structure and biocompatibility of an injectable bone regeneration composite

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Structure and Biocompatibility ofan Injectable Bone RegenerationCompositeRongwei Tan a , Qingling Feng b , He Jin c , Jinyu Li d ,Xing Yu e , Zhending She f , Mingbo Wang g & Huanye Liu ha State Key Laboratory of New Ceramics and FineProcessing, Department of Materials Science andEngineering, Tsinghua University, Beijing 100084, P. R.Chinab State Key Laboratory of New Ceramics and FineProcessing, Department of Materials Science andEngineering, Tsinghua University, Beijing 100084, P. R.China. [email protected] Department of Orthopaedics, Dongzhimen HospitalAffiliated to Beijing University of Chinese Medicine,Beijing 100700, P. R. Chinad Department of Orthopaedics, Dongzhimen HospitalAffiliated to Beijing University of Chinese Medicine,Beijing 100700, P. R. Chinae Department of Orthopaedics, Dongzhimen HospitalAffiliated to Beijing University of Chinese Medicine,Beijing 100700, P. R. Chinaf State Key Laboratory of New Ceramics and FineProcessing, Department of Materials Science andEngineering, Tsinghua University, Beijing 100084, P. R.China; Center for Advanced Materials and Biotechnology,Research Institute of Tsinghua University in Shenzhen,Shenzhen 518057, P. R. Chinag State Key Laboratory of New Ceramics and FineProcessing, Department of Materials Science andEngineering, Tsinghua University, Beijing 100084, P. R.Chinah Department of Prosthodontics, School of Stomatology,China Medical University, Shenyang 110002, P. R. China

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To cite this article: Rongwei Tan , Qingling Feng , He Jin , Jinyu Li , Xing Yu , ZhendingShe , Mingbo Wang & Huanye Liu (2011) Structure and Biocompatibility of an InjectableBone Regeneration Composite, Journal of Biomaterials Science, Polymer Edition, 22:14,1861-1879, DOI: 10.1163/092050610X528561

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Journal of Biomaterials Science 22 (2011) 1861–1879brill.nl/jbs

Structure and Biocompatibility of an Injectable BoneRegeneration Composite

Rongwei Tan a, Qingling Feng a,∗, He Jin b, Jinyu Li b, Xing Yu b, Zhending She a,c,

Mingbo Wang a and Huanye Liu d

a State Key Laboratory of New Ceramics and Fine Processing, Department of Materials Science andEngineering, Tsinghua University, Beijing 100084, P. R. China

b Department of Orthopaedics, Dongzhimen Hospital Affiliated to Beijing University of ChineseMedicine, Beijing 100700, P. R. China

c Center for Advanced Materials and Biotechnology, Research Institute of Tsinghua University inShenzhen, Shenzhen 518057, P. R. China

d Department of Prosthodontics, School of Stomatology, China Medical University, Shenyang110002, P. R. China

Received 1 April 2010; accepted 3 August 2010

AbstractWith the development of minimally invasive techniques, injectable materials have become one of the majorhotspots in the biomaterial field. We have developed an injectable bone regeneration composite (IBRC)using calcium alginate hydrogel as matrix to carry nano-hydroxyapatite/collagen particles. In this work,we evaluated the homogeneity of IBRC by dry/wet weight ratio test. The results showed that the structuralhomogeneity was determined by controlling the molar ratios of trisodium phosphate to calcium sulfate ratherthan alginate concentration in the studied ranges. Pore sizes of wet IBRC samples were characterized bythermoporometry. The pore properties of dried IBRC were tested by mercury porosimetry. Average pore sizeand porosity of dried IBRC declined with increasing alginate concentration. In contrast, surprisingly, poresize of wet homogeneous IBRC increased with increasing alginate concentration. Meanwhile, the swellingratio did not increase with varying alginate concentration, but the swelling degree increased with increasingalginate concentration. In vitro cell culture showed that IBRC had no obvious cytotoxic effect on the rat bonemesenchymal stem cells. The morphology and viability of cells were also related to MR value. IBRC hadgood histocompatibility with a mild short-term inflammatory response in rat dorsum muscle. In addition, theexcellent ability of IBRC to promote bone healing was confirmed by 5-mm-diameter cranial defects usinghistological analysis and bone mineral density measurement.© Koninklijke Brill NV, Leiden, 2011

KeywordsStructure, biocompatibility, injectable, bone regeneration

* To whom correspondence should be addressed. Tel.: (86-10) 6278-2770; Fax: (86-10) 6277-1160; e-mail:[email protected]

© Koninklijke Brill NV, Leiden, 2011 DOI:10.1163/092050610X528561

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1862 R. Tan et al. / Journal of Biomaterials Science 22 (2011) 1861–1879

1. Introduction

Hydrogels have been recognized as having a variety of applications in drug delivery,tissue engineering and regenerative medicine due to their advantageous biocompat-ibility, biodegradation and intrinsic cellular interaction [1, 2]. In particular, theymimic features of extracellular matrix, having highly hydrated three-dimensional(3-D) networks [3–5]. Injectable hydrogels have been explored extensively as cell-delivery systems with the advantage that cells and biomolecules can be readilyintegrated into the gelling matrix [6]. The injectable nature of the hydrogels pro-vides the attractive feature of facile and homogenous cell distribution within anydefect size or shape prior to gelation [7]. In addition, injectable hydrogels allowgood physical integration into the defect, shortening the surgical operation time,bringing compliance and comfort to patients for non- or minimal invasive surgery,and allow patients to recover rapidly in a cost-effective manner [7–9].

