Chemical self-assembly of multi-functional hydroxyapatite with coral

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Functional Inorganic Materials and Devices

Chemical self-assembly of multi-functional hydroxyapatite with coral-like nanostructure for osteoporotic bone reconstruction Hongyu Quan, Yuwei He, Jujiang Sun, weihu Yang, Wei Luo, Ce Dou, Fei Kang, Chunrong Zhao, Jian He, Xiaochao Yang, Shiwu Dong, and Hong Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09879 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 15, 2018

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ACS Applied Materials & Interfaces

Chemical self-assembly of multi-functional hydroxyapatite with coral-like nanostructure for osteoporotic bone reconstruction Hongyu Quan1, Yuwei He1, Jujiang Sun1, Weihu Yang2, Wei Luo3, Ce Dou4, Fei Kang1, Chunrong Zhao1, Jian He5, Xiaochao Yang1, Shiwu Dong1,6*, Hong Jiang1* 1. Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Chongqing 400038, PR China. 2. Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, PR China. 3. Department of Orthopedics, Guizhou Provincial People’s Hospital, Guiyang 550002, PR China. 4. Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China. 5. College of Pharmacy, Third Military Medical University, Chongqing 400038, PR China. 6. State Key Laboratory of Trauma, Burns and Combined Injury, Third Military Medical University, Chongqing 400038, China.

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KEYWORDS: functionalization, hydroxyapatite, bisphosphonate, magnetic nanoparticles, osteoporotic bone reconstruction.

ABSTRACT Bone defects/fractures are common in older people suffering from osteoporosis. Traditional hydroxyapatite (HA) materials for osteoporotic bone repair face many challenges, including limited bone formation and aseptic loosening of orthopedic implants. In this study, a new multifunctional HA is synthesized by spontaneous assembly of alendronate (AL) and Fe3O4 onto HA nanocrystals for osteoporotic bone regeneration. The chemical coordination of AL and Fe3O4 with HA does not induce lattice deformation, resulting in a functionalized HA (Func-HA) with proper magnetic property and controlled release manner. The Func-HA nanocrystals have been encapsulated in polymer substrates to further investigate their osteogenic capability. In vitro and in vivo evaluations reveal that both AL and Fe3O4, especially the combination of two functional groups on HA, can inhibit osteoclastic activity and promote osteoblast proliferation and differentiation, as well as enhance implant osseointegration and accelerate bone remodeling under osteoporotic condition. The as-developed Func-HA with coordinating antiresorptive ability, magnetic property and osteoconductivity might be desirable biomaterial for osteoporotic bone defect/fractures treatment.

INTRODUCTION Synthetic hydroxyapatite (HA), Ca10(PO4)6(OH)2, has attracted great interest as orthopedic biomaterial due to its chemical and structural similarity to bone mineral. Common applications of HA include bone cements, bone fillers, as well as coating of orthopedic implants 1. Because of


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the biocompatibility and osteoconductive properties, HA-based materials have been utilized to improve osteoblast (OB) responses and accelerate bone repair under normal circumstances

2-3

.

However, the balance between osteoclastic bone resorption and osteoblastic bone formation is interrupted in disease states, such as osteoporosis, the most prevalent metabolic bone disease among post-menopausal women, elderly males and malnourished children 4. In the osteoporotic condition, excessive osteoclast (OC) activity induces skeletal destruction and reductions of bone mass, leading to the loosening and displacement of implants at the insertion site 5. Therefore, the healing of osteoporotic bone defect/fractures with conventional HA-based materials faces difficulties and challenges. Various approaches have been developed to regulate the osteoblast-osteoclast balance and enhance implant-bone integration, among which the incorporation and local release of bioactive components have been widely used in clinical studies 6-7. Bioactive molecules like growth factors (e.g. BMPs, TGF-β, FGF, IGF) can stimulate bone formation by regulating osteogenesis-related gene expression 8, but the high cost and short half-life in the human body restrict their application. Alternatively, bisphosphonates (BPs) are recognized as potent inhibitors of osteoclast-mediated bone resorption. The two phosphonate groups of BPs have a particular affinity for HA crystals, whereas the two side chains (termed R1 and R2) bound to the central carbon determine their efficiency (Figure S1) 9. For most of BPs in current clinical use (such as alendronate, pamidronate and zoledronate), R1 is a hydroxyl group, which enhances the affinity for HA via tridentate binding to calcium

10

. R2 is the primary determinant of antiresorptive

potency of BPs, in particular, a nitrogen-containing R2 side group can increase antiresorptive potency by inhibiting the key enzyme in the mevalonate pathway

11

. Among the nitrogen-

containing BPs, alendronate (AL) has been commonly used as the first-line therapy for


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osteoporosis. Recent studies demonstrated that AL is able to inhibit endothelial cells and osteoclasts differentiation, as well as promote osteoblastic activities and peri-implant bone formation even when incorporated in calcium phosphate nanocrystals, microspheres, cements or coatings 12-15. In fact, the in vivo bone remodeling occurs in a dynamic and complex 3D microenvironment, successful osseointegration of implant must be assisted by cell colonization and substantial angiogenesis. AL-incorporated implants have been shown to regulate bone metabolism and 16

enhance early mechanical fixation under osteoporosis

. However, without a continuous and

prolonged stimulation, cell penetration is limited at the implant/bone interface and the inner implant parts become necrotic, which leads to insufficient stability and functionality of newlyformed bone. To address this problem, the material design should improve cell ingrowth and proliferation by combinations of mechanical and biochemical stimulations. Static or pulsed magnetic fields are effective stimuli that induce mechanical stress to promote bone ingrowth, spinal fusion and fracture healing

17-18

. Several studies have demonstrated that

magnetic nanoparticles (MNPs) can cause shear stresses at the cellular level to stimulate osteogenesis with or without external magnetic field

19-21

. In particular, iron (II, III) oxide

nanoparticles (NPs) have been widely investigated for biomedical applications due to their favorable biocompatibility and intrinsic magnetic property

22-23

. It has been reported that the

Fe3O4 NPs embedded within HA nanocrystals could stimulate proliferation and differentiation of human osteoblasts

24

, and the dispersing Fe3O4 NPs in polymeric scaffolds has been found to

promote the angiogenic responses of endothelial cells and enhance bone regeneration in a mouse calvarial defect model 21.


