavior is benefi cial to the mechanical strength, acting as a radial compact.

The pore size and porosity are of great importance since they greatly affect the mechanical properties. According to the research on elastomeric CPC-chitosan composites, Chow et al. [ 48 ] attached importance to the porosity-dependent mechanical

Scheme 2 . Schematic representation the effect of shrinkage on self-reinforced Dex/CPC hybrid.

strength, especially compressive strength. In fact, considering the solubility of the polymer, the P/L value is always lower than that of pure CPC, which results in the generation of more intergranular voids and higher porosity, but, surprisingly, this does not seem to entirely match the CPC/Dex-MA system. Data obtained from our pore-size study showed that with the addition of Dex-MA, the pore volume in the smaller size range increases. It was expected that a lower P/L value would cause a larger pore radius, however, the total porosity decreases along with the Dex content. The Dex-based composites had evi-dently smaller pore volume than the CPC-base hybrids. This is attributed to the extraor-dinary deswelling behavior of the hydrogel


network. It appears that the competitive volume shrinkage may partially compensate for the effect of having higher inter-stices. As it is clearly visible that higher Dex-MA contents have an increased volume contractibility (Figure 7 B), interstices will be reduced with decreasing polymeric network content. Fur-thermore, the radial shrinkage not only produces smaller pore volumes and a more compact entangled network, but favors self-reinforcement, seemingly as if stressed ( Scheme 2 ).

3.4. Mechanical Properties

With respect to the effects of incorporating a polymer into the CPC cement, diverse results have been reported via different strategies. Combinations of pre-hardened CPCs and nano-silica-fused whiskers in bis-GMA resin might be a representative example for enhancement. [ 27 ] In other cases, the introduction of polymers has decreased the mechanical performance. [ 21 ] Overall, the connection between different compositions and the microscopic porosity plays a decisive role. In our study, we took advantage of both an IPN strategy and volume contraction to solve these problems. Though there is no chemical interac-tion between the chains but only physical interactions, the dual networks entangle each other to enhance adhesion at the interface.

It is worth noting that the introduction of a Dex-MA hydrogel into calcium phosphate cement possibly can be used to improve the mechanical properties of the cement, i.e. to overcome brit-tleness, as a higher strain-at-yield can be obtained with an increasing percentage of polymeric moiety. Also the tough-ness of the cement increases: composites do not fracture after reaching the yield strength, but are kept together by the elon-gated hydrogel network. As shown in Figure 10 , at higher Dex content, the stress after the yield was not a constant value but was enhanced slightly with increasing strain, which is ascribed to the incorporation of the elastomeric hydrogel component. Similar mechanical characteristics are also observed using fi ber-containing calcium phosphate cements. [ 27 , 40 ] Another feature of concern is that, distinct from those general pre-compacted cements, improved compressive strength can be attained by manually applying fi nger pressure without any additional axial compactions. Compared to these fi ber-modifi ed cements, the


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in situ formation of the CPC/hydrogel hybrid is more facile to process with respect to injectability and polymer degradation and therefore can form an attractive alternative.

Weaker interfacial bonding between inorganic granules and a polymeric matrix is always the crucial factor resulting in a decrease in desirable mechanical properties, as well as infl u-encing the respective reaction processes. The diffi culties of such an approach are usually the potential incompatibilities between the soft hydrogel and the rigid calcium phosphate-based cement, and the critical challenge is controlling the formation of the second network in the presence of the dissimilar fi rst one. Indeed, in the case of blending calcium phosphates into the crosslinked Dex-MA hydrogel rather than using the in situ IPN method, it is not only diffi cult to mix homogeneously but it also retards the hydration of CPC owing to the higher water-absorption, given the large amount of liquid required for the transition of phosphates. This leads to a decrease in compres-sive strength (data not shown), even with the same composition as used in the IPN method. Surprisingly, no visible decrease of mechanical properties was observed for CPC/Dex-MA prepared by in situ hybridization, despite the composition-dependent fl uctuant. There are two possible mechanisms which may have brought about the reinforcement. First, as above-mentioned, notwithstanding the fact that the sequential two-step process between inorganic and organic moieties is different from the unitary CPC hydration, the trend in pH variation indicates that the primary hydrogel network does not obstruct the secondary transformation of the calcium phosphate-based network. The evidence from the increased strength demonstrates that, despite existing in the interstices of CPC powders or covering as hydrogel, the concomitant polymerization does not completely retard the CPC reaction of CPC/Dex composites. Strengthening of the specimen can probably be explained by the DCPA and TECP particles phase transferring into HA and interconnecting with each other. Apropos those residual untransformed DCPA and TECP, which have been shielded from the hydrogel and survived from washout, would serve as the inorganic fi llers con-tributing to the enhanced strength. The second hypothesis is in regard to the distinct behavior of self-reinforcement during the drying process, as if it had suffered radial compactions, which reduces the porosity.

