Incorporation of calcium sulfate dihydrate into hydroxyapatite

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Incorporation of calcium sulfate dihydrate into hydroxyapatite microspheres to improve the release of bone morphogenetic protein-2 and accelerate bone regeneration Jaeuk Baek, Hyun Lee, Tae-Sik Jang, Juha Song, Hyoun-Ee Kim, and Hyun-Do Jung ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00715 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

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ACS Biomaterials Science & Engineering

Incorporation of calcium sulfate dihydrate into hydroxyapatite microspheres to improve the release of bone morphogenetic protein-2 and accelerate bone regeneration

Jaeuk Baeka, Hyun Leea, Tae-Sik Jangb, Juha Songb, Hyoun-Ee Kima,c, Hyun-Do Jung d,1

a

b

Department of Materials Science and Engineering, Seoul National University, Seoul, South Korea

Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, 637457, Singapore c

Biomedical Implant Convergence Research Center, Advanced Institutes of Convergence Technology, Suwon, South Korea

d

Liquid Processing & Casting Technology R&D Group, Korea Institute of Industrial Technology, Incheon, 21999, Korea

1

Corresponding author. Tel.: +82 32 850 0437; fax.: +82 32 850 0410.

E-mail address: [email protected] (H.-D. Jung)

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Abstract In this study, hydroxyapatite (HA)-based microspheres with the ability to deliver bone morphogenetic protein-2 (BMP-2) were developed for accelerating bone regeneration. The incorporation of calcium sulfate dihydrate (CSD) in the HA matrix improved the rate of BMP-2 release from the microspheres. Under physiological conditions, the CSD fully degraded within seven days and generated pore channels in the microspheres. The porosity and pore size of the HA–CSD microspheres after CSD degradation were 34.3% ± 4.2% and 11.5 ± 2.4 µm, respectively, significantly larger than those of the HA microspheres (23.9% ± 3.1% and 8.7 ± 0.9 µm, respectively). The increased porosity directly affected the rate of BMP-2 release from the microspheres. An in vitro experiment showed that both the BMP-2 release rate and the total amount of BMP-2 released increased considerably when incorporating the HA microspheres with CSD. BMP-2 was released slowly from the HA microspheres for up to six weeks. BMP-2 release was notably improved in the HA–CSD biphasic microspheres compared to the microspheres without CSD; the rate of release was 2.4-times faster due to the pores created by CSD dissolution after seven days. Prior to animal testing, in vitro cell tests were performed to evaluate the biocompatibility of the HA–CSD microspheres. During CSD dissolution, biocompatible bone-like apatite precipitated on the cell surfaces, and pre-osteoblasts grew on the microspheres. In vivo experiments using a rabbit lateral femoral condyle defect model demonstrated that the level of bone regeneration was significantly enhanced by mineralization on the surface, generated additional pores as well as improved BMP-2 release behavior. The HA–CSD microspheres accelerated new bone growth to fill the entire defect in six weeks, corresponding to a 170% improvement in performance compared to the HA microspheres. Keywords: Hydroxyapatite microspheres, Bone morphogenetic protein, Bone regeneration,


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Calcium sulfate dihydrate, In vivo animal test



1. Introduction Hydroxyapatite (HA) is one of the most widely used synthetic bone grafting materials for bone reconstruction in dentistry and orthopedics because of its outstanding performance and its osteoconduction in particular.1-4 The excellent biological properties of HA are primarily attributed to its chemical composition, which is similar to that of the inorganic phase of human bone.3, 5-6 Bone is an organic–inorganic composite constituting approximately 70% inorganic calcium phosphate.7-8 When implanted into defective bone, HA exhibits negligible immunoreaction and releases calcium ions at a rate appropriate to stimulate osteoblastic cells and promotes bone regeneration.3, 9-10 HA is particularly effective as a bone grafting material when prepared in the form of microspheres. Their spherical shape and small size allow microspheres to homogeneously fill complex-shaped defects without particle agglomeration.11-12 In addition, the pore channels built between microspheres play a critical role in accelerating bone recovery; the cells and nutrients needed for recovery are delivered by circulating blood through the interconnected space, facilitating new bone growth along the microspheres.11, 13-15 A water-in-oil emulsion technique using calcium phosphate cement (CPC) was recently used to synthesize HA microspheres with incorporated therapeutic agents for improved bone healing.13-14, 16 Since CPC spontaneously hardens and transforms into HA under physiological conditions, biological molecules such as drugs or growth factors can be directly loaded in the HA matrix with negligible denaturation.13, 17 Recombinant human bone morphogenetic protein type 2 (rhBMP-2) is one of the most widely used growth factors in bone remodeling. This growth factor has been approved by the Food and Drug Administration (FDA) for spinal fusion surgery18-19 and exhibits remarkable osteoinductive performance.17, 20