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Inspired by the above knowledge, we explored the design of a multi-functional HA, (AL and Fe3O4)-loaded HA, coordinating antiresorptive ability, magnetic property and osteoconductivity for osteoporotic bone regeneration. Herein, the multi-functional HA was synthesized by chemical self-assembly of AL molecules and Fe3O4 NPs onto HA. Chemical self-assembling is an effective method to fabricate hybrid nanomaterials.25-26 AL could bind with HA due to the particular affinity of two phosphonate groups. However, the inactive Fe3O4 did not interact with HA in aqueous solution. To address this issue, the synthesized Fe3O4 NPs were modified by PEG ligands, with carboxyl groups at distal ends of the linked chains, which provided a unique strategy to form chemical bonds with calcium ions of HA. The functionalized HA (Func-HA) as AL-loaded HA, Fe3O4-loaded HA, (AL and Fe3O4)-loaded HA were assessed for their physicochemical properties, including chemical composition, particle morphology, crystal structure and magnetization. Furthermore, the Func-HA nanocrystals-added 3D porous scaffolds were fabricated and investigated for the osteogenic capability in vitro and in vivo. We demonstrated that the grafting of AL and Fe3O4 on HA nanocrystals can provide biochemical and magnetic functionalities, which not only inhibit osteoclast function but also promote osteoblast activity and stimulate osteogenesis in osteoporotic condition.

RESULTS AND DISCUSSION Chemical constitution and crystal structure of Func-HA nanocrystals. In this study, 6 types of Func-HA nanocrystals (HA-AL1, HA-AL2, HA-Fe5, HA-Fe10, HA-AL1-Fe5 and HAAL2-Fe10) were synthesized by chemical self-assembly of AL or/and Fe3O4 onto HA. To investigate the quantity of functional groups linked on HA, AL and Fe3O4 contents were calculated from their relative absorbance. As could be seen from Table 1, AL/HA molar ratio of


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HA-AL1-Fe5 was close to that of HA-AL1, so did HA-AL2-Fe10 and HA-AL2. Since AL grafting onto HA was conducted before Fe3O4, it indicated that the grafted AL groups were not influenced by Fe3O4-loading reaction. On the other hand, Fe3O4 content of HA-AL1-Fe5 was lower than that of HA-Fe5 owing to steric hindrance by the grafted AL groups on HA nanocrystals. This explanation could also apply to HA-AL2-Fe10 and HA-Fe10. Table 1. AL/HA and Fe3O4/HA molar ratios of Func-HA samples Theoretical values

Experimentally values

AL/HA

Fe3O4/HA

AL/HA

Fe3O4/HA

HA-AL1

01/10

--

0.58/10

--

HA-AL2

02/10

--

0.90/10

--

HA-Fe5

--

05/10

--

4.18/10

HA-Fe10

--

10/10

--

8.19/10

HA-AL1-Fe5

01/10

05/10

0.58/10

4.09/10

HA-AL2-Fe10

02/10

10/10

0.90/10

8.01/10

TEM photographs (Figure 1A) exhibited the morphologies of synthetic HA, surface-modified Fe3O4, HA-AL2, HA-Fe10 and HA-AL2-Fe10. HRTEM images (upper right corner insets) showed the continuous lattice fringes of HA and Fe3O4, implying their single crystalline nature. The lattice spacings along c-axis and a-axis of needle-like HA crystals were determined to be 0.34 nm and 0.27 nm, which corresponded to the lattice constants of (002) plane and (300) plane of hydroxyapatite, respectively. Spherical-shaped Fe3O4 nanoparticles demonstrated lattice fringes with d-spacings of 0.25 nm, indicating the (311) plane of ferroferric oxide. In our study,


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the functionalization of HA was conducted by grafting AL or/and Fe3O4. According to TEM observation, the morphologies of Func-HA nanocrystals (HA-AL2, HA-Fe10 and HA-AL2-Fe10) had changed after grafting treatment. HA-AL2 crystals appeared to be thinner than HA, which suggested that the presence of AL could influence the crystallinity of hydroxyapatite. At variance, HA-Fe10 and HA-AL2-Fe10 crystals showed reduced mean dimensions with respect to HA; many spherical particles (inferred to be Fe3O4) were covered on the surface of hydroxyapatite crystals, forming a coral-like nanostructure. To estimate the compositions of the spherical particles, energy dispersive X-ray spectroscopy (EDS) analysis was performed. As expected, Fe and O elements were detected in Fe3O4, HA-Fe10 and HA-AL2-Fe10 samples, suggesting that the spherical particles anchored on coral-like nanocrystals were Fe3O4; the identified Ca and P elements were attributed to hydroxyapatite. It revealed that the surface-modified Fe3O4 NPs were successfully anchored onto HA-Fe10 and HA-AL2-Fe10, and certainly affected the growth of hydroxyapatite crystals. The crystallinities of different samples were determined by XRD (Figure 1B and Figure S2). HA crystals exhibited the characteristic diffraction peaks of a synthesized hydroxyapatite single phase (JCPDS card no. 09-0432). These peaks also appeared in the XRD patterns of Func-HA grafted with AL or/and Fe3O4, suggesting that the main constituent phases of Func-HA samples were crystalline hydroxyapatite. As evidenced in XRD patterns of HA-Fe5, HA-Fe10, HA-AL1Fe5 and HA-AL2-Fe10, the small peaks (2θ=35.5˚) pointed with spots (●) designated as Fe3O4 from (311) plane indicated that the Fe3O4 phase existed in these samples. A qualitative estimation of the size of coherently scattering domains (τhkl) was calculated using the Scherrer equation: τℎ𝑘𝑙 =