Hydrogels formed from alginate, a well-known natural polysaccharide com-posed of 1,4-linked β-D-mannuronate (MM-blocks) and 1,4-linked α-L-guluronate(GG-blocks) residues in variable proportions, have many attractive features for bio-medical applications. An important feature of alginate is that it is freely soluble inwater, and can be rapidly gelated in the presence of divalent cations, such as Ca2+[10, 11]. Due to its good biocompatibility, low toxicity, abundance in source andrelatively low cost [12–15], it has been widely used in the food industry as thick-ener and emulsification agent and in the medicine field as wound dressing [16, 17],and in drug delivery [14, 18, 19], cell encapsulation and tissue engineering [12, 20–22]. For medical applications, an alginate hydrogel is usually produced by drippingalginate solution into a CaCl2 bath [23] or by mixing alginate solution with CaSO4slurry [10, 24]. The major disadvantages of these systems are that the gelation rateis hard to control, the resulting structure is inhomogeneous, the mechanical prop-erty is usually poor, and the complex-shaped 3-D structure is difficult to be achieveddue to the rapid gelation [25–31]. To solve this problem, many strategies have beenused to control alginate gel degradation, such as manipulating alginate molecularweight and composition [32, 33], partial oxidation [34, 35] and forming compositescombined with other materials [36, 37].

Natural bone is a complex biomineral system with an intricate hierarchical struc-ture. It is assembled through the orderly deposition of hydroxyapatite mineralswith low crystallinity and nanometer size within a type-I collagenous matrix. Thebone-like nano-hydroxyapatite/collagen (nHAC) composite was synthesized bio-mimetically by mineralizing type-I collagen in our lab [38–40], which possessedfeatures similar to natural bone both in composition and hierarchical structure. Ex-tensive studies on nHAC confirmed that it is biodegradable, biocompatible andbioactive [38–40]. However, pure nHAC is difficult to be molded, and its me-chanical property is relatively low. Therefore, a porous nHAC/poly(L-lactic acid)composite [38–42], chitin fibre reinforced nHAC/poly(L-lactic acid) scaffold [41,43, 44] and controlled growth factors release from these scaffolds [45–47] havebeen successfully developed in our lab. These composites, however, are solid im-

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R. Tan et al. / Journal of Biomaterials Science 22 (2011) 1861–1879 1863

plants and need invasive surgical operations in clinical applications. Recently wehave developed an injectable bone regeneration composite (IBRC) using calciumalginate hydrogel as matrix to carry nHAC particles with controllable setting timeand tunable injectability, which provides a possible injectable material for bone re-pair and bone tissue engineering [48].

In tissue engineering, for cell distribution, well-controlled properties through-out the materials, diffusion of nutrients, removal of metabolic wastes, and structureproperties in scaffolds are highly important [26]. In this study, therefore, the firstobjective was to improve the structural homogeneity of IBRC and evaluate the porestructure. For tissue engineering applications, swelling behaviour and cross-linkingdegree are also important structural parameters for hydrogels, because structuralstability and mechanical property are determined by the swelling property, cross-linking density, properties of main polymer chain and cross-linking molecules. Thesecond aim was to investigate swelling behavior, cross-linking degree and effect ofpolymer concentration on them. Moreover, we assessed the in vitro cytocompat-ibility on 1,1-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil)labelled rBMSCs co-cultured with IBRC. Finally, the in vivo biocompatibility wasevaluated by embedding IBRC into rat dorsum muscle and repairing the rat calvarialdefects.

2. Materials and Methods

2.1. Material Preparation

The IBRC preparation method has been described in our previous paper [48]. Inbrief, the nHAC particles (50 ± 6.6 µm) were fabricated by mineralizing col-lagen using the method developed in our lab [38, 39]. Sodium alginate (Wm =(2–3) × 105 g/mol, Protanal LF 10/60) with 65–75% GG-blocks, trisodium phos-phate (Na3PO4 ·12H2O, Chemical Agents) and nHAC or hydroxyapatite (HA,prepared by a wet chemical reaction with a crystal size of 18 nm, and Ca/P =1.67; Osartis) were mixed in deionized water to form colloidal suspension A. Thefunction of trisodium phosphate is to control the gelation rate. Calcium sulfate ascross-linking agent (CaSO4 ·2H2O, Chemical Agents) was magnetically stirred for1 h to form CaSO4 slurry in deionized water, and then statically placed for morethan 24 h. Before mixing CaSO4 slurry with colloidal suspension A, CaSO4 slurryneeded to be stirred again for 1 min. Colloidal suspension A and CaSO4 slurry weremixed together for 1 min, and this pre-gelled colloidal suspension was defined ascolloidal suspension B. The colloidal suspension B was put rapidly into the moldto form a 2.0 cm diameter × 1.6 cm high cylindrical hydrogel. Here we define themolar ratio of Na3PO4 ·12H2O/CaSO4 ·2H2O as MR. Alginate was sterilized byhigh-pressure steam, the other materials were irradiated with a 25 kGy from a 60Cosource. For cell culture, the colloidal suspension B with 3% alginate and varied MRvalues was injected into 24-well cell-culture plates (1 ml/well) through a syringe to

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form a gel with a smooth surface. Then these plates were incubated in a cell-cultureincubator for 24 h before cells were seeded.