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Even though HA microspheres synthesized from CPC delivered BMP-2 to the defective bone and notably improved bone regeneration rate, the release rate was slow and the total released amount was relatively low compared to the initial loading amount.21 Since CPC-based materials lack macro-pores and exhibit high binding affinities for proteins, the BMP-2 trapped within CPC is not actively released.17,

22

Incorporating BMP-2-loaded

poly(lactic-co-glycolic acid) (PLGA) in a CPC matrix can improve BMP-2 release from the CPC scaffold; however, PLGA degradation causes the pH of phosphate buffered saline (PBS) to decrease, and the resulting acidic environment can damage the surrounding tissues.23 Although several studies have been conducted on the release of growth factor from CPC scaffolds, few have focused on improving BMP-2 release from CPC-based HA microspheres. Surgical-grade calcium sulfate is an FDA-approved bone graft material with a rodlike shape. Compared to HA, which requires at least a few years for resorption, the resorption of calcium sulfate is predictable and rapid.24 Calcium sulfate also encourages angiogenesis and osteogenesis as it dissolves.25-26 Little change in pH is observed around calcium sulfatecontaining composite materials, demonstrating their biocompatibility.27-28 In addition, calcium sulfate cements have been used to treat osteomyelitis since they sustain the release of antibiotics for several weeks.29-30 In the present study, we used calcium sulfate to create pores in HA microspheres. The pores generated due to dissolution of calcium sulfate create pathways to prolong and steady the release of BMP-2, while the calcium sulfate embedded in the HA microspheres acts as a physical barrier to delay and decelerate release in the initial stage. In this study, we propose novel biphasic microspheres comprising HA and calcium sulfate dehydrate (CSD) that release BMP-2 at an accelerated rate and promote a high degree of bone regeneration. CSD-incorporated HA microspheres were fabricated using a water-in-



oil emulsion method, and BMP-2 was directly incorporated in situ into a mixture of CPC and CSD. The changes in microstructure, chemical composition, and cellular response caused by the degradation of CSD were evaluated using scanning electron microscopy (SEM), X-ray diffraction (XRD), and pre-osteoblast cell attachment tests, respectively. The release behavior of BMP-2-loaded HA–CSD microspheres was evaluated to identify the role of CSD. The results indicated that the HA–CSD biphasic microspheres accelerated BMP-2 release and in vivo bone regeneration.

2. Materials and methods

2.1. Preparation of α-tricalcium phosphate, tetracalcium phosphate, and CSD powders

A mixture of α-tricalcium phosphate (α-TCP) and tetracalcium phosphate (TTCP) powders was used as the CPC powder. The α-TCP and TTCP powders were prepared by mixing and heat treating anhydrous dicalcium phosphate (CaHPO4, Sigma Aldrich, USA) and calcium carbonate (Sigma Aldrich, USA), as described in our previous study.21 CSD (CaSO4·2H2O, Sigma Aldrich, USA) was sieved down to 90 µm.

2.2. Preparation of CPC–CSD paste CSD powder (25 wt%) was mixed with the CPC powder constituting α-TCP and TTCP (molar ratio = 1:1). To obtain a self-hardening paste containing BMP-2 produced from Escherichia coli, a BMP-2 solution and a hardening liquid medium comprising 10 wt% citric acid in 1.0 M Na2HPO4 were hand-mixed with the CPC–CSD powder at a powder-to-liquid


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ratio of 1.67.

2.3. Synthesis of HA–CSD microspheres Before the CPC–CSD paste was solidified, it was transferred to olive oil containing a surfactant (Labrafil®,, 1944 CS, Gattefossé, France) under magnetic stirring at 300 rpm to form spherical particles. After emulsification for 10 min, the TCP and TTCP phases of the microspheres transformed into HA by treating the microspheres for 3 d in an oven at 37°C. The HA–CSD microspheres in oil were then washed with acetone and ethanol and air-dried. The BMP-2-loaded HA–CSD microspheres were sieved to be between 250 and 500 µm to narrow the size distribution. For comparison, BMP-2-loaded HA microspheres were fabricated using the same procedure but without CSD. Unloaded HA and HA–CSD microspheres were also produced to analyze the basic properties of the microspheres.

2.4. Microstructural characterization The pore structures and microstructures of the HA and HA–CSD microspheres were observed via SEM (JSM-6360, JEOL, Japan). Porosity and pore size were evaluated from the SEM images of each sample using the National Institutes of Health Image J 1.36b imaging software (National Institutes of Health, Bethesda, MD, USA). The crystalline phases of the HA and HA–CSD microspheres were characterized via XRD (D8-advance, Bruker Co., Germany) using Cu Kα radiation at a scanning rate of 1°/min from 25° to 35°. The microspheres were also analyzed via SEM and XRD after immersion in PBS solution at 37 °C for 4 and 7 d.