𝑘𝜆 𝛽𝑚 cos𝜃


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where k is a constant depending on crystal habit (chosen as 0.9), λ is the wavelength of X-ray radiation (1.5418 Å), βm is the full-width at half maximum of peak height (rad) and θ is the diffraction angle. The line broadening of the 002 and 300 reflections was used to evaluate τ002 and τ300. τ002 refers to the mean crystallite size along the c-axis, whereas τ300 is related to the mean crystal size along a direction perpendicular to it. The crystallinity degree (Xc), corresponding to the fraction of crystalline phase present in the investigated volume, was evaluated by the following equation: 𝛽002 × 3√𝑋𝑐 = 𝐾𝐴 where β002 is full-width at half maximum of peak height from (002) reflection (degree), KA is a constant set at 0.24. Table 2. Calculated lattice parameters, coherent lengths (τhkl) of the perfect crystalline domains, crystallinity degree (Xc) of HA and Func-HA samples a/Å

c/Å

β002(˚)

β300(˚)

τ002(H)/Å τ300(L)/Å H/L

Xc(%)

HA

9.448(2)

6.904(2)

0.166(1)

0.520(2)

490(2)

159(1)

3.08(1)

3.00(3)

HA-AL1

9.452(1)

6.904(3)

0.168(1)

0.579(3)

484(2)

143(1)

3.39(2)

2.90(3)

HA-AL2

9.455(3)

6.904(2)

0.174(1)

0.588(3)

470(2)

141(1)

3.33(1)

2.64(3)

HA-Fe5

9.449(1)

6.904(1)

0.178(1)

0.495(3)

475(2)

177(1)

2.69(2)

2.44(2)

HA-Fe10

9.450(1)

6.904(3)

0.192(1)

0.479(2)

469(3)

185(1)

2.53(1)

1.95(3)

HA-AL1-Fe5

9.449(1)

6.905(2)

0.172(1)

0.469(3)

457(1)

167(1)

2.73(2)

2.73(3)

HA-AL2-Fe10

9.451(1)

6.906(3)

0.174(1)

0.448(3)

425(2)

173(1)

2.45(2)

2.62(4)


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The results of full profile fitting of the XRD patterns (Table 2) indicated that the values of the cell parameters did not vary with AL or/and Fe3O4 content, suggesting that the chemical assembly of AL or/and Fe3O4 onto HA did not induce major structural modifications. However, τ002 and τ300 changed with the content of AL or/and Fe3O4, indicating that the presence of AL and Fe3O4 had a crucial influence on HA crystallization. The H/L ratios of HA-AL1 and HA-AL2 were slightly greater than that of HA, whereas Fe3O4-loaded HA samples (HA-Fe5, HA-Fe10, HA-AL1-Fe5 and HA-AL2-Fe10) showed a lower H/L ratio, in agreement with the results of TEM observations. Furthermore, the decrease in Xc value of Fun-HA samples further confirmed that the incorporation of AL or/and Fe3O4 inhibited the crystallization of HA. Basis on the above analysis of Func-HA nanocrystals structural modification, we suggested that the interaction of AL or/and Fe3O4 with calcium ions of HA could occur without greatly affecting the crystal structure of HA. Figure 1C gave a schematic diagram of the possible interaction of AL and Fe3O4 at the HA surface. The bisphosphonate anion interacted with calcium cation through deprotonated oxygen atoms, with an added contribution from R1-OH group, the binding of AL to calcium on the HA surface showed a tridentate coordination. In addition, the amino group (in the R2 side chain of AL) could form N-H-O hydrogen bonds with hydroxyl, which led to an increase in HA binding. In contrast to AL which exhibited high affinity for HA surface, Fe3O4 were generally inert to inorganic crystals and insoluble in water. To improve the surface properties, Fe3O4 NPs used in this study were modified by PEG ligands (Figure S3), with carboxyl groups at distal ends of the linked chains. According to TG analysis (Figure S4), the PEG ligand weight percentage for Fe3O4-DPA-PEG600-COOH (in thermal decomposition range of 200-500°C) was 33.27%. A ligand/nanoparticle ratio was calculated to be 515, for simplicity, only one ligand binding to Fe3O4 nanoparticle was presented in Figure 1C.


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When a surface-modified Fe3O4 nanoparticle was adjacent to HA surface, the two oxygen ions of the distal carboxyl group formed new chemical bonds with calcium ion of HA. In this way, AL and Fe3O4 have been successfully assembled onto HA surface, resulting in multi-functional HA nanocrystals. Topographies, magnetization and release properties of Func-HA added scaffolds. FuncHA nanocrystals-added 3D porous scaffolds were fabricated by incorporating Func-HA into PCL framework. Figure 2A showed the synthesis procedure and macroscopic photographs of nanocrystal slurries and scaffolds. HA and HA-AL samples appeared in white color, however, with the increase of Fe3O4 content, the color of the slurries (scaffolds) turned from light brown to dark brown. The surface topographies of the scaffolds and the distribution of HA or Func-HA particles were observed by SEM (Figure 2B). Solvent-casting/particle-leaching process generated an open pore microstructure with good interconnectivity. During the process, the extended PCL chains entwined into a polymer matrix framework, in which the HA or Func-HA formed hydrogen bonds with the polymer chains and produced a three dimension network. The pure PCL scaffold displayed a spongy structure, a multitude of interconnected micropores spread within macropore walls, with the pore size ranged from 10 to 300μm. When polymer scaffolds were reinforced by HA or Func-HA nanocrystals, the surfaces became rough and compact, most pores were in the range of 150~400μm, inorganic particles were uniformly distributed in the scaffolds and no agglomeration was detected. Scaffold mean pore size plays a critical role in cell growth and phenotypic expression. Hulbert et al. proposed that the optimum pore size for osteoconduction was 100~150μm

