An osteoclast-responsive, injectable bone of bisphosphonated

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Article

An osteoclast-responsive, injectable bone of bisphosphonated-nanocellulose that regulates osteoclast/osteoblast activity for bone regeneration Akihiro Nishiguchi, and Tetsushi Taguchi Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01767 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28 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

Biomacromolecules

An

osteoclast-responsive,

injectable

bisphosphonated-nanocellulose

that

bone

of

regulates

osteoclast/osteoblast activity for bone regeneration Akihiro Nishiguchi†*, Tetsushi Taguchi†* †

Biomaterials Field, Research Center for Functional Materials, National Institute for Materials

Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan)

KEYWORDS. Bone regeneration, Injectable bone, Nanocellulose, Drug delivery system, Bisphosphonate

ACS Paragon Plus Environment

1

Biomacromolecules 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

Page 2 of 28

ABSTRACT.

An injectable bone may serve as a minimally invasive therapy for large orthopedic defects and osteoporosis and an alternative to allografting and surgical treatment. However, conventional bone substitutes lack the desirable biodegradability, bioresponsibility, and functionality to regulate the bone regeneration process. Here, we report an injectable, bioresponsive bone composed of bisphosphonate-modified nanocellulose (pNC) as a bone substitute for bone regeneration. Composites composed of nano-fibrillated cellulose and β-tricalcium phosphate (βTCP) mimic bone structures in which apatite reinforces collagen fibrils. Bisphosphonate groups on nanocellulose provide reversible, physical crosslinking with β-TCP, apatite formation, binding property to bone, and pH responsiveness. When the pH drops to ~4.5, which corresponds to an osteoclast-induced pH decrease, pNC-β-TCP composite degrades and releases pNC. pNC suppresses osteoclast formation and pit formation. This osteoclast-responsive property allows for controlling the degradation rate of the composite. Moreover, the composite of pNC, α-tricalcium phosphate (α-TCP), and β-TCP enhances osteoblast differentiation. This injectable bone substitute of pNC that regulates osteoclast/osteoblast activity has enormous potential for the treatment of bone diseases and prevention of locomotive syndrome.


2

Page 3 of 28 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

Biomacromolecules

Introduction. Bone-defect management has gained much importance for the therapy of large orthopedic defects and bone diseases, such as osteoporosis, Paget’s disease, osteolytic tumors, and sequential locomotive syndrome.1 Although the use of autograft and allograft bones is standard treatment owing to their ideal mechanical properties and bioactivities, there are potential drawbacks, including a need of secondary invasive surgery, limitation of available bone size, and infection.2 Since the 1980s, many types of bone substitutes using calcium phosphates, such as hydroxyapatite (HA; Ca10(PO4)6(OH)2) and α- or β-tricalcium phosphate (α/β-TCP, Ca3(PO4)2) have been developed.3-6 Bioactivity, degradation rate, and cell invasion can be controlled by surface coating, the degree of porosity, and mechanical properties.7 To realize minimally invasive therapy, an injectable bone which can be delivered to a defect using a syringe is promising to reduce the burden on operations and patients.8 α-TCP and β-TCP pastes can be a candidate as an injectable bone. Although, due to low mechanical property of β-TCP paste, αTCP pastes have been commercially employed, α-TCP are quickly degraded due to their high solubility and are often resorbed before bone formation is complete.9 Several researchers have reported organic-inorganic composite materials of calcium phosphates and biocompatible polymers, including poly(lactic acid) (PLA),10 collagen,11 pullulan,12 bacterial cellulose nanofiber,13 hyaluronan,14 and poly(ethylene glycol)15 to control the degradation rate and mechanical properties. Despite numerous research efforts focusing on composite materials, it is still challenging to design highly biofunctional bone substitutes because PLA composites cause inflammatory response and delayed bone regeneration,16 and highly crystalline HA is not bioresorbable.5 Therefore, an injectable, degradation-controlled, and biocompatible bone substitute needs to be developed for promoting bone regeneration.