2.5. In vitro tests The in vitro biocompatibilities of the HA and HA–CSD microspheres were evaluated using a pre-osteoblast cell line (MC3T3-E1; ATCC, CRL-2593, Rockville, MD, USA). The microspheres were fixed to carbon tape attached on cell culturing plates. The cells were then seeded on the microspheres at a density of 2 × 104 cells/mL and cultured at 37 °C in a humidified incubator with 5% CO2. For comparison, the in vitro cytocompatibility of the microspheres after CSD degradation was also evaluated. Prior to cell seeding, the CSD in the microspheres was completely dissolved in PBS at 37°C for 7 d, and the cells were then placed and cultured on the CSD-free microspheres under identical conditions. After 24 h of culturing, the morphologies of the cells on the microspheres were observed via SEM. Before SEM observation, the cells were fixed with 2.5% glutaraldehyde for 10 min and dehydrated in 70%, 95%, and 100% grade ethanol. The specimens were immersed in hexamethyldisilazane for 10 min, followed by air-drying.

2.6. Characterization of BMP-2 release The BMP-2-loaded microspheres were immersed in PBS at 37°C for up to 42 d. 20 mg of microspheres was immersed in 1 ml of PBS. The PBS solution was periodically exchanged to mimic physiological conditions. The amount of BMP-2 released from the microspheres was calculated by measuring the optical absorbance of the PBS solution prior to each exchange using ultraviolet (UV) spectroscopy (UV-1700, Shimadzu, Japan) at a wavelength of 220 nm. A standard curve was obtained by measuring the optical absorbance values of BMP-2 solutions with various concentrations in the range of 4–2048 ng/mL.31 Absorbance showed a linear relationship with BMP-2 concentration (y) as follows: y =


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1.1099x (R2 = 0.9998). The theoretical initial amount of BMP-2 in each microsphere was calculated using the initial concentration of BMP-2 in solution and the volume of the microsphere.

2.7. In vivo animal experiment To evaluate the bone regenerative performance of the BMP-2-loaded HA–CSD microspheres, an in vivo animal experiment was conducted using the rabbit lateral femoral condyle defect model.11, 32 For comparison, HA microspheres with and without BMP-2 were also examined. Thirteen-week-old healthy New Zealand white rabbits with weights between 2.7 and 3.0 kg were used in the animal tests. Prior to the surgical procedure, the rabbits were anesthetized using an intramuscular injection having a combination of 0.7 mL 2% Xylazine HCl (Rompun, Bayer Korea, Korea), 1.4 mL Tiletamine HCl (Zoletil, Virbac Laboratories, France), and Lidocaine (Yuhan Corporation, Korea). A cylindrical defect (diameter = depth = 6 mm) was created on a lateral side of the femoral condyle of each leg using trephine drills. The defects were filled with approximately 0.12 g of microspheres, and the endothelium and epidermis were then sutured. After surgery, the rabbits were administered an intramuscular injection of gentamycin. After six weeks of recovery, the rabbits were sacrificed to extract the femoral condyles with the implants. To estimate the extent of new bone formation, the extracted bone fragments were scanned using micro computed tomography (µ-CT) (resolution = 15 µm; voltage = 130 kV; current = 60 µA). Two-dimensional images of the microspheres implanted in femoral condyles were obtained using post-processing software (Data Viewer 1.4, Bruker,



Kontich, Belgium). The three-dimensional images were constructed using CTVox software (Bruker, Kontich, Belgium). The level of bone regeneration was evaluated by calculating the areas of the defects covered by the microspheres and newly grown bone. More than three specimens for each sample were used for statistical analysis. For histological evaluation, the bone tissues were fixed in a neutral 10% formaldehyde solution, followed by decalcification in an ethylenediaminetetraacetic acid solution. Paraffin blocks containing the specimen were prepared to obtain 5-µm-thick histological sections stained with Masson’s trichrome. Microscopic images of representative regions of the stained tissues were obtained using Axioskop microscopy (Olympus BX51, Olympus, Japan). The mature bone matrix was stained red by Biebrich scarlet-acid fuchsin, and immature new bone and collagen fibers were stained blue by aniline blue.33-34 The boneregeneration abilities of the microspheres were investigated by calculating the area of new bone in the histological images. Three specimens for each sample were examined for statistical analysis.

2.8. Statistical analysis Statistical Package for the Social Sciences (SPSS 23; SPSS Inc., USA) was used for statistical analysis, and the data are presented as the mean ± standard deviation. Prior to data analysis, a Shapiro–Wilk test was conducted to evaluate variable normality. In the animal test, one-way analysis of variance was performed, followed by a least significant difference (LSD) post hoc comparison test. A p-value of less than 0.05 was considered statistically significant.