27

. Boyan et al. reported that a pore size of 200~400μm was

compatible for osteoblasts migration, attachment and proliferation 28. Accordingly, the pore size and an interconnective porous structure of Func-HA added scaffolds were suitable for bone


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ingrowth and remodeling. When the surface topographies of the scaffolds changed considerably with the addition of inorganic particles, the porosities of the samples decreased from 84.1±5.4% (PCL) to 65.2±6.6%~76.7±8.4% (HA and Func-HA), as shown in Figure 2C. Since the deformation and swelling behavior of PCL polymer were different from inorganic particles, in this case, more micropores formed in pure PCL scaffold during solvent evaporation process, resulting in a higher porosity. However, the porosities of Func-HA added scaffolds showed an increasing trend when compared to HA (although the changes were not significant), which was probably due to chemical interaction between grafted functional groups and PCL molecules. Table 3. Magnetic parameters obtained from hysteresis cycles of different samples

Nanocrystals

Scaffolds

Ms (emu/g)

Mr (emu/g)

Mr/Ms

Hc (Oe)

Fe3O4

63.29

0.67

0.01

14.16

HA-Fe5

5.55

0.74

0.13

337.21

HA-Fe10

9.62

1.35

0.14

366.15

HA-AL1-Fe5

5.96

0.92

0.15

430.82

HA-AL2-Fe10

9.76

1.22

0.13

351.36

HA-Fe5

3.27

1.26

0.39

3256.06

HA-Fe10

4.81

1.33

0.28

1261.83

HA-AL1-Fe5

3.61

1.38

0.38

2953.20

HA-AL2-Fe10

4.99

1.40

0.28

1290.80


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Figure S5 and Figure 2D depicted magnetic hysteresis loops of the synthesized nanocrystals and scaffolds. The corresponding magnetic parameters, saturation magnetization (Ms), remanence magnetization (Mr), relative remanence (Mr/Ms) and coercive force (Hc), were summarized in Table 3. Fe3O4 NPs exhibited the magnetic property with Ms of 63.29emu/g. However, the magnetic saturation values of Fe3O4-loaded Func-HA samples reduced remarkably, which confirmed the formation of functionalized hydroxyapatite hybrid nanocrystals. Owing to grafting treatment of Fe3O4, HA-Fe5, HA-Fe10, HA-AL1-Fe5 and HA-AL2-Fe10 nanocrystals showed ferrimagnetic behaviors with relatively high values of Hc and Mr/Ms. The magnetic properties could be inﬂuenced by the content of Fe3O4 NPs and microstructure of the samples. As a consequence, the scaffold samples of Fe3O4-loaded Func-HA exhibited lower Ms and even higher Hc and Mr/Ms values than nanocrystals. Magnetic scaffolds have been reported to improve implant fixation and stimulate bone formation by exploiting magnetic forces at the scaffold/bone interface

29-31

. Therefore, the synthesized Fe3O4-loaded Func-HA nanocrystals

with magnetic properties have the potential to accelerate bone regeneration when used as biomaterial components. The release properties of Func-HA added scaffolds were investigated in PBS solution at 37°C. As shown in Figure 2E, the profiles of AL release over 28 days from different samples indicated comparable shapes, where all the curves showed linear kinetics, excepted for a minimal burst release during the initial 4 days. In this study, AL was loaded onto hydroxyapatite nanocrystals via strong BP-Ca chelation and the AL-loaded Func-HA nanocrystals were further encapsulated in PCL porous scaffolds. Consequently, AL was released from the scaffolds in a controlled manner, and the initial bursting might be ascribed to the AL-loaded hydroxyapatite bound on the surfaces of porous scaffolds. At 28 days, AL release amounts of HA-AL1 and HA-AL1-Fe5


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were found at a similar level, HA-AL2 with the higher loading content released 18.1±0.9mmol AL/mg scaffold, whereas HA-AL2-Fe10 released 15.3±0.9mmol AL/mg scaffold at an identical time. The released AL amount of HA-AL2-Fe10 was slightly lower than that of HA-AL2 although their loading contents were similar. It indicated that the steric effects caused by grafted Fe3O4 NPs influenced the release profile of AL, and also improved the biostability of HA-AL2Fe10 hybrid nanocrystals. During the whole release process, no iron was detected in PBS solutions (data not shown). One possible explanation was that the released Fe3O4 NPs from hydroxyapatite were wrapped by polymer matrix due to their large size and surface chemical properties, and the isolated iron particles or ions were below the detection limit. It is well known that PCL has a slow hydrolytic degradation rate because of its hydrophobicity and crystallinity, the interpenetrating network only allows delivery of small hydrophilic molecules (e.g. alendronate). In this condition, the PEG-modified Fe3O4 NPs that fixed in polymer networks were hard to be released into PBS solutions. Cellular compatibility and proliferation. To investigate the effect of Fun-HA on cellular behavior in different cell types, osteoblast monoculture, osteoclast monoculture, osteoblast and osteoclast co-culture were established as described in Figure 3A. MC3T3-E1 cells induced by differential medium including ascorbic acid and β-glycerophosphate were used as osteoblast model, and RAW264.7 cells induced by RANKL plus M-CSF were used as osteoclast model. The morphologies of osteoblasts and osteoclasts on different 3D porous scaffolds were shown in Figure 3B. It could be seen that osteoblasts were highly flattened and formed a near continuous layer on the PCL scaffold. Interestingly, osteoblasts attached to HA and HA-AL2-Fe10 scaffolds showed a star-shaped morphology with numerous filopodia, lamellipodia and cytoplasmic extensions, and mineralized collagenous extracellular matrices were observed. Most importantly,