3


Page 4 of 28

The bone is a dynamic organ which undergoes a cooperative remodeling process comprising of osteogenesis by osteoblasts and bone resorption by osteoclasts.17 An advanced bone substitute dynamically functions in response to physiological changes in bone microenvironments and regulates the bone remodeling process. During the remodeling process, osteoclasts play a central role in skeleton formation through bone resorption.18 We consider that suppression of bone resorption leads to control of degradation rate of bone substitutes. To this end, the use of bisphosphonate, which is a class of bone resorption inhibitors for the treatment of osteoporosis and metastatic bone diseases,19 is promising approach. Bisphosphonates induce apoptosis of osteoclast precursors and suppress osteoclast adhesion and formation.20 However, massive systemic administration of bisphosphonates to patients causes adverse effects, such as osteonecrosis of the jaws, ulcers, and abdominal pain.21 Although the incorporation of bisphosphonates into bone substitutes allows for local administration,22-24 water-soluble bisphosphonate may be rapidly released, which impairs the balance of bone remodeling due to over suppression of bone metabolism. Therefore, local administration of bisphosphonates with controlled release kinetics needs to be performed, ideally when osteoclasts actively resorb bone. Here, we report an injectable, bioresponsive bone substitute that can regulate osteoclast and osteoblast activity in response to bone microenvironment for bone regeneration (Figure 1). To recapitulate bone structures comprising organic fibrils (collagen type I) reinforced with inorganic particles (minerals, such as HA),25 we employed nano-fibrillated cellulose, called nanocellulose (NC). NC, such as cellulose nanofiber and cellulose nanocrystal, is a naturallyoccurring crystalline fibrous nanomaterial derived from plant cell wall.26 Bisphosphonatemodified nanocellulose (pNC) was used as an organic fibrous component to fabricate structurally mimicking bone substitute. Conjugation of alendronate (AL) as a bisphosphonate with NC


4

Page 5 of 28 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

Biomacromolecules

provides functionality to the bone substitute. Bisphosphonate groups can form reversible, physical crosslinking with inorganic components, such as β-TCP, which may enhance the mechanical property and impart thixotropic property.14 Thixotropic property enables the single syringe injection of materials to bone defects. We evaluated the ability of pNC-β-TCP composites involved in bone adhesiveness, such as apatite formation and bonding strength to bone. Since bisphosphonate groups in pNC form ion-crosslinking with calcium ions at pH 7 (pK1=2.5, pK2=6.5), a pH drop to ~4.5, which is induced by osteoclasts, inhibits composite crosslinking. Therefore, nanofibers bearing pharmacologically active bisphosphonates are released in response to osteoclasts, called osteoclast-responsive, and locally delivered to osteoclast precursors. We evaluated the pH responsiveness of pNC-β-TCP composites and the effect of pNC on osteoclast formation. Moreover, the effect of pNC-α/β-TCP composites on osteoblast function was also evaluated. Osteoclast-responsive composites that regulate osteoclast/osteoblast activity and control degradation rate may serve as novel injectable bones for the treatment of bone diseases and prevention of locomotive syndrome.


5


Page 6 of 28

Figure 1. Schematic illustration of preparation of bone substitutes of pNC for bone regeneration. pNC was prepared by oxidation of cellulose and sequential reductive amination with alendronate (AL). Induration by crosslinking between pNC and β-TCP formed an injectable composite (pNC-β-TCP composite). Bisphosphonate groups imparted thixotropic property by reversible, physical crosslinking, bone adhesive property, and pH responsiveness. When the active osteoclasts resorbed pNC-β-TCP composite by the release of proton (1), the composite released pNC loading pharmacologically active bisphosphonates in response to a pH drop to ~5 (2). pNC taken up by osteoclast precursor cells suppressed osteoclast formation (3). Moreover, pNC-α/βTCP composites enhanced osteoblast differentiation (4). This composite allows for the regulation of bone remodeling process and control of degradation rate by modulation of osteoclast/osteoblast activity.


6

Page 7 of 28 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

Biomacromolecules

MATERIALS AND METHODS.