In this study, the CPC/Dex-MA hybrid is composed of inter-penetrating double networks by integrating Dex-MA hydrogel with HA cement. Methyacrylated dextran chains are introduced into calcium phosphate particles and crosslinked to generate the fi rst hydrogel network via rapid free radical polymerization. The second inorganic network of HA cement subsequently forms by hydration of calcium phosphate in the presence of the previously formed Dex-MA network. The improved mechanical performance of the hybrid results from the DN structure. The stress-strain profi les indicate that the ductile polymeric network contributes to the elastic stress, whilst the other brittle inor-ganic network contributes to the strain.

In particular, the two individual networks of the CPC/Dex system are formed in succession, containing one soft-but-ductile fi rst network and one stiff-but-brittle second network. This sequence is noteably inverse to those mechanically strong double-network hydrogels: [ 52–54 ] however, it still has exceptional mechanical strength and toughness. The inorganic cement


network has a higher modulus but is rather brittle. Under com-pression, stress could easily develop locally inside the network, leading to the formation of cracks. Nevertheless, the loosely crosslinked hydrogel effectively dissipates the crack energy by deforming the network conformation and/or by sliding the physical entanglement points along the chains to prevent the crack growing to a macroscopic level, (Figure 11 c), and the free calcium phosphate particles acting as fi llers still have positive effects on the mechanical strength. The intervallic dis-parity between hard cement and soft hydrogel subtly comple-ment one another. In other words, the increased mechanical strength of the CPC/Dex hybrid is largely attributable to the effective relaxation of locally applied stress and dissipation of the crack energy through the combination of two networks with discrepant structures.

Two crucial structural parameters affect the properties of this DN hybrid, i.e., the mass ratio of dual networks, and the pore parameters of the hybrid. The dependence of mass ratio on com-pressive strength (Figure 11 a and 11 b) shows a similar tendency for both CPC-based and Dex-based composites. Increasing the Dex content results in a gradual enhancement of the compres-sive strength, but a further increase leads to a gradual decline. Higher porosity and pore area might be obtained by introducing more polymeric moiety, which correlates with a weakening of the mechanical strength. This concords with the results of the effect of P/L on the compressive strength. However, owing to shrinkage during the drying process, a greater water uptake brings about further radical compression. The compressive strength neither increases nor decreases linearly, but fl uctuates with Dex-MA content. The infl uence of the crosslinking den-sity on the fi rst polymeric network is not prominent. As shown in Figure 12 B, enhancing the degree of substitution from 0 to 8.8 leads to a substantial increase in the crosslinking density, resulting in a progressive increase of compressive strength of the hybrid. However, further increasing the DS would form a stiffer network with a limited capacity to dissipate the stresses imposed during compression, as well as restricting the radical contraction during drying, thus leading to a reversal of the DN effect.

There are optimal values for both the mass ratio and the porosity of the separated network. The mass ratios of 16.67% ( r = 5) and 66.7% ( r = 0.5) provide the highest compressive strength for the CPC/Dex hybrid formulations. It should be emphasized that, although both of the two individual networks are mechanically weak, that is, the fi rst one is soft-and-ductile and the second stiff-and-brittle, their combined DN gels are stiff-but-not-brittle, ductile-but-not-soft. Compared to common cements, the advantages of the CPC/Dex hybrid include its moldability, ability to be injected and to self-harden in situ in the bone cavity, its ability to conform to complex and irregular cavity shapes without machining, and bioresorbability.