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the surface of HA-AL2-Fe10 scaffold was completely covered with cell multilayers and calcification, osteoblasts presented a connect structure bridging across the 3D pores. On the other hand, most of osteoclasts on PCL and HA scaffolds had flattened central areas and numerous prominent filopodia, displaying typical appearance of mature osteoclasts. However, osteoclasts exhibited a round or small polygonal shape on the HA-AL2-Fe10 scaffold, and the number of osteoclasts was obviously decreased when compared with that on the PCL and HA scaffolds. These phenomena were further confirmed by CCK-8 assay (Figure 4A). For the osteoblast monoculture, Func-HA (HA-AL1, HA-AL2, HA-Fe5, HA-Fe10, HA-AL1-Fe5 and HA-AL2Fe10) groups induced higher cell proliferation than HA group, and the highest value was observed in the HA-AL2-Fe10 group. However, an opposite trend was found in a monoculture of osteoclasts. Func-HA scaffolds showed pronounced inhibitory effect on osteoclasts, and this effect was enhanced with the increase of AL or Fe3O4 content. For osteoblast and osteoclast coculture, the proliferation of osteoblasts was remarkably enhanced in the HA-Fe10 and HA-AL2Fe10 groups, whereas osteoclast proliferation was inhibited in all the Func-HA groups. The above results suggested that PCL-based 3D porous scaffolds had good biocompatibility, and the introduction of HA and Func-HA component could modulate the initial attachment and subsequent behavior of osteoblasts and osteoclasts. Some published studies, including our preliminary tests, found that AL could improve osteoblast activity at low concentration, but the concentrations of AL greater than 10-4 M inhibited the proliferation of osteoblasts

32-33

.

Accordingly, we optimized AL/HA molar ratios of AL-grafted HA during synthesis, samples were selected only when AL/HA < 2/10. In the present study, HA-AL1, HA-AL2, HA-AL1-Fe5 and HA-AL2-Fe10 had relatively low AL contents, and thus they showed a positive influence on osteoblast proliferation. Recently, it was reported that Fe3O4 NPs dispersed in carbonated HA


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microspheres could promote adhesion, proliferation and osteogenic differentiation of BMSCs 34. Moreover, HA-coated iron oxide NPs were found to enhance osteoblast proliferation and function 24, which was a good support for our current study. We have demonstrated that both AL and Fe3O4 grafted on HA could accelerate the proliferation of osteoblasts, but inhibit osteoclast proliferation. This effect could be enhanced when the two functional groups were used in conjunction. Cellular activity and differentiation. To evaluate the effect of Func-HA added scaffolds on the activity and differentiation of osteoblasts and osteoclasts, the following parameters in culture supernatants were assessed: alkaline phosphatase (ALP) and collagen type I (Col-I) as differentiation markers of osteoblast, osteoprotegerin/receptor activator for nuclear factor ΚB ligand (OPG/RANKL) as the anti-osteoclast capacity of osteoblast, and tartrate-resistant acid phosphatase (TRAP) as osteoclast differentiation marker (Figure 4B). For the osteoblast monoculture, ALP values were observably higher onto HA-AL1, HA-AL2, HA-Fe10 and HAAL2-Fe10 when compared to HA. Col-I production was significantly increased in HA-AL2-Fe10 group in comparison to HA group. OPG/RANKL showed increased values in HA-AL2, HA-Fe5, HA-Fe10, HA-AL1-Fe5 and HA-AL2-Fe10 groups as compared to HA group. These results indicated that the functional groups of AL and Fe3O4 exerted their beneficial action when loaded onto HA crystals, which promoted osteoblast differentiation and activity. For the osteoclast monoculture, the values of TRAP production, an indicator of osteoclast differentiation, were obviously lower in all the Func-HA groups than HA group. The suppression of osteoclasts was enhanced with higher contents of AL or/and Fe3O4. It was noteworthy that, the tendencies of ALP, Col-I, OPG/RANKL and TRAP continued in osteoblast and osteoclast co-culture. ALP is an early marker of osteoblast differentiation, and Col-I is one of the main components to form


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bone extracellular matrix 35-36. Hence, the increase of ALP activity and Col-I deposition in FuncHA groups were associated to the progressive differentiation of osteoblasts. Moreover, it is known that the ratio of OPG and RANKL play a key role in osteoclastogenesis regulation, and high levels of OPG/RANKL inhibit the differentiation of osteoclasts 37-38. For the osteoblast and osteoclast co-culture, OPG/RANKL ratio had the highest value in HA-AL2-Fe10 group when compared to other groups; TRAP activity, a known marker for osteoclast, was significantly lower in HA-AL2, HA-Fe10, HA-AL1-Fe5 and HA-AL2-Fe10 groups than HA group. Statistical analysis confirmed a correlation between OPG/RANKL ratio and TRAP (Pearson test inverse correlation -0.716, p HA-AL2 > HA-Fe10. According to the in vitro results, AL molecules were gradually released from the scaffolds, which could inhibit osteoclast function and promote osteoblast activities. The in vivo microenvironment could accelerate degradation of the scaffolds, thus the faster release of AL enhanced bone formation around the AL-loaded HA scaffolds. On the other hand, Fe3O4-loaded HA scaffolds have positive effects on osteogenesis due to constant magnetic stimulation, and accelerated Fe3O4 release would have little influence on their magnetic properties. Hence, the osteogenic potency of HA-AL2 is better than HA-Fe10 under in vivo condition. To further investigate osteogenesis within the 3D porous scaffolds, cross sections in the middle of the defects were submitted for histological and immunohistochemical evaluation (Figure 5C), and the quantitative evaluation of immunohistochemistry images was shown in Figure S7. At 4 weeks post-surgery, a few islands of new bone tissue were formed inside the degraded HA, HA-AL2, HA-Fe10 and HA-AL2-Fe10 scaffolds, whereas the defects of the Blank and PCL groups were filled with fibrous connective tissue, shown by H&E and Masson’s trichrome staining (Figure 5C). In the Func-HA groups, more newly formed bone tissue in the defective zones were observed compared to HA group, and the HA-AL2-Fe10 scaffold residues were well integrated into the surrounding tissue, which implied that the AL/Fe3O4-loaded FuncHA materials could promote bone regeneration and osseointegration in osteoporotic condition. Col-I immunohistochemical staining (Figure 5C) of the samples presented a wide range of positive stained area distributed in the newly regenerated tissue, and a general increase of Col-I