2.2. Structural Property Tests

Firstly, the homogeneity of the cylindrical IBRC samples was characterized withdry to wet weight ratios [26]. The wet IBRC samples (diameter × height =2.0 cm × 1.6 cm) were cut perpendicular to the cylinder axis into 5 slices withapproximately the same thickness. The slices were labelled number 1–5 from topto bottom for homogeneity test. After measuring their wet weights, the slices werefrozen in a refrigerator at −20◦C for 12 h and then lyophilized in a freeze drier. Thespecimens were weighed again after drying, and their dry/wet weight ratios werecalculated. The averages and the standard deviations of triplets were reported.

In order to understand the pore structural properties of IBRC, wet IBRC anddried IBRC samples were tested. The pore sizes of wet IBRC samples were char-acterized by the reported thermoporometry technique [10] after 12 h of samplegelation. The principle of thermoporometry is based on the lowering of the triplepoint temperature of the liquid in a porous material, thus providing a unique methodfor porous characterization of the hydrogels [49]. The triple point temperature ofthese materials depends on the solid–liquid and the liquid–gas interfaces; thus, theshift in equilibrium temperature of the liquid–solid transformation can be used todetermine the pore size distribution [50]. The shift in freezing and melting temper-ature of water held in IBRC was determined by differential scanning calorimetry(DSC) (Seiko DSC 6200 differential scanning calorimeter equipped with a liquidnitrogen-cooling accessory). The following procedures were taken to avoid the un-dercooling effect. Firstly, 10–12-mg samples were placed in a sealable aluminiumpan and cooled to −30◦C, held for 10 min, then heated up to 20◦C at a rate of2.0◦C/min, held for 10 min, and cooled down from 20◦C to −30◦C at a rate of2.0◦C/min. From the DSC thermograph and the equations below, the pore size andpore size distribution of IBRC could be determined [49]:

Rp = −(

64.47

�T

)+ 0.57, (1)

Wa (J/g) = −5.56 × 10−2�T 2 − 7.43�T − 332, (2)

�V

Rp=

(k�T

64.67Wa

)Y, (3)

where Rp is the pore radius, Wa the apparent energy of solidification of water, �T =T − T0 the shift in the triple point temperature, V the volume of the pores andk a factor depending on the rate of cooling and the weight of the sample. The heat(Y ) recorded by calorimetry during the experiment is proportional to the amount ofenergy involved [49]. The pore size averages and the standard deviations of tripletsare reported.

In addition, the samples were dried by freezing in a high vacuum and stored indesiccator for porosity and pore sizes tests. The porosity of dried IBRC was tested

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by mercury porosimetry (Micromeritics AutoPore IV 9510 Pore Size Analyzer).The porosity averages and the standard deviations of triplets were reported.

The swelling behaviours of IBRC at equilibrium were determined by measuringthe weight of completely swollen IBRC after incubation in deionized water at 37◦Cfor 24 h and the weight of dried IBRC. The swelling degree (Q) of the alginatehydrogel was defined as the reciprocal of the volume fraction of a polymer in ahydrogel (ν2) in previous report as follows [33]:

Q = 1

ν2=

(1

ρp

(Qm

ρs+ 1

ρp

)−1)−1

, (4)

where ρp is the polymer density, ρs is the water density (0.9971 g/cm3 at 25◦C),and Qm is the swelling ratio, defined as the mass ratio of absorbed water to thedried gel.

However, the swelling property of nHAC particles and inorganic salts in IBRCsystem could not be ignored compared with alginate molecules, so we redefined theequation (4) as follows:

Q = 1

ν2=

(1

K × ρGC

(Qm

ρs+ 1

K × ρGC

)−1)−1

, (5)

where ρGC is the density of dried IBRC samples, which was determined by au-tomatic pycnometers (AccuPyc 1330, Micromeritics Instrument) and the densitycoefficient K was defined as mass ratio of alginate to total components.

The degree of cross-linking of IBRC was characterized with effective number ofcross-links (N0) calculated using the rubber elastic theory that defined by Anseth etal. [51] as follows:

N0 = (GQ1/3/RT ), (6)

where R is the gas constant (8.314 J/mol K), T is the absolute temperature (K)at which the modulus was measured, Q is the swelling degree and G is the shearmodulus tested in our previous study [48].

2.3. In Vitro Cell Culture

Rat bone mesenchymal stem cells (rBMSCs, purchased from Chinese Academyof Medical Sciences), passage number 3–5, were cultured and expanded in cul-ture medium (89% DMEM containing 4500 mg/l D-glucose, 10% FBS, 1% peni-cillin/streptomycin; Sigma). The cultured cells were trypsinized with trypsin-EDTA(Sigma) and washed twice with DPBS (Sigma). Of the cell suspension 1 mlwas seeded onto the surface of IBRC in 24-well culture plates at a density of3.25×105 cells/ml in comparison with cells seeded on tissue-culture plates as stan-dard control group. The 6 IBRC samples with different alginate concentrations ordifferent MR values were used for cell culture. Four h after seeding, non-adherentcells were removed and the culture medium was replaced by fresh medium. There

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were at least 5 samples per condition and time point. All samples were harvested at4 h, 1, 2, 4 and 7 days for viability and proliferation test.