4. Conclusions

In the present work, we have demonstrated that in situ for-mation of Dex-MA-modifi ed calcium phosphate cement via a double-network interpenetrating strategy is feasible. Both separated networks form in the presence of the other dissimilar


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Scheme 3 . Chemical structure of glycidyl methacrylated dextran (Dex-MA).

phases. The apparent setting time decreased obviously and could be modulated. The generation of dual networks was carried out as a sequential two-step process, wherein the Dex-MA crosslinked network was formed fi rst, and then hydration occured in the hydrogel sheltering. Though the HA conver-sion was imperfect, it did not obstruct the essential hydration process of CPC slurry. Interestingly, the shrinkage of the hydrogel during the deswelling process serves to reinforce the network, which plays an important role in the enhancement of mechanical strength. Due to the synergistic effect of the DN structure, the hybrid possesses improved mechanical proper-ties as compared to the single inorganic cement: the loosely crosslinked fi rst hydrogel network dissipates stress during compression and contributes to an increased ductility, and the inorganic cement network supplies the increased stiffness. In the optimal condition, the compressive strength is improved remarkably to 98.3 MPa without additional compaction.

One of the key contributions of the present work is the achievement of a universal method of in situ hybridization for a wide range of matrices, from inorganic-based to polymer-based. Our study sheds some light on the problem of whether the transformation of HA occurs in the two-phase system. The implication is that the hydration of calcium phosphate can be conducted gradually in spite of the hydrogel shelter.

To the best of our knowledge, these hydrogel-cement inter-penetrating network methods have not been previously applied to the design of calcium phosphate bone cements. The protocol is not costly, since no sophisticated technology is required. The method is interesting from a methodological point of view, because most additives used previously have worked to rein-force the CPC by, for example, modifi cation with intensifi ers such as fi bers, whereas virtually no attention has been paid to the introduction of soft moieties and their potential to reinforce the CPC as a synergetic complement. Such a strategy affords a versatile platform to develop novel inorganic-organic hybrids based on the combination of different building blocks: although here we have chosen the glycidyl methacrylated dextran as an example, some other hydrogel could also be integrated using this protocol. Thus, based on the above results, we envisage that the CPC/Dex DN hybrid has great potential for bone regen-eration. Further investigation will be directed at exploring its degradation behavior, good cell affi nity, and osteoconductivity.

5. Experimental Section Materials : All reagents used were available from commercial

sources. Dextran (from Leuconostoc mesenteroides , T40, Mn = 40000), 4-dimethylaminopyridine (DMAP), ammonium persulfate (APS), N,N,N ′ N ′ -tetramethylethylenediamine (TEMED) were obtained from Shanghai Sinopharm Chemical Reagent Co. (Shanghai, China), glycidyl methacrylate (GMA) was purchased from Acros (New Jersey, USA), dimethyl sulfoxide (DMSO) was purchased from Shanghai Linfeng Chemical Reactant Co. (Shanghai, China), dialysis tubes (cellulose, MW cutoff 14000) were supplied by Shanghai Yuanju Bio-Tech Co. Ltd. (Shanghai, China), isopropanol, acetone, disodium hydrogen phosphate and all other reagents were form Shanghai Feida Chemical Co. (Shanghai, China), and used as received.