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expression was found for Func-HA groups in comparison to Blank, PCL and HA groups. This trend was further evidenced by the MOD values of Col-I (Figure S7A). Col-I is considered as the basic initial bone matrix protein in bone formation 53, the higher amount of Col-I deposition in Func-HA groups would be beneficial for cells attachment and migration in tissues, and subsequent skeletal reconstruction through cytoskeleton and matrix synthesis and degradation 54. Osteoclasts in the histological sections were identified by TRAP staining (Figure 5C). As expected, the Blank, PCL and HA groups exhibited a large number of TRAP-positive multinucleated osteoclasts at the defect sites (Figure S7B). This is because of the excessive osteoclast activity under osteoporotic condition 4. However, very few osteoclasts were present at the bone-implant interfaces in Func-HA groups due to the antiresorptive ability of the materials, and the lowest numbers of TRAP-positive cells were found in the HA-AL2-Fe10 group. These findings provided strong evidence that the AL/Fe3O4-loaded Func-HA materials, especially HA-AL2-Fe10, could regulate cell function, accelerate matrix deposition and osteogenesis in an osteoporotic model, which were in good agreement with our in vitro results. AL is a powerful antiresorptive agent that widely used for the treatment of systemic metabolic bone diseases. Many studies have claimed that AL could inhibit bone resorption via suppression of osteoclast formation and function, meanwhile, it may also improve osteoblast activity and osseointegration between implants and native bone 55-57. On the other hand, Fe3O4 NPs have been demonstrated to promote osteoblast proliferation and differentiation, due to their innate magnetic properties

58-59

. However, previous research has shown that unconjugated AL is rapidly cleared

from the blood (95% in 6h) reticuloendothelial system

62

60-61

, and monodisperse Fe3O4 NPs are easily uptaken by the

, which reduces their therapeutic efficacy. In the current study,

loading of AL and Fe3O4 onto HA crystals prevented short burst release of functional


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components into the defect site, hence the long-term release of bioactive molecules and the sustained magnetic stimulation by MNPs accelerated healing of osseous defects in osteoporotic situation. Interestingly, we have also found that a combination of two functional groups (AL and Fe3O4) on HA, could enhance bone remodeling and osseointegration when compared with monofunctionalized HA. However, our present work mainly focuses on investigating the functionality of the AL/Fe3O4loaded Func-HA, and more research is required to optimize the morphological, mechanical and degradation properties of the 3D porous scaffolds for better bone healing and controlled release of functional components. Another limitation of current work is that the in vivo study is performed for a relatively short period after implantation. Future study will present a long-term animal experiment to explore the potential effects of Func-HA added scaffolds on bone regeneration and osseointegration at different implantation period.

CONCLUSIONS A multi-functional HA was successfully fabricated by chemical self-assembly of AL and Fe3O4 onto HA nanocrystals. The resultant AL/Fe3O4-loaded Func-HA achieved a desirable combination of osteoconductive, antiresorptive and magnetic properties. In vitro and in vivo evaluations demonstrated that the Func-HA could inhibit osteoclastic bone resorption and promote osteoblast proliferation and differentiation, as well as enhance osseointegration and accelerate bone remodeling under osteoporotic condition. We believe that this multi-functional HA can be further applied as a biomaterial component to promote bone reconstruction, which may open new avenues for the prevention and healing of osteoporotic bone defect/fractures.


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EXPERIMENTAL SECTION Synthesis and surface modification of Fe3O4 nanoparticles. The monodisperse Fe3O4 nanoparticles were synthesized by high-temperature thermal decomposition

63

. 3mmol of

Fe(acac)3 (Sigma, USA) was dissolved in a mixture of 15ml benzyl ether and 15mL oleylamine. The solution was dehydrated at 110°C for 1h, and was quickly heated to 300°C and kept at this temperature for 1h. 50mL of ethanol was added into the solution after it was cooled to room temperature. The precipitate was collected by a permanent magnet and was washed with ethanol 3 times. Finally, the product was redispersed in 12mL hexane, and 200μl oleic acid was added to stabilize the particles. To convert the nanoparticles from hydrophobic to hydrophilic, dopamine (DPA, Sigma, USA) was first linked with one COOH group in PEG600 diacid (Sigma, USA) via the EDC/NHS chemistry to give DPA-PEG600-COOH, which was then used to replace oleate/oleylamine around the Fe3O4 nanoparticles in DMF/CHCl3 solution