Viability and proliferation of rBMSCs were analyzed using MTT assay. In or-der to clarify the cells attached to the gels but not to bottom of wells, the gels weretransferred into another 12-well culture plates. Then MTT (0.5 mg/ml) solution wasadded into wells (60 µl/well), thereafter the cells were incubated for another 4 h at37◦C, and then dimethyl sulphoxide was added into wells (450 µl/well) to resolveformazan. The samples were transferred to 5 ml plastic tubes, which were cen-trifuged for 5 min at 8000 rpm. The supernatant fluid was extracted and put into an-other 96-well cell-culture plate for the MTT test. The optical density (OD) of the so-lution is proportional to the mitochondrial activity of viable cells. OD was measuredat a wavelength of 570 nm (Bio-Rad 680). For morphological observation of livingcells, cells were labeled by 1,1-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate (Dil) before cultured on IBRC surface. For Dil labeling and detection,rBMSCs of the third passage were incubated in 5.0 µg/ml DMSO solution in Dilbuffer at 37◦C for 30 min [52]. After washing twice in PBS, the cells were culturedon the IBRC surface. The cells were observed under a fluorescence microscope(Leica).

2.4. In Vivo Biocompatibility

All animals for biocompatibility tests were treated in accordance with the nationalanimal care guidelines. Firstly, 12 male SD rats (320–350 g) were used to test thehistocompatibility of IBRC. The rats were housed in sterilized cages with sterilefood, water and filtered air. The rats were anesthetized by intraperitoneal injection(50 mg/kg body weight) of ketamine (China National Medicines) in combinationwith local anesthesia of xylocain (10 mg/kg body weight) (China National Medi-cines). The pre-gelled IBRC was zygomorphously injected using a 16-gauge needleinto the rats’ dorsum muscle of the spinal column where IBRC was fixed in situ.At different time points after injection, 3 rats were anaesthetized and the IBRC incombination with around muscle tissue was removed. Subsequently, all the sam-ples were fixed with 10% formalin for 24 at 4◦C, and embedded in paraffin wax.Paraffin-embedded samples were sectioned at 10 µm thickness with a microtomeand stained with hematoxylin and eosin (H&E). The ensuing sections were ex-amined under an optical microscope (Leica). IBRC samples with 3% alginate and0.260 MR were used for all in vivo biocompatibility assessments.

After evaluation of histocompatibility, 24 male SD rats (320–350 g) were dividedinto two groups (group A and group B) to test bone regeneration ability. Under anes-thesia (by intraperitoneal injection ketamine at a dose of 50 mg/kg body weight incombination with local anesthesia of xylocain at a dose of 10 mg/kg body weight),an incision was made in the skin over the sagittal suture and the parietal bones wereexposed. Then two craniotomy defects (5 mm diameter) were created with trephine,carefully avoiding dural perforation. The surgical site was flushed with saline to re-move bone debris and the composite was injected into one defect (group A with

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IBRC, group B with hydroxyapatite (HA) instead of nHAC in IBRC in comparisonwith group A), the other defect as blank control was used to compare with group Aand B. Rats were anesthetized and killed at 4 and 8 weeks after surgical implanta-tion (6 animals per time point in A and B group). Calvarial samples were harvestedfor gross observation, and fixed in 10% formalin for 24 at 4◦C for bone mineraldensity (BMD) test and histological evaluation. Calvarial samples were decalcifiedovernight with decalcifying solution, then trimmed, processed and embedded inparaffin wax. Paraffin embedded samples were sectioned at 10 µm thickness witha microtome. Sections were stained with H&E and Masson’s trichrome stain forhistology study. The BMD of the defective regions was measured by dual energyX-ray absorptiometry (DEXA) (Lunar) using the small animal scan mode with asample resolution of 0.03 cm × 0.06 cm. The method for measurement of interestpositions and regions has been described in a previous report [53].

2.5. Statistical Analysis

Three examples were used in each test and their average values were calculatedfor statistical analysis. All data were statistically analyzed by analysis of variance(ANOVA) software and expressed as mean ± SD. P � 0.05 was considered to bestatistically significant.

3. Results

3.1. Material Preparation and Its Homogeneity

To determine the homogeneity of IBRC, cylindrical slices with varying alginateconcentrations or varying MR were prepared. With varying alginate concentrations,dry/wet weight ratios of slices per IBRC sample, which were numbered 1–5 fromtop to bottom, have no significant difference (Fig. 1a). The results of Fig. 1a indicatethat alginate concentrations have no significant influence on gel homogeneity inthe studied concentration ranges of 2–5% alginate. In contrast, a MR of 0.180 and0.388 affected gel homogeneity significantly (P < 0.05) except the MR of 0.260(Fig. 1b). The groups not gelated after 24 h and the groups gelated too rapidly arenot displayed here, because they cannot be used in clinical applications.

3.2. Pore Structural Properties

In spite of the MR values affecting gel homogeneity significantly (Fig. 1b), driedIBRC samples with same alginate concentration have similar average pore sizeswith varying MR values, but pore size distribution of dried IBRC samples with MR0.260 is smaller than that with MR 0.180 and 0.388. Meanwhile, average pore sizesof dried IBRC with the same MR value declined with increasing alginate concentra-tion (Table 1). Porosities of dried IBRC with the same alginate concentration haveno significant difference with varying MR values (P > 0.05) (Table 1). Porositiesof dried IBRC with the same MR value declined with increasing alginate concentra-tion (Table 1). In contrast, pore sizes of wet IBRC increased with increasing alginate

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(a) (b)

Figure 1. (a) Dry/wet weight ratios of IBRC slices of samples (MR = 0.260) numbered 1–5, fromtop to bottom of the cylinder with varying alginate concentrations; (b) dry/wet weight ratios of IBRCslices of samples numbered 1–5, from top to bottom of the cylinder with 3% alginate and varying MR.