CPC Powder : Generally, the hybrid cement consisted of CPC powder and a liquid component which contained Dex-MA solution. The CPC powder prepared in our laboratory [ 44 ] was composed of tetracalcium


phosphate (TECP, Ca 4 (PO 4 ) 2 O) and dicalcium phosphate anhydrous (DCPA�CaHPO 4 ) in an equivalent molar ratio, using preparation methods obtainable from the relevant literature. Briefl y, TECP was synthesized by a solid-to-solid reaction between calcium phosphate and calcium carbonate at a temperature of 1500 ° C for 8h. Dicalcium phosphate dehydrate (DCPD, CaHPO 4 · 2H 2 O) was prepared from ammonium hydrogen phosphate ((NH 4 ) 2 HPO 4 ) and calcium nitrate (Ca (NO 3 ) 2 ) in an acidic environment. DCPA was obtained by evaporation of the crystallization water in DCPD at 120 ° C.

Synthesis of Glycidyl Methacrylated Dextran (Dex-MA) : Dex-MA ( Scheme 3 ) with different degrees of substitution were synthesized according to published procedures. [ 35 , 55 ] Briefl y, dextran (5g) was dissolved in DMSO (45mL) in a three-neck fl ask under argon atmosphere. After dissolution of DMAP (1g), a calculated amount of glycidyl methacrylate (GMA) was added. The solution was stirred at 30 ° C for 48 h, after which the reaction was stopped by adding an equimolar amount of concentrated HCl to neutralize the DMAP. The product was precipitated with cold isopropyl alcohol, fi ltered, washed several times with isopropyl alcohol and acetone, and then dried in a vacuum oven at room temperature. After that, the crude product was dissolved in demineralized water and dialyzed for at least 3 days against demineralized water at 4 ° C, and then a white fl uffy solid was obtained after lyophilization. The Dex-MA product was stored at − 20 ° C before use. Proton nuclear magnetic resonance ( 1 HNMR) was used for quantitative determination of the degree of substitution (the number of functional methacrylate groups per 100 glucopyranose residues in dextran) according to the literature [ 35 ] using Bruker Avance 500 MHz instrument (Bruker, Germany) with D 2 O as the solvent.

Fabrication of CPC/Dex-MA Hybrid : Unless otherwise noted, the Dex-MA with stoichiometric ratio GMA/Dex = 1:6 (DS = 12.5%) was used for the specimen fabrication, and was dissolved in disodium hydrogen phosphate (Na 2 HPO 4 , 4 wt%) with a known concentration. The viscous polymeric aqueous solution acted as the CPC liquid phase.

The liquid was then mixed with CPC powder according to the desired mass ratio r ( r = W CPC : W Dex-MA ). The compositions of different hybrid cements are listed in Table 2 . Then appropriate amounts of initiator APS (50 mg mL − 1 ) and accelerator TEMED (23 mg mL − 1 ) were added sequentially and rapidly mixed to form an homogeneous paste that was cast into a Tefl on mold to produce cylindrical samples of 6mm diameter and 12 mm height without additional compaction. The specimens were set into an incubator with 100% relative humidity at 37 ° C for 24h. The off-white specimens were demolded and immersed into a simulated physiological solution (SBF buffered to a pH of 7.4) for 24 h, both for removing the remnant of the unreacted monomer and initiator/accelerator and for further hydration. The rod specimens were then taken out, rinsed with demineralized water, and dried.

Characterization of CPC/Dex-MA Hybrid : Fourier transform infrared (FTIR) spectra were obtained on a Nicolet AVATAR-360 spectrometer


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Table 2. Compositions of the CPC/Dex-MA mixture for hybrid fabrication.

Mass ratio, r [w/w]

CPC [g] Dex-MA [g] APS [ μ L] TEMED [ μ L] Na 2 HPO 4 [ μ L] P/L

10:1 1 0.1 40 100 250 2.56

5:1 1 0.2 40 100 400 1.85

5:2 1 0.4 80 200 400 1.14

5:3 0.5 0.3 40 100 600 0.68

5:4 0.5 0.4 80 200 600 0.57

1:1 0.5 0.5 80 200 800 0.46

1:2 0.25 0.5 80 200 800 0.23

1:3 0.2 0.6 80 200 800 0.19

(Thermo Scientifi c Inc., Waltham, MA). All samples of CPC, Dex-MA and CPC/Dex-MA with different r values were made into particles and analyzed using KBr pellets. For each sample, 16 scans were recorded between 4000 and 500 cm − 1 .