64

. Briefly, 80mg

PEG600 diacid, 13mg EDC, 8mg NHS and 5mg dopamine hydrochloride were dissolved in a mixture solvent containing 2mL DMF, 4mL CHCl3 and 40mg anhydrous Na2CO3. The mixture was stirred at room temperature for 5h. After the reaction, 20mg Fe3O4 nanoparticles were added, and the solution was stirred at 80°C under Ar for 24h. The modified Fe3O4 nanoparticles were precipitated by adding hexane, collected by a permanent magnet and dried under vacuum. The particles were then dispersed in water. The extra surfactants and other salts were removed by dialysis using a dialysis bag (MWCO=10000) for 72h in water. Well-dispersed Fe3O4 nanoparticles were obtained as a transparent aqueous suspension. Synthesis of Func-HA nanocrystals. HA: HA nanocrystals were synthesized by the wet chemical technique according to the following reaction: 10Ca(NO3)2 + 6(NH4)2HPO4 + 8NH4OH → Ca10(PO4)6(OH)2 + 20NH4NO3 + 6H2O. 100mL (NH4)2HPO4 solution (0.3M) was dropped


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into 100mL Ca(NO3)2 solution (0.5M) under stirring at 400rpm. The reaction was performed at 70°C for 2h. During the process, 25% ammonia solution was added drop-wise into the mixture under stirring to keep the pH at ~12. After 72h aging at room temperature, the resultant deposits were washed with water until the pH reached ~7. HA@AL: 10mL alendronate (AL, Aladdin, China) solution was dropped into 10mL HA slurry (40mM) under stirring at 400rpm. The precipitate was maintained in contact with the reaction solution for 16h at 37°C under stirring, then centrifuged at 6000rpm for 3min and washed repeatedly (5~6 times) with water. HA-AL1 and HA-AL2 were prepared using AL concentrations of 4mM and 8mM. HA@Fe3O4: 10mL Fe3O4 aqueous suspension was dropped into 10mL HA slurry (5mM) under stirring at 400rpm. The precipitate was maintained in contact with the reaction suspension for 3h at 37°C under stirring, then centrifuged at 6000rpm for 3min and washed repeatedly (5~6 times) with water. HA-Fe5 and HA-Fe10 were prepared using Fe3O4 concentrations of 2.5mM and 5mM. HA@AL@Fe3O4: 10mL Fe3O4 aqueous suspension was dropped into 10mL HA-AL1 or HAAL2 slurry (5mM) under stirring at 400rpm. The precipitate was maintained in contact with the reaction suspension for 3h at 37°C under stirring, then centrifuged at 6000rpm for 3min and washed repeatedly (5~6 times) with water. HA-AL1-Fe5 and HA-AL2-Fe10 were prepared using Fe3O4 concentrations of 2.5mM and 5mM. Fabrication of Func-HA nanocrystals-added 3D porous scaffolds. Polycaprolactone (PCL, Mn~80,000, Sigma, USA) was used as polymer matrix to construct a 3D porous scaffold, while HA and Func-HA (HA-AL1, HA-AL2, HA-Fe5, HA-Fe10, HA-AL1-Fe5 and HA-AL2-Fe10) nanocrystals were encapsulated in the polymer network to form a uniform composite. The 3D


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porous scaffolds were fabricated by a solvent-casting/particle-leaching method using NaCl particles as the porogen. In brief, 5mL tetrahydrofuran (THF) was added into the 1mL Func-HA (or HA) slurry and mixed well. The precipitate was collected by centrifugation and was washed with THF 5 times. Then 76mg PCL pellets were dissolved in 3mL THF which containing the prepared Func-HA (or HA) nanocrystals (40:60 weight ratio of Func-HA (or HA) to PCL). After a complete dispersion of the Func-HA (or HA) nanocrystals, 4g NaCl sieved particles (200~500μm in diameter) were added into the suspension and the final dispersion was casted into a flat-plate column mold. The samples were air-dried for 24h to allow the solvent to evaporate completely. In order to leach NaCl particles out, the sample were soaked in water for 72h. The water was replaced 6 times by fresh water during this period. Salt-removed samples were freeze-dried, and 3D porous composite scaffolds were obtained. A pure PCL scaffold was prepared without Func-HA (or HA) nanocrystals as a control. Physicochemical characterization. Alendronate contents of Func-HA nanocrystals were determined spectrophotometrically via complex formation with Fe(III) ions using a BioTek Synergy H4 microplate reader (λ=300nm) 65. Fe3O4 contents were determined by ferrozine-based colorimetric assay at the wavelength of 550nm 66. XRD studies were run on Puxi XD-2/XD-3 Xray diffractometer using Cu Kα radiation (λ=0.15418nm). TG analysis was done by TA instruments Q50 thermogravimetric analyzer. TEM measurements were taken on JEOL JEM 2010 (200KV) transmission electron microscopy. SEM observation was performed using a Hitachi S-3400N II scanning electron microscopy. The porosity of 3D porous scaffolds (6.5×0.3mm) was measured by gravimetric procedure using ethanol (density 0.7893g/cm3, 20°C) as the displacement liquid 67. Magnetization measurements were carried out with Quantum Design Versalab vibrating sample magnetometer (VSM) at 295K. In vitro release experiments of