3. Results and discussion


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3.1 Microstructures and crystalline phases of the microspheres BMP-2-loaded HA–CSD microspheres were successfully fabricated by incorporating BMP-2 into CSD and CPC powders to form a BMP-2 containing CPC–CSD paste, followed by emulsification of the paste in oil; then, TCP and TTCP transform to HA. Figure 1 shows representative SEM images of the synthesized HA and HA–CSD microspheres. Both these microspheres exhibited well-defined spherical shapes with sizes ranging from 250 to 500 um [Figures 1(A) and (B)], indicating that the hardening liquid for setting reaction in CPC materials could be carried out in hydrophobic oil. Thus, HA–CSD microspheres could be successfully prepared with a negligible change in morphology, even though CSD was mixed with CPC. The fracture surfaces of the HA and HA–CSD microspheres are shown in Figures 1(C) and (D). The microspheres exhibited porous structures generated by volume reduction during the phase transformation, as reported in our previous study.21 All the synthesized microspheres had uniform microstructures without unexpected cracks or voids. Figure 2 shows high-magnification cross-sectional SEM images of the assynthesized HA and HA–CSD microspheres and those incubated for 7 d. The as-synthesized HA microspheres demonstrated well-distributed homogeneous pores in the HA matrix, as shown in Figure 2(A). The rod-like CSD crystals were well incorporated in the porous HA matrix, and the HA–CSD microspheres had a lower porosity compared with that of the HA microspheres [Figure 2(B)]. These results indicate that the CSD crystals mixed into the CPC powder were well preserved during fabrication process, including phase conversion. The changes in the internal microstructures of the HA–CSD microspheres were observed after immersing the microspheres in PBS for 7 d at 37 °C since CSD undergoes degradation in the human body after implantation. For comparison, HA microspheres were also incubated under the same conditions. After immersion in PBS, the HA microspheres maintained their initial



microstructures because the HA matrix is chemically stable under physiological conditions [Figure 2(C)]; in contrast, the internal microstructures of the HA–CSD microspheres changed dramatically after immersion because the dissolution of CSD generated rod-like pores [Figure 2(D)]. CSD degradation forms pore channels within the HA microspheres, and as more CSD is degraded the degradation directly affects the rate of BMP-2 release from the spheres. We expect that the degradation rate can be controlled by adjusting the amount of the incorporated CSD. The porosities and pore sizes of the microspheres before and after immersion were estimated from SEM images, as shown in Table 1. The porosity of the HA microspheres was 24.5% ± 2.5%, similar to the value obtained in our previous study.21 The porosity of the assynthesized HA–CSD microspheres was 20.1% ± 3.0%, which was lower than that of HA microspheres. The additional pores generated by CSD dissolution caused the porosity of the HA–CSD microspheres to increase to 34.3% ± 4.2%. Conversely, the porosity of the HA microspheres incubated for 7 d was comparable (23.9% ± 3.1%) with that of the assynthesized HA microspheres. Moreover, the pore size of the HA–CSD microspheres increased from 8.7 ± 0.9 µm to 11.5 ± 2.4 µm after incubation, whereas the pore size of the HA microspheres after incubation remained similar to the initial pore size. These results suggest that the HA–CSD microspheres will become highly porous in the body as a result of the pore sites provided by the dissolution of CSD crystals. To examine the changes in the chemical compositions of the microspheres under physiological conditions, the as-formed microspheres along with the microspheres incubated in PBS at 37 °C for 4 and 7 d were characterized via XRD [Figure 3]. The XRD patterns of the HA microspheres showed typical broad peaks corresponding to the low-crystalline HA phase at around 26° and 32°; no significant changes in composition were observed after


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physiological incubation [Figure 3(A)]. These results indicate that TCP and TTCP were successfully converted into HA with high conversion efficiency. On the other hand, the XRD patterns of the HA–CSD microspheres showed strong peaks associated with CSD phase at approximately 29°, 31°, and 33° along with the peaks of low-crystalline HA observed in the patterns of the HA microspheres [Figure 3(B)]. The relative intensities of the CSD peaks of the HA–CSD microspheres incubated for 4 d decreased slightly more than those of the assynthesized microspheres, and they decreased substantially upon incubation in PBS for 7 d. After the CSD crystals completely dissolved under physiological conditions (7 d), only the peaks of HA were detected in the XRD pattern. Thus, the CSD crystals within the HA–CSD microspheres biodegraded within 7 d, and the HA–CSD biphasic microspheres were eventually converted into monophasic HA microspheres.