Preparation of bisphosphonated-nanocellulose. Bisphosphonated-nanocellulose (pNC) was prepared by periodate oxidation and sequential amination with AL in the presence of 2-picoline borane, according to a previous report.27 Filter paper (1 g) (cotton linter; mesh size: 40–100, fiber length: 150–400 μm, cellulose content: ~99%; Advantec, Japan) was dispersed in 100 mL of ultrapure water, and sodium periodate (NaIO4; Nakarai Tesque, Japan) was added at 50–200 mol% to glucopyranose unit. The reaction proceeded at 50 °C for 4 h under light-shielding conditions. The products were purified by repeating suction filtration and washing with ultrapure water three times and freeze-dried for 3 days to obtain dialdehyde cellulose (DAC). The amount of aldehyde groups was quantified by neutralization titration method.28 Next, 1 g of DAC was dispersed in 100 mL of 0.5 M sodium hydrogen carbonate solution (NaHCO3 aq., pH=8), and 50–1000 mol% AL (Tokyo Chemical Industry, Japan) to aldehyde groups was dissolved in the solution. After 1 h of stirring, 2-picoline borane (the same molar ratio as AL) dissolved in 50 mL of ethanol (purity: 98%; Fujifilm Wako Pure Chemical Corporation, Japan) was added (water:ethanol=2:1) and reacted for 24 h at room temperature. The products were collected by centrifugation at 4000 rpm for 30 min and washed with a mixed solvent of 0.5 M NaHCO3 aq. and ethanol. The samples were purified by repeating these steps three times and then washed with phosphate-buffered saline (PBS) twice to remove ethanol. The samples were nanofibrillated using an ultrasound homogenizer (150 W, 40 kHz, 60% amplitude, Branson, USA) for 30 min under stirring in an ice bath to avoid increase in temperature. pNC dispersion was stored at 4 °C. The introduction of AL to DAC was confirmed using 1H- and

31

P-nuclear magnetic

resonance spectroscopy (1H-NMR, 31P-NMR, AL300; JEOL, Japan) and energy dispersive X-ray spectroscopy (EDX) with field emission scanning electron microscopy (FESEM, S-4800


7


Page 8 of 28

ultrahigh-resolution SEM; Hitachi, Japan) and detector (EMAX Evolution X-Max; Horiba, Japan). The residual aldehyde groups were evaluated using Fourier transform infrared spectroscopy (FT-IR, FTIR-8400S; Shimadzu Ltd, Japan). The morphology of the obtained pNC was observed by FESEM. Platinum was sputtered on the fibers with 10 nm of thickness. The accelerating voltage and working distance were set to 10 kV and 8 mm.

pNC composite. pNC composite was prepared by crosslinking the bisphosphonate groups with calcium ions. Three types of calcium salts were used in this study: (1) calcium chloride (CaCl2; Fujifilm Wako Pure Chemical Corporation, Japan), (2) alpha- or beta-tricalcium phosphate (αTCP, β-TCP, approximately 3 µm in diameter, Taihei Chemical Industrial Co., Ltd. Japan). (1) pNC (0.8–6.5 wt%) was immersed into 100 mM CaCl2 solution. Rapid gelation occurred soon after immersion, and gelation was completed after 1 h of incubation at 37 °C. (2) α-TCP and βTCP were dispersed in PBS and added to 3–9 wt% pNC solution and incubated for 24 h at 37 °C. The solid-liquid ratio of α-TCP was fixed at 1.8 and the final concentration of β-TCP was in the range of 5–100 wt%.

Rheology. To evaluate the viscoelastic properties of pNC hydrogels, rheological measurement was performed using a rheometer (MCR301; Anton Paar GmbH, Austria). The pNC pre-gel solution (100 μL) was placed on a stage. The measurement was conducted with a 10 mm diameter jig. The storage modulus and loss modulus were measured after 30–60 min of incubation on the stage (angular frequency: 1 Hz, strain: 1%, temperature: 25 °C). To evaluate the effect of the surface functional groups in NC on viscoelastic property, 2,2,6,6tetramethylpiperidine 1-oxyl (TEMPO)-oxidized NC (COOH-NC), mechanically disintegrated NC (OH-NC), pNC, and mono-pNC were used. TEMPO-oxidized NC was prepared according to