Table 1.Pore sizes and porosity of dried IBRC with varying alginate concentrations and MR

MR Pore size (µm), porosity (%)

2% Alginate 3% Alginate 4% Alginate 5% Alginate

0.180 185 ± 97, 90.3 ± 3.6 150 ± 91, 89.7 ± 2.3 125 ± 76, 88.3 ± 2.7 105 ± 97, 87.5 ± 2.10.260 183 ± 29, 90.5 ± 2.7 152 ± 21, 89.5 ± 2.1 127 ± 19, 88.4 ± 1.6 102 ± 22, 87.9 ± 1.90.388 184 ± 91, 90.0 ± 3.2 151 ± 90, 89.5 ± 2.0 125 ± 95, 88.0 ± 2.4 103 ± 84, 87.5 ± 1.7

Values represent means ± SD.

Table 2.Pore sizes of IBRC with varying alginateconcentrations, MR = 0.260

Alginate concentration Pore size(%) (nm)

2 6.5 ± 2.13 7.1 ± 2.74 8.1 ± 3.15 8.4 ± 3.3

concentration (Table 2). When the concentration of alginate increased from 2% to5%, the corresponding pore size increased from 6.5 nm to 8.4 nm. Because sampleswith MR 0.388 and 0.180 do not have good homogeneity, Table 2 only lists poresizes of IBRC with MR 0.260.

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Table 3.Swelling properties and degree of cross-linking of IBRC with varying alginate concentrations

Alginate Density of Density Swelling Swelling Number of effectiveconcentration dry IBRC coefficient ratio Qm degree cross-links(%) ρGC (g/cm3) K Q (N0 × 106 mol/cm3)

2 1.57 ± 0.02 0.31 20.5 ± 0.9 11.7 ± 0.3 22.6 ± 0.23 1.79 ± 0.02 0.41 22.1 ± 1.5 17.1 ± 0.5 42.3 ± 0.34 2.01 ± 0.04 0.48 22.0 ± 0.8 22.2 ± 0.2 53.6 ± 0.15 2.23 ± 0.03 0.53 22.1 ± 1.4 27.4 ± 0.3 59.6 ± 0.3

Values represent means ± SD. The concentration of Na3PO4 ·12H2O is 5.0 mg/ml, the MR is0.260.

3.3. Swelling Properties and Cross-Linking Degree

Because samples with MR 0.388 and 0.180 did not show good homogeneity, andalginate concentrations had no significant influence on gel homogeneity (Fig. 1),we mainly measured influence of polymer concentration on swelling properties andcross-linking degree of homogenous IBRC. Densities of dry IBRC (ρGC) had asimilar trend to mechanical properties with varying alginate concentration in ourprevious study [48]. However, the swelling ratio (Qm) increased with increasing al-ginate concentration from 2% to 3%, but there were no obvious changes from 3% to5% (Table 3). The swelling degree (Q) increased with increasing alginate concen-tration (Table 3). The number of effective cross-links (N0) was determined basedon the rubber elastic theory; equation (6) was used to calculate N0. As expected,the N0 value was the lowest in IBRC with 2% alginate, and the highest in IBRCwith 5% alginate in the studied ranges.

3.4. In Vitro Cell Culture

The viability and proliferation of rBMSCs on cultured IBRC for 4 h, 1, 2, 4 and 7days were assessed by MTT assay. The data are shown in Fig. 2. There is no statisti-cally significant difference among all groups until 2 days. On the fourth day, there isno statistically significant difference among 4 groups (c, d, e and f groups in Fig. 2)of IBRC with MR value 0.260 and the control group (g group in Fig. 2). However,these groups displayed a higher level of viability than IBRC with a MR value of0.180 or 0.388 (P < 0.05). On the seventh day, there were four groups of IBRCwith different alginate concentration, but the same MR value (0.260), displaying ahigher level of viability and proliferation than the standard control group and theIBRC groups with MR value 0.180 and 0.388 (P < 0.05).

Using fluorescent microscopy, Dil-labeled rBMSCs on IBRC surface were ob-served in this study (Fig. 3). As for IBRC with MR value 0.260 and 3% alginate,the number and morphology of rBMSCs changed significantly at 2, 7 and 14 days,respectively. A small amount of rBMSCs can be observed at 2 days (Fig. 3a), butthe cell number increased obviously after 7 days culture. The partial cellular mor-

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Figure 2. MTT assay for viability and proliferation of rBMSCs cultured on IBRC or on tissue cultureplate as the standard control group. This figure is published in colour in the online edition of thisjournal, that can be accessed via http://www.brill.nl/jbs

phologies were elongated and a small amount of cells showed rounded morphology(Fig. 3b). Up to 2 weeks, the number of cells further increased, the cells adhered,spread and survived on IBRC surface very well, and nearly all cells became elon-gated (Fig. 3c). There was no obvious difference on morphologies among the 4groups with MR 0.260 on day 7 (Fig. 3b, d–f). Interestingly, the cells on the IBRCwith MR 0.180 or 0.388 agglomerated (Fig. 3g and 3h). The non-Dil-labeled rBM-SCs as standard control group are shown in Fig. 3i. The cells adhered, spread andsurvived very well on the tissue culture plate.