3.2 In vitro biocompatibilities of the HA and HA–CSD microspheres To assess their potentials as bone substitutes, the in vitro biocompatibilities of the HA and HA–CSD microspheres were evaluated via in vitro cell tests using MC3T3-E1 cells [Figure 4]. The pre-osteoblasts adhered to and spread on the surfaces of the as-synthesized microspheres, demonstrating the non-cytotoxicity of the specimens [Figures 4(A) and (B)]. The cells spread less on the HA–CSD microspheres than on the HA microspheres; this might be attributed to the hindrance of the initial cell attachment by Ca2+ and SO42− ions released from CSD. To simulate the physiological environment in which microspheres are continuously immersed in circulating body fluid, the HA–CSD microspheres were incubated in PBS at 37°C for 7 d, and the cellular response was re-evaluated. For the HA microspheres, the cell morphology and cell spreading were similar for the as-synthesized and incubated



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microspheres [Figure 4(C)]. This result can be attributed to the fact that the microstructures and chemical compositions of the HA microspheres hardly changed during incubation However, for the HA–CSD microspheres, the microspheres incubated for 7 d exhibited better cell attachment than the as-synthesized microspheres, as evidenced by the greater degree of focal adhesion with elongated spindle-like cell morphology of the former [Figure 4(D)].35 The significantly improved cellular response of the HA–CSD microspheres was attributed to the formation of precipitates on the microsphere surfaces and the change in chemical composition resulting from the degradation of CSD [inset of Figure 4(D)]. During the dissolution of CSD, spherical clusters mineralized on the surfaces of the HA–CSD microspheres from the Ca2+ ions released from CSD. Energy dispersive X-ray spectrometer (EDS) analysis indicated that the Ca/P ratios of the precipitates were 1.4–1.5 (data not shown), corresponding to calcium-deficient HA.6, 36 During immersion in PBS, dissolved CSD was ionized and reacted with phosphates [e.g., sodium phosphate dibasic (Na2HPO4)], which are the main component of PBS, to form calcium phosphates, as shown in Eqs. (1)– (3):37 CaSO4 · 2H2O → Ca2+ + SO42− + 2H2O,

(1)

Na2HPO4→ 2Na+ + H+ + PO43−,

(2)

3Ca2+ + 2 PO43−→ Ca3(PO4)2.

(3)

The calcium phosphate crystals formed on the microsphere surfaces had a chemical composition similar to that of tricalcium phosphate (TCP), and they increased the micro-level surface roughness, which may have improved cellular response. These data suggest that the HA–CSD microspheres can induce bone-like mineral formation and stimulate cellular response.


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3.3 Release of BMP-2 from HA and HA–CSD microspheres In vitro BMP-2 release from the HA and HA–CSD microspheres was assessed by monitoring the BMP-2 concentrations in the PBS solutions containing the microspheres. The cumulative amounts of BMP-2 released from the HA and HA–CSD microspheres as functions of time are shown in Figure 5(A). Both types of microspheres exhibited sustained BMP-2 release during the six-week monitoring period; however, the release rates were considerably different. The HA microspheres exhibited precipitous release behavior of BMP2 for 2 d because the rapid diffusion of BMP-2 near the microsphere surfaces accounted for a large portion of the total BMP-2 release. After the initial release period, the amount of the BMP-2 released from the HA microspheres continuously increased at a slow rate of 1.0 ng/day. The total amount of BMP-2 released during the monitoring period was approximately 10% of the initial loading amount, similar to the results of previous studies on the in vitro release of BMP-2 from CPC implants.38-39 On the other hand, BMP-2 was released more rapidly from the HA–CSD microspheres. After 2 d of the initial burst, BMP-2 was steadily released from the HA–CSD microspheres at a rate of 2.4 ng/day, and 20% of the initial loading amount was released after six weeks. The difference in the release behavior of BMP-2 from the HA and HA–CSD microspheres resulted from different release kinetics. To compare the release mechanisms of the two microspheres, we hypothesized that the release system of the microspheres is a diffusion controlled release system and that the PBS medium permeated into the pores of microspheres. The dissolved BMP-2 then diffused to the medium outside the microspheres driven by the concentration gradient. Higuchi explained this physico-chemical phenomenon



in mathematical terms,40 and the equation has been applied to diffusion-controlled release from a porous structure having pores and capillaries: =

( )

,

where Q is the amount of drug released after time t, D is the diffusivity of the drug in the homogeneous matrix medium, A is the amount of drug present in the matrix, C is the solubility of the drug in the permeating fluid, and τ is the tortuosity factor of the capillary system. This equation can be expressed simply as = √, where k is the Higuchi release rate coefficient. Thus, the cumulative amount of BMP-2 released is directly proportional to the square root of time. These equations are shown to demonstrate the parameters capable of affecting release behavior. The releases of BMP-2 from the HA and HA–CSD microspheres are plotted against the square root of time [Figure 5(B)]. The HA microspheres fell upon the trend line with a near-unit slope (y = 7.4x + 2.4; R2 = 0.9864), indicating that the release behavior was fit well by the Higuchi model. In contrast, the HA–CSD microspheres exhibited two-stage release behavior: a slow-release stage from immersion to day 7, followed by a fast-release stage from day 7 onward. The Higuchi release coefficient of the second stage was 19.7 ng/day1/2 (R2 = 0.9862) which is a 1.5 times greater than that of the first stage (12.4 ng/day1/2, R2 = 0.9966). When the release behavior of the HA microspheres was divided into the same two stages, the Higuchi release coefficients of the first stage (k = 7.2, R2 = 0.8881) and second stage (k = 8.7, R2 = 0.9493) were not significantly different, and the coefficient of determination (R2) of each stage was lower than that of entire curve corresponding to the initial burst.