8

Page 9 of 28 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

Biomacromolecules

a previous report.29 Cellulose powder (1 g; Nacalai Tesque, Japan), 0.1 mmol of TEMPO (Fujifilm Wako Pure Chemical Corporation, Japan), and 1 mmol of sodium bromide (Tokyo Chemical Industry, Japan) were suspended in 100 mL of ultrapure water. TEMPO-mediated oxidation was initiated by adding 5 mmol of sodium hypochlorite (NaClO) solution (Nacalai Tesque, Japan). The reaction was performed at room temperature under stirring for 4 h, and the pH was maintained at 10 by adding 0.5 M NaOH using a pH stat. The cellulose dispersion was centrifuged at 4000 rpm for 5 min and washed with ultrapure water three times to obtain COOHNC. OH-NC was purchased from Sugino Machine (BiNFi-s, Japan). Mono-pNC was prepared using aminopropyl phosphonic acid (Sigma-Aldrich, USA) in the same manner as pNC.

Adhesion strength to hard tissue. A dentin plate (elephant tusk, lot No.: LKP3854, 10 mm diameter, 300 μm thickness, pre-treated with ethanol and UV irradiation for sterilization; Fujifilm Wako Pure Chemical Corporation, Japan) was used for the adhesion test of pNC. The adhesion strength was measured using a Texture Analyzer (TA-XT2i; Stable Microsystems, UK). Two pieces of ivory plates were used for one measurement. One plate was fixed to the stage and the other one was fixed to a probe (10 mm diameter of jig) at a dry state with a cyanoacrylate adhesive (Loctite; Henkel, Germany). pNC dispersion (100 μL) containing 100 mg of β-TCP was placed on the ivory plate on the stage. As a control, 100 mg of β-TCP were dispersed in 100 μL of PBS and used for adhesion measurement. The top probe with ivory plate approached to the bottom at 10 mm/min tracking speed and contacted at 10 N for 1 min. The top probe returned to the original position at 10 mm/min speed and then the adhesion strength was measured.

Apatite formation. To check the ability to form apatite in vitro, simulated body fluid (SBF) was used. The composition of SBF was determined according to a previous report: Na+: 142 mM, K+:


9


Page 10 of 28

5 mM, Mg2+: 1.5 mM, Ca2+: 2.5 mM, Cl–: 148.8 mM, HCO3–: 4.2 mM, PO43–: 1 mM, and SO42–: 0.5 mM. pNC film was prepared by casting pNC dispersion on a glass substrate (boroslicate glass, Matsunami, Japan) and added SBF solution and incubated at 37 °C for 2 weeks. α-TCP and α-TCP-pNC composites were immersed in SBF and incubated at 37 °C for 2 weeks. After washing the substrates with ultrapure water, the surface structure was analyzed by FESEM (S4800 ultrahigh-resolution SEM; Hitachi, Japan).

Osteoclast formation. RAW264.7 cells (European Collection of Authenticated Cell Cultures, UK) were pre-cultured in α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, USA) and 1% penicillin/streptomycin (P/S) (Thermo Fisher Scientific, USA) at 37 °C in a 5% CO2 incubator. RAW264.7 cells (1 x 103) were seeded onto a 96-well plate and cultured in media containing 0.32-2.5 mg mL-1 of pNC for 1 day. The media were changed to differentiation media: α-MEM containing 10% FBS, 1% P/S, and 50 ng mL–1 of receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL, Fujifilm Wako Pure Chemical Corporation, Japan). The cells were cultured for 4 days and the media were changed every 2 days. Differentiation to osteoclasts was confirmed using a TRAP assay kit (Fujifilm Wako Pure Chemical Corporation, Japan). Briefly, the samples were fixed with 4% paraformaldehyde (PFA) for 10 min at 4 °C. After washing with PBS three times, cells were permeabilized with a mixture of ethanol/acetone for 1 min at –20 °C. After washing with PBS three times, TRAP reagent was added and incubated for 30 min at 37 °C. After washing with water three times, cells were visualized by optical microscopy in bright field mode (Thermo Fisher Scientific, USA), and the area of TRAP-positive osteoclasts was measured using ImageJ. Pit formation was analyzed using a calcium phosphate plate (bone resorption assay plate, PG Research, Japan). After 4 days of culture of differentiated RAW264.7 cells exposed to 1.2 or 2.5 mg mL-1 of pNC, cells on the


10

Page 11 of 28 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

Biomacromolecules

plate were removed by trypsinization. The pit area was then analyzed by optical microscopy in bright field mode and measured using ImageJ. For cell viability assay, the cell number was counted based on mitochondrial activity (WST-8 assay; Dojindo, Japan). Briefly, 10 μL of WST8 reagent was added to RAW264.7 cells cultured in a 96-well plate and incubated for 2 h. The absorbance of media was measured at 450 nm by a microplate reader (Spark 10M; Tecan, Switzerland). The cell number was calculated from a standard curve.