3.5. In Vivo Biocompatibility

To assess the histocompatibility of IBRC, firstly, all rats were observed grosslyevery day. There was no obvious inflammatory response. Then the rats were anaes-thetized and the injected IBRC in combination with around muscle tissue wasremoved for H&E staining. As for IBRC, the H&E stained histological sectionsshow that after 1 week a number of macrophages and neutrophils can be seen at theborder between IBRC and muscle (marked by arrows in Fig. 4a). After 2 weeks,there were few cells at the interface of IBRC and tissue (marked by arrows inFig. 4b). After 4 weeks, many cells invaded into IBRC, and there is a dense ac-cumulation of cells around IBRC bulks (marked by arrows in Fig. 4c). Similarly,after 6 weeks, IBRC was further divided by cells (IBRC is marked by arrows inFig. 4d). In addition, no fibrous capsule was observed for IBRC at 1, 2, 4 and 6weeks, respectively (Fig. 4).

In order to test the bone regeneration ability of IBRC in repairing a 5-cm cal-varial defect, group A (IBRC), group B (the nHAC in IBRC was replaced by HA)and blank control were used to compare. The rat in IBRC surgical implantation

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Figure 3. Dil-labeled rBMSCs were cultured on IBRC surface and observed by fluorescent mi-croscopy. The cell membrane is red after Dil labeling. (a–c) The IBRC with 3% alginate and MR0.260 observed on days 2, 7 and 14, respectively. (d–f) IBRC with MR 0.260 but different alginateconcentration on day 7. (h, i) IBRC with different MR value but same 3% alginate on the seventhday. (i) Non-Dil labeled rBMSCs were cultured on tissue-culture plate as standard control group onday 7. This figure is published in colour in the online edition of this journal, that can be accessed viahttp://www.brill.nl/jbs

is shown in Fig. 5. After 4 and 8 weeks surgical implantation, calvarial sampleswere harvested for gross observation (Fig. 5). After 4 weeks surgical implanta-tion, there are apparent differences between composites-filled defects and ambientnatural bone (see the narrow arrows in Fig. 5a and 5c). H&E and Masson stainingconfirmed that composites were divided into ‘islands’ and some newly formed bonein group A (Fig. 6a), but the central regions of defects in group B were mainly com-posites, a large number of osteoblasts grew into the composites, and a few newlyformed bone cells mainly existed on the edge of the defects (Fig. 6b). In addition,no fibrous capsule was observed at interface of composite and injured natural bone,but some fibrous capsule formed on the upper and lower surfaces of composite.All blank defects formed a kind of translucent fibrous tissue membrane (Fig. 6).After 8 weeks surgical implantation, the differences between A or B filled defectsand ambient natural bone were not distinctive, but blank defects were still markeddifferent in comparison with ambient natural bone from gross observation (see the

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Figure 4. H&E staining results of rBMSCs on IBRC (3% alginate and 0.260 MR) under opticalmicroscope (200×) at (a) 1, (b) 2, (c) 4 and (d) 6 weeks, respectively. This figure is published incolour in the online edition of this journal, that can be accessed via http://www.brill.nl/jbs

narrow arrows in Fig. 5b and 5d). All blank defects still covered with the translucentfibrous tissue membrane (wider arrows in Fig. 5b and 5d; fibrous tissue in Fig. 6c).Compared with histology results of group A and group B at 8 weeks, newly formedbone filled most of defects in group A, but most region did not calcified in group B,indicating that IBRC has better bone repair capability than composite B. This resultis consistent with BMD tested at 8 weeks, in which group A filled defects havehigher BMD than that of group B. There is significant difference among group A,group B, blank control group (P < 0.05) (Fig. 7).

4. Discussion

4.1. Structural Properties

The major disadvantages for alginate hydrogel cross-linked with CaCl2 or CaSO4to form hydrogels are that the gelation rate is hard to be controlled, the resultingstructure is inhomogeneous, the mechanical property is usually very poor and thecomplex-shaped 3-D structure is difficult to obtain [25–29]. In order to prepare aforming alginate hydrogel in combination with nHAC with homogeneous structure

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Figure 5. Observation of 5-mm-diameter defects in calvarial bone after a healing period of (a, c) 4and (b, d) 8 weeks, respectively. (A) IBRC; (B) composite in which the nHAC in IBRC was replacedby HA. Scale bar = 1 cm. This figure is published in colour in the online edition of this journal, thatcan be accessed via http://www.brill.nl/jbs

and controlled injectability in situ, trisodium phosphate was added to pre-gelled so-lutions in order to manipulate the gelation rate [48]. Structural uniformity in tissueengineering scaffolds is necessary, not only for uniform cell distribution, but alsofor well-controlled material properties. Meanwhile, mechanical properties are moreconsistent throughout the gel and among samples if the structure is homogeneous[26]. The dry/wet weight ratios would vary if IBRC formed an inhomogeneousstructure. Therefore, the results of Fig. 1a indicate that alginate concentrations haveno significant influence on gel homogeneity in the studied concentration ranges of2–5% alginate. On the contrary, the MR of 0.180 and 0.388 affected gel homogene-ity significantly (P < 0.05) except the MR of 0.260 (Fig. 1b). These results can beexplained by gelation mechanism of IBRC in our previous study [48]. The gela-tion mechanism is that the Ca2+ from sparingly soluble CaSO4 reacted with PO4