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In the initial stage, the BMP-2 embedded in the HA–CSD microspheres was released slowly into PBS at a rate similar to that observed for the HA microspheres. In contrast, the BMP-2 bound to the HA matrix diffused from the HA–CSD microspheres at a relatively higher rate as a result of the pore channels generated due to the degradation of CSD after day 7. As the BMP-2 concentration gradient decreased with time, the driving force for BMP-2 diffusion (i.e., the driving force to overcome the binding force with the HA matrix) weakened, and some of the loaded BMP-2 remained in the microspheres. These findings imply that the incorporation of CSD into the HA microspheres greatly improved the rate of BMP-2 release and the total amount of BMP-2 released. This proposed mechanism is schematically shown in Figure 6. Through the suggested mechanism of BMP-2 release from microspheres, BMP-2 was diffused from the microspheres with sustained and controlled release kinetics. All the release data indicated that the addition of CSD improved the ability of the HA microspheres to release BMP-2 over long periods of time, a result that has not been reported until now. Based on these findings, an in vivo test was conducted.

3.4 In vivo bone regeneration ability The bone regeneration abilities of the BMP-2-loaded HA and HA–CSD microspheres were evaluated via in vivo animal tests using a rabbit lateral femoral condyle defect model. For comparison, HA microspheres without BMP-2 were also tested as a reference. A schematic drawing of the bone defect and an image showing animal preparation are shown in Figure 7. All animals exhibited healing at the surgical site, and no infections were observed. Furthermore, all animals were in good health with no noticeable change in body weight or diet. At the time of sample retrieval, the microspheres were stable at the defect site and were covered by a thick layer of new soft tissue that formed after the operation.



Figure 8 shows representative two-dimensional, cross-sectional µ-CT images of rabbit femoral condyle after six weeks of healing. Since the implanted microspheres and bones have similar densities, the two components could hardly be distinguished in the µ-CT images. However, the grey area within the surgical site (outlined in red) excluding the circular parts that are presumed to be microspheres denote newly generated bone. In the HA microspheres without BMP-2, although osteoconduction was observed at the border of the defect, most of the defect area was not covered by new bone [Figure 8(A)]. When the HA microspheres were loaded with BMP-2, a large amount of new bone grew from the boundary to the central region of the defect as a result of the synergistic effect of the osteoconductivity of HA and the osteoinductivity of BMP-2 [Figure 8 (B)]. Surprisingly, the BMP-2-loaded HA–CSD microspheres exhibited excellent bone regeneration ability compared to the BMP2-loaded HA microspheres [Figure 8(C)]. The defect sites were almost completely filled by the new bone and the microspheres. This result was attributed to the accelerated BMP-2 release from the HA–CSD microspheres and suggests that the embedded BMP-2 had a stimulatory effect on the osteoblast cells. To compare the formation of new bone more quantitatively, three-dimensional µ-CT images were constructed, as shown in Figure 9. The exteriors of the HA microspheres showed small amounts of bone formation, and minimal bone–cell interaction was observed at the boundary between the microspheres and the defect wall [Figure 9(A)]. The BMP-2loaded HA microspheres showed enhanced bone formation on the outer surfaces of the microspheres compared with those of HA microspheres without BMP-2, and a minimal amount of new bone was generated in the inner region [Figure 9(B)]. In contrast, a substantial amount of bone growth was observed on the BMP-2 loaded HA–CSD


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microspheres, as shown in Figure 9(C). Bone formation on both the interior and outer regions of these microspheres was significantly improved compared the other two samples. The percentages of newly grown bone and implanted microspheres within the defect boundary are shown in Figure 9(D). The defect coverage was estimated by dividing the volume of new bone and microspheres by the defect volume based on the reconstructed threedimensional µ-CT images. The HA microspheres without BMP-2 had the lowest defect coverage of 66.2% ± 6.5%; the HA microspheres with BMP-2 exhibited a coverage of 80.1% ± 3.7%, demonstrating the effect of BMP-2. The bone regeneration level was significantly higher in the HA–CSD microspheres loaded with BMP-2, as evidenced by the defect coverage of 90.5% ± 3.5% (p < 0.05 compared to the HA microspheres; p < 0.005 compared to the BMP-2-loaded HA microspheres). This suggests that the accelerated release of BMP-2 from the biphasic microspheres effectively facilitated bone regeneration, resulting in complete bone healing in approximately six weeks. Figures 10 present typical histological cross-sectional images of the tissues formed around the microspheres after six weeks of recovery. The mature bone and immature new bone appear red and blue, respectively, and osteocytes are indicated with yellow arrows in Figure 10. Collagen fibers are also stained blue, but they do not contain osteocytes. The microspheres are absent in the histological images because they were removed during decalcification, which was part of the process for preparing the histological specimens. Collagen fibers with thicknesses of approximately 20 µm formed on the surfaces of the unloaded HA microspheres, and the spaces between microspheres were mainly filled with adipose tissue [Figure 10(A) and (D)]. In the BMP-2-loaded HA microspheres, new bone tissue, which was clearly distinguished from collagen fibers by the presence of osteocytes, was clearly observed, and adipose tissue occupied the remaining space [Figure 10(B) and