Osteoblast culture and ELISA assay. Human osteoblast-like cells (MG-63) and mouse osteoblast-like cells (MC3T3-E1) were used to evaluate the effects of pNC on osteoblast activity. Both cells were cultured in α-MEM supplemented with 10% FBS and 1% P/S as growth medium or α-MEM supplemented with 10% FBS, 1% P/S, 50 μg/mL ascorbic acid (Nacalai Tesque, Japan), and 10 mM β-glycerophosphate (Nacalai Tesque, Japan) as differentiation medium at 37 °C in a 5% CO2 incubator. pNC-α/β-TCP composites were prepared by mixing 3 wt% pNC, 40 wt% α-TCP, and 40 wt% β-TCP, followed by incubation for 24 h at 37 °C and sterilization by UV irradiation for 1 h. MG-63 cells (3 x 104) were seeded on 10 mm diameter pNC-α/β-TCP composites placed in a 48-well plate and cultured in growth media for 24 h. Then, the media were changed to differentiation media and cells were cultured for 3 weeks. After cultivation, the supernatants were collected, and the secretion of osteocalcin from osteoblasts was quantified using an ELISA assay kit (R&D Systems, USA), according to the manufacturer’s protocol. The cells were fixed with 4% PFA for 15 min, and treated with 0.1% Triton-X solution for 15 min to perform permeabilization. After washing with PBS three times, cells were incubated in 1% bovine serum albumin for 1 h and stained with phalloidin labeled with tetramethyl rhodamine B isothiocyanate (Sigma-Aldrich, USA) (1:100) for 1 h. After washing with PBS three times, the


11


Page 12 of 28

nuclei were stained with DAPI (Dojindo, Japan). The cells were visualized using fluorescence microscopy (BZ-1; Keyence, Japan).

Statistical analysis. All data were expressed as means ± standard deviation (SD) unless otherwise specified. The values represent the means ± SD from three independent experiments. Statistical analysis was performed by unpaired t-test or one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. A P value < 0.05 was considered to be statistically significant.


12

Page 13 of 28 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

Biomacromolecules

RESULTS AND DISCUSSION. Preparation of pNC composites and their viscoelastic properties There is a growing interest in the use of NC, which exhibits unique structural characteristics (high specific surface area, high aspect ratio, high mechanical strength, and light-weight) for potential applications, such as composite material, gas barrier film, and microelectronics.[24] NC consists of cellulose microfibrils (~3 nm in width) in which fully extended 30–40 cellulose chains are hierarchically aligned via strong intermolecular hydrogen bonding. Although various kinds of NCs, such as mechanically homogenized NC,30 bacterial cellulose,31 and TEMPOoxidized NC29, 32 have been reported, NC is not considered to be a desirable biomaterial source due to its poor biodegradability resulting from the high crystallinity of cellulose.33 We employed biodegradable DAC to prepare NC. Cellulose is first oxidized with sodium periodate to convert 1,2-dihydroxy(glycol) groups to paired aldehyde groups by cleaving the C2-C3 bond. The DAC obtained forms NC by surface functionalization with charged groups and homogenization.34 DAC-derived NC degrades at physiological pH in vivo and the resulting glycolic acid and 2,4dihydroxybutyric acid are metabolized.35 Therefore, we expect DAC-based NC such as pNC possesses biodegradability. Moreover, introduction of aldehyde groups in NC enhances the availability of NC and introduces various NC-bearing carboxy,34 sulfonyl,36 and cationic groups.37 Here, pNC was prepared by oxidation of cotton linter and sequential reductive amination with AL.27 Filter paper powder was treated with sodium periodate (0.5–2 equivalent to glucopyranose unit mol) in water to form DAC. The amount of aldehyde groups ranged from 0.78 to 6.93 mmol/g (Table S1). DAC was reacted with AL (0.5–10 equivalent to aldehyde groups) in the presence of 2-picoline borane in 0.5 M NaHCO3 aq./ethanol mixed solvent and homogenized using an ultrasonic homogenizer to produce pNC dispersion. Introduction of