3−first, and produced insoluble calcium phosphate because the solubility constant ofCa3(PO4)2 that is much lower than that of CaSO4. Then the surplus CaSO4 un-ceasingly produced Ca2+, which cross-linked carboxyl groups of alginate to forma calcium alginate hydrogel [48]. When MR = 0.180, excessive free Ca2+ resultsin partial gelation and hindering Ca2+ to access the uncross-linked alginate regionsbefore CaSO4 and colloidal suspension A mixed thoroughly, which resulted in for-mation of a heterogeneous structure. When MR = 0.388, Ca2+ from CaSO4 mainlyreacted with PO4

3− to produce too much insoluble Ca3(PO4)2, which deposited onCaSO4 particles to hinder dissolving of CaSO4 and producing of Ca2+; therefore,IBRC cannot gel in a relative short time, which leads to phase separation for for-mation of heterogeneous structure. When MR = 0.260, the reaction for producingCa3(PO4)2 could provide a short time for colloidal suspension A to mix thoroughly.

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Figure 6. HE and Masson’s trichrome staining. (a) Defects repaired by IBRC; (b) defects repaired bycomposite that nHAC in IBRC replaced by HA; (c) blank control defects. (A) IBRC; (B) composite inwhich nHAC in IBRC was replaced by HA. b, Newly formed bone; f, fibrous tissue; N, natural bonetissue. This figure is published in colour in the online edition of this journal, that can be accessed viahttp://www.brill.nl/jbs

Then the surplus CaSO4 dissolved and produced Ca2+, which cross-linked carboxylgroups of alginate to form homogeneous IBRC.

In spite of MR values affected gel homogeneity significantly (Fig. 1b), there isno obvious influence of MR values on pore sizes of dried IBRC (Table 1). But poresize distribution of dried IBRC samples with MR 0.260 was smaller than that withMR 0.180 and 0.388. These results confirmed that pore size distributions dependedon MR values and reflected homogeneity degree of IBRC. Pore sizes and porositiesof dried IBRC with same MR value declined with increasing alginate concentration(Table 1), so the variation trend of pore sizes and porosities of dried IBRC can bepredicted by alginate concentration. Interestingly, pore sizes of wet IBRC increasedwith increasing alginate concentration (Table 2). It may be contributed to inter-molecular forces enlarged with increasing alginate concentration. It was reported

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Figure 7. BMD of newly formed bone in defects repaired by different materials was tested by DEXA.(A) IBRC; (B) composite in which nHAC in IBRC was replaced by HA; (C) blank control; (D) BDMof natural calvaria. Data are means ± standard error.

that pore sizes of a 2% alginate hydrogel cross-linked by CaSO4 slurry are 5.8 ±0.2 nm [10]. It can be inferred that pore sizes of wet IBRC reflected the strength ofinter-molecular forces increased with increasing of alginate concentration.

Densities of dry IBRC (ρGC) had similar trend to mechanical properties withvarying alginate concentration in our previous study [48]. The mechanical proper-ties of IBRC increased with alginate concentration due to the increase of polymerchain density and entanglement [48]. The swelling degree (Q) also increased withincreasing alginate concentration (Table 3) may due to that higher alginate concen-tration of IBRC had absorbed water lead to increasing of volume. However, theswelling ratio (Qm) did not increase with alginate concentration increasing (Ta-ble 3). In other words, the net quantity of absorbed water in same volume of IBRCdid not increase with alginate concentration increasing, which may be due to the ca-pability of absorbed water limited by increased strength of inter-molecular forces.Combined with the pore sizes of wet IBRC, it can be inferred that the variation trendof swelling ratio and pore size of wet IBRC also can be predicted by alginate con-centration. The number of effective cross-links (N0) was the lowest in IBRC with2% alginate, and highest in IBRC with 5% alginate in the studied ranges, whichis due to that more interaction between alginate and Ca2+ led to the increase ofcross-links with alginate concentration increasing.

4.2. Biocompatibility

Dil, a long-chain dialkylcarbocyanine, has been used to label the cell membranesince 1980s. Once the cells were labeled, Dil can be detected by fluorescent mi-croscopy for as long as 7 weeks in vitro, even if some had transferred to daughtercells during proliferation [54]. Using fluorescent microscopy, the number and mor-phology of Dil-labeled rBMSCs on the IBRC surface were observed. In this study,the number of cells further increased, the cells adhered, spread and survived on

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IBRC with MR 0.260 in comparison with standard control group or IBRC with MR0.180 or 0.388. These results confirmed that IBRC with MR 0.260 had a good cy-tocompatibility, which will be benefit to cellular adhesion and biological activity.However, the cells were well-distributed on IBRC with MR 0.260. In contrast, thecells agglomerated on IBRC with MR 0.180 or 0.388, because the IBRCs with MR0.260 have better structural homogeneity than the one with MR 0.180 or 0.388. Theagglomeration may result from structural inhomogeneity. Although the swellingbehaviours and pore sizes of dry/wet IBRC mainly depended on alginate concen-tration, the viability and proliferation of cells on IBRC surface were related to theirMR values. Also, structural homogeneity of IBRC was determined by MR value.Thus, it can be concluded that the structural homogeneity of IBRC influences theviability, proliferation and morphology of cells in 2-D culturing.