(E)]. Compared to the HA microspheres, the BMP-2-loaded HA–CSD microspheres induced a greater amount of new bone, and the defect area not occupied by the implant was almost completely filled with new bone [Figure 10(C) and (F)]. As shown in the Figure 10(D)-(F), the ratio of mature bone and immature bone was different. In particular, the histological image of HA-CSD microspheres [Figure 10(F)] indicated fastest bone regeneration because the ratio of mature bone was highest among three groups. To quantify the degree of bone regeneration, the area of new bone was calculated from the histological images [Figure 11]. New bone occupied 13% of the defect area in the HA microspheres without BMP-2 and 21% of the defect area in the HA microspheres with BMP-2 (p < 0.01). For the HA–CSD microspheres loaded with BMP-2, the proportion of new bone reached 35%, which is close to the maximum amount that can be formed in the empty spaces between microspheres. This suggests that the enhanced release of BMP-2 from the HA–CSD microspheres increases new bone formation by approximately 170% compared to BMP-2-loaded HA microspheres. From the in vitro results we confirmed that the CSD was almost fully dissolved under physiological condition within 7 days. The rapid degradation is thought to have accelerated the release of BMP-2 from the microspheres and stimulated bone regeneration. We confirmed that the system with HA-CSD with BMP-2 addition has a higher BMP-2 delivery efficiency than that of HA with BMP-2.

4. Conclusions Rod-like CSD crystals were successfully incorporated into HA microspheres, resulting in biphasic microspheres with homogeneous internal structures. As the CSD degraded under physiological conditions, rod-like pores were generated in the microspheres,


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resulting in increases in pore size and porosity. The in vitro cytocompatibility of the HA– CSD microspheres in osteoblastic cells was enhanced after CSD dissolution, which induced the precipitation of bone-like precipitates on the microspheres. In addition, BMP-2 loaded into the HA–CSD microspheres was released at a rate faster than that from HA microspheres, and the release was sustained for six weeks because of the increased microsphere porosity. HA-CSD microspheres with an enhanced BMP-2 release behavior stimulated bone regeneration in a rabbit lateral femoral condyle defect model. The HA–CSD microspheres with enhanced BMP-2 delivery capability have great potential for bone regeneration in vivo.

Acknowledgements This research was supported by Basic Science Research Program (No. 2015R1D1A1A01057311) through the National Research Foundation of Korea and Technology Innovation Program (10037915, WPM Biomedical Materials–Implant Materials) funded by the Ministry of Education and the Ministry of Trade, Industry & Energy.

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52 (12), 1145-1149.



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Tables

Table 1. Pore characteristics of the as-synthesized HA and HA–CSD microspheres and those incubated in PBS for 7 d. HA microspheres

HA–CSD microspheres

As-synthesized

Incubated

As-synthesized

Incubated

Porosity (%)

24.5 ± 2.5

23.9 ± 3.1

20.1 ± 3.0

34.3 ± 4.2

Pore size (µm)

8.2 ± 0.5

8.9 ± 1.1

8.7 ± 0.9

11.5 ± 2.4


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Figures

Figure 1. SEM images of the as-synthesized (A and C) HA and (B and D) HA–CSD microspheres. (A) and (B) are low-magnification images, while (C) and (D) are highermagnification images of the fracture surfaces.



Figure 2. Representative cross-sectional SEM images of the as-synthesized HA (A) and HA– CSD (B) microspheres and the HA (C) and HA–CSD (D) microspheres incubated in PBS for 7 d.


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Figure 3. XRD patterns of the as-synthesized microspheres and those incubated in PBS for 4 and 7 d: (A) HA and (B) HA–CSD microspheres (●: HA, ■: CSD, ◆: β-TCP).



Figure 4. SEM images of MC3T3-E1 cells cultured for 24 h on the as-synthesized HA (A) and HA–CSD (B) microspheres and the HA (C) and HA–CSD (D) microspheres incubated for 7 d. The inset in (D) shows a high-magnification image of a HA–CSD microsphere.


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Figure 5. Cumulative amount of BMP-2 released from the HA and HA–CSD microspheres as functions of (A) time and (B) the square root of time.



Figure 6. Schematic showing the mechanism of BMP-2 release from a HA–CSD microsphere after implantation.


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Figure 7. (A) Schematic illustration and (B) optical image of microsphere implantation into the femoral condyle of a rabbit.