The crystalline compositions of the binary hybrids with different mass ratios were determined by wide-angle X-ray diffraction (WAXD). The measurements were performed by powder X-ray diffractometry (D/MAX 2550 VB/PC, Rigaku Co. Japan) with Cu K α radiation ( λ = 1.5406 Å, 40 kV, 450 mA) in a continuous scan mode. The 2 θ range was from 3 ° to 70 ° at a scanning speed of 6 ° min − 1 .

Fractured surface morphology of the composites was observed under scanning electron microscopy (SEM) (JSM-6360LV, JEOL, Japan). Fractured cross-sections of the samples were obtained by cutting from a whole cylinder using a saw. Pieces of the specimens were mounted onto an aluminum stub, sputter coated with gold-palladium, and images were recorded at 20 kV acceleration voltage. The infl uence of polymeric additions on the crack is examined by observation of compressed samples to obtain information regarding reinforcement mechanisms.

Thermogravimetric analysis (TGA) was detected using a thermogravimetric analyzer (WRT-2P, TA Instruments, Shanghai Precision & Scientifi c Instrument Co., China). Heating was performed in a platinum crucible from 10 to 800 ° C at a rate of 10 ° C min − 1 in fl owing nitrogen (20 cm 3 min − 1 ).

Setting Time Measurement : The premixed CPC and CPC/Dex-MA paste was fi lled into the glass tube ( Φ 6 × 8 mm 3 ) and kept in 100% relative humidity at 37 ° C. The setting time was determined at various intervals by using Vicat apparatus bearing a 300 g needle with a diameter of 1.13 mm according to standard Gillmore needles method. [ 56 ] The time of setting was presented as the number of minutes elapsed from the addition of TEMED to the time when the needle failed to create an indentation of 1 mm in depth on the surface of the specimen. Triplicate parallel experiments were carried out and the average value was calculated for each specimen.

For Dex-MA, the Gilmore needle method was not used because the pure hydrogel was relatively soft even after crosslinking. Therefore, the time to form a gel (denoted as gelation time) was determined by the vial tilting method following previous studies on similar hydrogels. [ 57 ] When the sample showed no fl ow within 5 s upon inverting, it was regarded as gel-forming.

Water Uptake and Volume Shrinkage : Water uptake of Dex/CPC composites with different Dex-MA contents were determined as followed. The dried gels (weight W 0 ) were immersed in PBS at 37 ° C in an incubator. At predetermined time intervals the weights of the samples in swollen state ( W t ) were measured immediately after the removal of the excess water by blotting between two pieces of fi lter paper. All experiments were conducted in triplicate. Water uptake ratio ( W a %) at time t was calculated using the following equation:

Wa % =

W t − W0

W0× 100%


The volume shrinkage ( S %) is a measure of the volume contractibility in the course of deswelling. The samples were allowed to reach equilibrium in ultrapure water or buffer solution and then dried. The temperature was maintained at 37 ° C. The specimens were retrieved at regular time intervals and the diameter was measured. S % was defi ned as the percentage of radial constriction after drying.

S% = (D 2t − D 2

d )/D 2d × 100%

where D t is the diameter of specimen at time t and D d is the diameter of the thoroughly dried sample. Each experiment was performed in triplicate and results were reported as the mean ± standard deviation.

Pore Size and Distribution Determination : [ 44 ] The hydrated specimens were broken into small fragments or particles and dried before measurement. The specifi c surface, pore size and distribution of the hybrid cement were performed via mercury porosimetry (AutoPore IV 9500 V1.06, Micromeritics Co., USA) and multipoint Brunauer-Emmett-Teller (BET) nitrogen adsorption method (Tristar 3000, Micromertics Co., USA) at liquid nitrogen temperature and then desorption.

pH Measurement : [ 58 ] The slurry containing CPC (2 g), Dex-MA (0.4 g), and the other additives as described above were mixed rapidly and then placed in physiological saline (50 mL). The samples were incubated at 37 ° C with stirring speed 200 r min − 1 . The fl uctuation of the pH in the slurry during the setting process was detected with pH meters ( n = 3) to investigate the effects of the polymerization of Dex-GMA on the hydration process.