Following biomaterial implantation, the healing response consists of inflamma-tion, wound healing and the foreign body reaction [55]. In addition, the foreignbody reaction to an implanted material follows a stereotypical sequence of events[56, 57]: nonspecific protein adsorption and adhesion of cells (such as monocytes,leukocytes and platelets) on the biomaterial surface, which leads to giant cell for-mation and cytokine release. Ultimately, the implant is encapsulated by a fibrouscapsule [55]. For the IBRC system, the H&E stained histological sections showthat after 1 week a number of macrophages and neutrophils are notable at the bor-der between IBRC and muscle (marked by arrows in Fig. 4a). The inflammatoryphase is a necessary prerequisite for healing [58]. Therefore, it is difficult to assesswhether the inflammatory response is due to the normal healing process or the ma-terial’s effect during early stage of wound healing. After 2 weeks, few cells existedon the interface of IBRC and tissue (marked by arrows in Fig. 4b). These findingssupport that IBRC has good biocompatibility in vivo with a mild short-term inflam-matory response. After 4 weeks, many cells invaded into IBRC, and there is a denseaccumulation of cells around IBRC bulks (marked by arrows in Fig. 4c). Up to 6weeks, IBRC was further divided by cells, IBRC is marked by arrows in Fig. 4d. Itwas reported that, as a consequence of macrophage–biomaterial interaction, there isa fusion of adherent macrophages, leading to formation of multinucleated foreignbody cells (FBGC) on the biomaterial surfaces [59–61]. This process is accom-panied by FBGC-mediated biomaterial degradation [62]. As for IBRC, however,the cells concentrated at the interface only in the first week (Fig. 4a), then thecell number decreased obviously at material surface (Fig. 4b), a large number ofcells assembled inside of IBRC, and the material was divided into ‘islands’ (Fig. 4cand 4d). This phenomenon may be contributed to the hydrated structure of IBRCthat is in favour of migration and spread of cells. The end stage of healing responseto biomaterials is generally fibrosis or fibrous encapsulation [56]. However, theremay be exceptions to this general statement such as porous materials inoculatedwith parenchymal cells or porous materials implanted into bone [56]. Similarly, nofibrous capsule was observed for IBRC at 1, 2, 4 and 6 weeks, respectively (Fig. 4),maybe due to its porous structure and a large number of cells growing into IBRC.

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The bone regeneration ability of IBRC was characterized by repairing a 5-cmcalvarial defect. In addition, group B (the nHAC in IBRC was replaced by HA)and blank control were used to compare with IBRC (group A). Calvarial sampleswere harvested for gross observation after 4 weeks surgical implantation; an appar-ent difference existed between composites filled defects and ambient natural bone(see the narrow arrows in Fig. 5a and 5c) due to that the amount of newly formedbone is not enough. Histological results confirmed that the material in group A hada higher degradation rate than that of group B. Grossly observation after 8 weekssurgical implantation, the defective regions filled with composite A or B were notmarkedly different from ambient natural bone, which confirmed that the bone den-sity in defects increased, but blank defective regions were still markedly differentin comparison with ambient natural bone (see the narrow arrows in Fig. 5b and 5d).Histological results and BMD further confirmed that the bone densities in defectiveregions filled with composites A or B were higher in comparison with blank control.Compared with histology results of group A and B at 8 weeks, newly formed bonefilled most space of the defective regions in group A, but most space of the defectiveregions in group B was not calcified, indicating that IBRC has better bone repaircapability than composite B. This result is consistent with BMD measurement thatthere is a significant difference between group A and group B (P < 0.05) (Fig. 7).The differences between group A and B are components: A is IBRC in that calciumalginate hydrogel as matrix vehicle to carry nHAC particles, but B is a composite inwhich nHAC was replaced by HA. The nHAC has better bone bioactivity than HA,possibly due to its natural intricate hierarchical structure and orderly deposition ofhydroxyapatite minerals with low crystallinity and nano-size within a type-I colla-gen matrix. The hierarchical structure, low crystallinity, nano-sized hydroxyapatiteand bioactive collage I matrix are favourable for osteoblasts to grow, proliferate andalso for new bone formation. All these results could support that IBRC possesses agood biocompatibility in vivo.

5. Conclusion

In this work, the results of dry/wet weight ratios have shown that the injectable andin situ forming IBRC has a good homogeneity with varying alginate concentrationand a molar ratio of trisodium phosphate to calcium sulfate of 0.260. The resultsshowed that the structural homogeneity was determined by controlling molar ratiosof trisodium phosphate to calcium sulfate rather than alginate concentration in thestudied ranges. Pore size and porosity of dried IBRC declined with increasing al-ginate concentration. In contrast, pore sizes of wet IBRC increased with increasingalginate concentration. In addition, the alginate concentration obviously influencedthe swelling property and cross-linking degree of IBRC. In vitro cell culture demon-strated that rBMCs adhered, spread and survived on IBRC with MR 0.260 verywell. The structural homogeneity of IBRC had influence on viability, proliferationand morphology of cells. In vivo results supported that IBRC has good biocom-

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patibility with a mild short-term inflammatory response. Histological analysis andBMD measurement confirmed that IBRC can promote bone regeneration for 5-mm-diameter cranial defects. In a word, the injectable and in situ forming IBRChas good structural homogeneity and biocompatibility for possible use as injectablematerial for non- or minimal invasive bone repair and bone tissue engineering.

Acknowledgements

This study was supported by National Natural Science Foundation of China(50772052) and Doctor Subject Foundation of the Ministry of Education of China(20070003004). We thank Prof. Qiang Lu for helpful discussions.

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structure and biocompatibility of an injectable bone regeneration composite

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