Figure 8. Representative µ-CT images of rabbit femoral condyles implanted with (A) the HA microspheres without BMP-2, (B) the HA microspheres with BMP-2, and (C) HA–CSD microspheres with BMP-2. The red dotted circles indicate the defect boundaries.


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Figure 9. Reconstructed three-dimensional images of rabbit femoral condyles implanted with (A) HA microspheres without BMP-2, (B) HA microspheres with BMP-2, and (C) HA–CSD microspheres with BMP-2. (D) Defect coverage calculated as the area of new bone and microspheres with respect to the defect area (*: p < 0.05 and ***: p < 0.005).



Figure 10. Representative histological cross-sectional images of rabbit condyle defects filled with (A,D) HA microspheres without BMP-2, (B,E) HA microspheres with BMP-2, and (C,F) HA–CSD microspheres with BMP-2 after six weeks of healing ((A) – (C) : low magnification; (D) – (F) : high magnification; osteocytes indicated with yellow arrows).


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****

40

New bone area [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


****

30

** 20

10

0

HA

HA with BMP-2

HA-CSD with BMP-2

Figure 11. Quantitative analysis of new bone calculated from the histological images. (**: p < 0.01 and ****: p < 0.001).



For Table of Contents Use Only

Incorporation of calcium sulfate dihydrate into hydroxyapatite microspheres to improve the release of bone morphogenetic protein-2 and accelerate bone regeneration

Jaeuk Baeka, Hyun Leea, Tae-Sik Jangb, Juha Songb, Hyoun-Ee Kima,c, Hyun-Do Jung d,2

a

b

Department of Materials Science and Engineering, Seoul National University, Seoul, South Korea

Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, 637457, Singapore c

Biomedical Implant Convergence Research Center, Advanced Institutes of Convergence Technology, Suwon, South Korea

d

Liquid Processing & Casting Technology R&D Group, Korea Institute of Industrial Technology, Incheon, 21999, Korea


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Figure 1. SEM images of the as-synthesized (A and C) HA and (B and D) HA–CSD microspheres. (A) and (B) are low-magnification images, while (C) and (D) are higher-magnification images of the fracture surfaces. 247x173mm (150 x 150 DPI)



Figure 2. Representative cross-sectional SEM images of the as-synthesized HA (A) and HA–CSD (B) microspheres and the HA (C) and HA–CSD (D) microspheres incubated in PBS for 7 d. 246x167mm (150 x 150 DPI)


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Figure 3. XRD patterns of the as-synthesized microspheres and those incubated in PBS for 4 and 7 d: (A) HA and (B) HA–CSD microspheres (●: HA, ■: CSD, ◆: β-TCP). 334x128mm (119 x 121 DPI)



Figure 4. SEM images of MC3T3-E1 cells cultured for 24 h on the as-synthesized HA (A) and HA–CSD (B) microspheres and the HA (C) and HA–CSD (D) microspheres incubated for 7 d. The inset in (D) shows a high-magnification image of a HA–CSD microsphere. 246x163mm (150 x 150 DPI)


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Figure 5. Cumulative amount of BMP-2 released from the HA and HA–CSD microspheres as functions of (A) time and (B) the square root of time. 333x127mm (123 x 121 DPI)



Figure 6. Schematic showing the mechanism of BMP-2 release from a HA–CSD microsphere after implantation. 271x154mm (150 x 150 DPI)


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Figure 7. (A) Schematic illustration and (B) optical image of microsphere implantation into the femoral condyle of a rabbit. 319x143mm (96 x 96 DPI)



Figure 8. Representative µ-CT images of rabbit femoral condyles implanted with (A) the HA microspheres without BMP-2, (B) the HA microspheres with BMP-2, and (C) HA–CSD microspheres with BMP-2. The red dotted circles indicate the defect boundaries. 83x189mm (150 x 150 DPI)


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Figure 9. Reconstructed three-dimensional images of rabbit femoral condyles implanted with (A) HA microspheres without BMP-2, (B) HA microspheres with BMP-2, and (C) HA–CSD microspheres with BMP-2. (D) Defect coverage calculated as the area of new bone and microspheres with respect to the defect area (*: p < 0.05 and ***: p < 0.005). 247x188mm (109 x 104 DPI)



Figure 10. Representative histological cross-sectional images of rabbit condyle defects filled with (A,D) HA microspheres without BMP-2, (B,E) HA microspheres with BMP-2, and (C,F) HA–CSD microspheres with BMP-2 after six weeks of healing ((A) – (C) : low magnification; (D) – (F) : high magnification; osteocytes indicated with yellow arrows). 385x196mm (96 x 96 DPI)


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Figure 11. Quantitative analysis of new bone calculated from the histological images. (**: p < 0.01 and ****: p < 0.001). 254x186mm (150 x 150 DPI)


Incorporation of calcium sulfate dihydrate into hydroxyapatite

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