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the scaffolds (6.5×0.3mm) were conducted in phosphate buffer solution (PBS, pH=7.4, 37°C), alendronate and iron concentration were detected at different time intervals. In vitro tests. Cell experiments were performed on disk-shaped scaffolds (6.5×0.3mm) of PCL, HA, HA-AL1, HA-AL2, HA-Fe5, HA-Fe10, HA-AL1-Fe5 and HA-AL2-Fe10. For the monoculture assays, MC3T3-E1 or RAW264.7 cells were plated at a density of 2×104cells/well on scaffolds in 24-well plates. To establish a co-culture system, the inserts containing 5×103cells/well of RAW264.7 on scaffolds were put together in the 24-well plates seeded with MC3T3-E1 (2×104cells/well). The morphology of different cells growing on the scaffolds was observed with SEM (Crossbeam 340, Zeiss, Germany). Cell proliferation was evaluated by CCK-8 assay. Cell activity and differentiation was evaluated by immunoenzymatic assays for alkaline phosphatase (ALP), collagen type I (Col-I), osteoprotegerin (OPG), receptor activator for nuclear factor ΚB ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP). Gene expression of Runx2, ALP, Col-I, IRF9, c-Fos, NFATc1, OSCAR, DC-STAMP, and CD9 were also evaluated by quantitative real time polymerase chain reaction (qRT-PCR). More details on in vitro tests were reported in supporting information. In vivo examination. Animal experiment was approved by the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University, China (SYXK(PLA)20170031). Female Sprague Dawley (SD) rats, 12 weeks of age (300-320g), were used in this study. Four weeks after ovariectomization, the osteoporotic rats were randomized into six groups: Blank, PCL, HA, HA-AL2, HA-Fe10 and HA-AL2-Fe10. After exposure of the lateral femoral condyle, a defect with the size of 2.5mm in diameter and 3mm in depth was created. The spiral-cylindrical scaffolds (2.5×3mm), fabricated according to our previously reported method

68

, were

implanted into the cavity defects. The blank group was operated without implantation. At


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postoperative week 4, the rats were euthanized, and femurs with implantations were harvested for micro-CT, histological and immunohistochemical examination. Further details were reported in supporting information. Statistical analysis. Quantitative data were presented as mean ± standard deviation (SD). Statistical analysis was carried out using one-way ANOVA with a Student-Newman-Keuls test. A significant difference was considered when *p < 0.05, **p < 0.01 and ***p < 0.001.

ASSOCIATED CONTENT Supporting Information Supplemental results and detailed experiment methods are included in the Supporting Information. Figure S1. Chemical structures of bisphosphonate and alendronate; Figure S2. Powder Xray diffraction patterns of alendronate and Fe3O4; Figure S3. Surface modification of Fe3O4 nanoparticles via DPA-PEG600-COOH; Figure S4. Thermogravimetric (TG) analysis of PEG600diacid and Fe3O4-DPA-PEG600-COOH; Figure S5. Magnetic hysteresis loops of Fe3O4; Figure S6. Establishment of rat osteoporotic model in femur. A) 2D representation and 3D reconstructed images of normal group and OVX group. B) Micro-CT parameters (BMD, BV/TV, Conn.D, Tb.Th, Tb.Sp and Tb.N) of the femurs. C) Representative H&E and Masson’s trichrome staining for normal and OVX bone; Figure S7. A) The MOD values of Col-I and B) the number of TRAP-positive multinucleated cells in the defect region.

AUTHOR INFORMATION


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Corresponding Author *E-mail: [email protected] (S. Dong) *E-mail: [email protected] (H. Jiang) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant No. 31500768) and the National Key Technology Research and Development Program of China (Grant No. 2017YFC1103300).

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Figure 1. Morphology, elemental composition and crystal structure of Func-HA. A) TEM images and EDS analysis of as-synthesized nanocrystals, and the insets are the high-resolution TEM images. B) Powder X-ray diffraction patterns of HA and different Func-HA samples; (▲) indicates the main peaks of HA, and (●) marks the most intense peaks of Fe3O4. C) Scheme of possible structural interaction between alendronate or surface-modified Fe3O4 and the crystalline structure of HA. 144x117mm (300 x 300 DPI)


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Figure 2. Fabrication and physicochemical characterization of Func-HA. A) Preparation of Func-HA nanocrystals and Func-HA added 3D porous scaffolds. B) SEM photographs of 3D porous scaffolds fabricated by solvent-casting/particle-leaching method. C) Porosities of different 3D porous scaffolds. D) Magnetic hysteresis loops of the Fe3O4-loaded HA nanocrystals and scaffolds. E) In vitro AL releasing profile of FuncHA added 3D porous scaffolds. 126x89mm (300 x 300 DPI)



Figure 3. In vitro cell culture model and morphology of osteoblasts and osteoclasts. A) Scheme of osteoblast monoculture, osteoclast monoculture and osteoblast-osteoclast co-culture system. B) SEM photographs of osteoblasts and osteoclasts cultured on different 3D porous scaffolds at 7 days. 205x236mm (300 x 300 DPI)


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Figure 4. In vitro cell proliferation, differentiation and gene expression. A) CCK-8 assay of osteoblasts and osteoclasts grown on different substrates. B) Osteoblast activity (ALP, Col-I and OPG/RANKL) and osteoclast activity (TRAP) measured in cell culture supernatants. C) The relative mRNA expression of osteogenesisrelated genes (Runx2, ALP and Col-I) and osteoclastogenesis-related (IRF9, c-Fos, NFATc1, OSCAR, DCSTAMP and CD9) genes analyzed by real-time PCR. 112x71mm (300 x 300 DPI)



Scheme 1. Schematic illustration of Func-HA added 3D porous scaffold with proper magnetic property and controlled release manner for promoting osteoblast differentiation and inhibiting osteoclast differentiation. 177x72mm (300 x 300 DPI)


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Figure 5. In vivo microscopic osteogenic capacity of Func-HA added scaffolds. A) 3D reconstructed images of mineralized bone formation in distal femur defects. The dashed-line circles indicate the original defect areas. B) Micro-CT parameters (BMD, BV/TV, Conn.D, Tb.Th, Tb.Sp and Tb.N) of bone regeneration within femur defects. C) Histological and immunohistochemical analysis (H&E, Masson’s trichrome, Col-I and TRAP) of the cavity defects in lateral femoral condyle at 4 weeks post-surgery. (Red triangle: implanted scaffolds; Black arrows: multinucleated osteoclasts; Scale bars: 200 µm) 201x228mm (300 x 300 DPI)



44x30mm (300 x 300 DPI)


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Chemical self-assembly of multi-functional hydroxyapatite with coral

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