Mechanical Strength : As for mechanical performance testing, samples of each group were molded into columns (6 mm in diameter and 10 mm in length). No additional precompaction was carried out except for the fi nger pressure. The specimens were removed from the molds and immersed into SBF for 24 h. After removing them, washing with deionized water and drying, specimens were uniformly polished on both sides. Compressive strength was measured with a universal testing machine (AG-2000A, Shimadzu Autograph, Shimadzu Co. Ltd., Japan) at a loading rate of 1 mm min − 1 . Unless otherwise specifi ed, the period of time between fabrication and measurement was about 72 hours including the whole process.

The Following Properties were Evaluated : compressive strength, compressive modulus, and work-of-fracture. The compressive strength value of the specimens was calculated from the formulation σ = 4 F max / π D 2 , where F max is the peak load in Newtons. and D is the diameter (mm) of the specimen. The maximal compression load at failure was obtained from the recorded load-defl ection curves. The compressive modulus was determined from the slope of the linear portion of the stress-strain curves, and the work-of-fracture was obtained from the area under the load-displacement curve normalized by the specimen’s cross-sectional area, which denoted the energy required to fracture the specimen. At least fi ve replicates were tested for each group, and the results were expressed as the mean ± standard deviation (mean ± SD).

Samples of the r = 5:1 group were selected to further investigate the compressive strength with various DS and P/L ratios. The stoichiometric ratios of GMA/Dex were assigned as 1:3, 1:4, 1:6, and 1:9. As to the various P/L ratios, samples in the fi ve groups were prepared using the parallel liquid phase. The amounts of APS/TEMED were fi xed in order to obtain similar initiating conditions in the various groups, but the amount of Na 2 HPO 4 was changed, which resulted in various P/L from 2.27 to 0.57 g mL − 1 . The optimum P/L value of pure CPC was 3.3 g mL − 1 . The details are listed in Table 3 .

With regards to the time-dependent compressive strength, CPC/Dex-MA ( r = 5) and CPC specimens were fabricated as above mentioned. Homogeneous pastes were cast into the Tefl on mold and placed into a setting environment of 100% relative humidity at 37 ° C without additional compaction. Then samples were taken out at designated time and immersed into acetone to terminate the setting process. Compressive strength was determined without further drying. At least fi ve replicates were tested for each group, and the results were expressed as the mean ± SD.

Washout Resistance Observation : Typical CPC/Dex-MA mixtures ( r = 5) were divided into two groups: the crosslinked group was fabricated


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as above noted, and the other uncrosslinked group was prepared in abinwcovi

geofva

A ThPr50Ch(NLa

Table 3. Compositions of CPC/Dex-MA mixture with various P/L.

r [w/w] CPC [g] Dex-MA [g] APS [ μ L] TEMED [ μ L] Na 2 HPO 4 [ μ L] P/L

300 2.27

350 2.04

5:1 1 0.2 40 100 400 1.85

500 1.56

700 0.57

sence of APS and TEMED. The premixed paste was manually shaped to a ball and placed into the deionized water immediately. Observation as carried out 10 min after putting into water. The material was nsidered to pass the washout resistance test if the paste ball did not

sibly disintegrate in the solution. Statistical Analysis : Results were expressed as mean ± SD. All data were

nerated in three or four independent experiments. One-way analysis variance (AVOVA) was used to evaluate the statistical signifi cance. A lue of P < 0.05 was considered to be statistically signifi cant.

cknowledgements e authors are indebted to the fi nancial support from the Major ogram of National Natural Science Foundation of China (No. 732002), the Program of National Natural Science Foundation of ina (No. 50973029), Program of Shanghai Subject Chief Scientist o. 07XD14008), and the Basic Research Foundation of the State Key boratory of Bioreactor Engineering.

Received: May 18, 2010 Published online: September 22, 2010

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