13


Page 14 of 28

negatively charged bisphosphonate groups can induce electrostatic repulsion to break hydrogen bonding, which facilitates nano-fibrillation. The introduction of AL was confirmed by 1H-NMR, 31

P-NMR, and EDX analysis (Figure S1–3). The degree of substitution (D.S.) of AL increased

with increasing of AL feeding ratio and D.S. at 3 equivalent of AL feeding ratio was 6% (Figure S4). Fourier transform infrared spectroscopy (FT-IR) showed disappearance of aldehyde groups after reductive amination of DAC with AL (Figure S5). Scanning electron microscopy (SEM) showed the successful formation of nano-fibrillated fibers with 20–30 nm in diameter, which was close to a single microfibril (~10 nm) (Figure 2a). The reaction with more than 1.0 equivalent of AL provided the nanofibers, which indicates that electrostatic repulsion between bisphosphonate groups is required for nano-fibrillation. To fabricate bone substitute, we started with the preparation of pNC gels using CaCl2 to check calcium ion-based crosslinking with pNC. When pNC dispersion (PBS without calcium and magnesium, pH=7.4) was immersed into 100 mM CaCl2 solution, a crosslinking reaction between pNC and calcium ions occurred, forming calcium ion-crosslinked pNC gels (swelling degree ~10). The obtained pNC gels possessed fibrous structures with nano-pores (Figure 2b). While injectable materials based on chemical crosslinking fail to promote cell infiltration due to their dense molecular network structure (pore size: ~5 nm), nanofiber-based materials may provide a porous space for cell infiltration. Rheological measurement of pNC gels was performed as a function of strain at a fixed frequency (1 Hz) (Figure 2c). pNC gels showed linear elastic property in the range of 0.1 to 10% strain and a rapid decrease above the critical strain region (γ=6%). The storage shear modulus (G’) was over 100 kPa. Shear elastic modulus values increased with increasing the concentration of pNC and feeding ratio of AL, while the amount of aldehyde groups in DAC did not affect the shear modulus (Figure 2d–e). The


14

Page 15 of 28

viscoelastic properties of pNC gels can be tuned by varying the concentration of bisphosphonates. Since bisphosphonates strongly interact with calcium ions, pNC gels displayed highest shear modulus compared to other NCs functionalized with carboxy (COOH-NC), hydroxy (OH-NC), and mono-phosphate groups (mono-pNC) (Figure S6). Therefore, the use of bisphosphonate groups is suitable for the fabrication of bone substitutes with high viscoelastic property. Based on these results, pNC prepared at 3 equivalent of AL feeding ratio (No. 8 shown in Table 1) was

a

b

c

Shear modulus (kPa)

used in the following experiments.

1000 100 10 1

G' G"

0.1 0.1

1

10

100

e

100 10 1 0.1 0.01 0

1

2

3

4

5

pNC (wt%)

6

7

f 1000 100 10 1 0.1 0.01 0

2

4

6

8

10

AL (eqv.)

Shear modulus (kPa)

d

Shear modulus (kPa)

Strain (%)

Shear modulus (kPa)

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

Biomacromolecules

1000 100 10 1 0.1 0.01 0

2

4

6

Aldehyde group (mmol/g)

Figure 2. Preparation and characterization of pNC composites using CaCl2. a) SEM image of pNC with nanofibrous structures. b) SEM image of pNC composite. pNC dispersion (4 wt%) was crosslinked with 100 mM CaCl2. c) Rheological measurement of pNC composite on strain sweep (γ=0.1% to 100%) at a fixed frequency (1 Hz). d) Shear modulus of pNC composites represented as a function of pNC concentration at a fixed strain and frequency (1%, 1 Hz). pNC prepared with 1.0 equivalent of AL was used for the measurements. e) Shear modulus of pNC composites represented as a function of AL feeding amount. pNC (5 wt%) was used for the measurements. f) Shear modulus of pNC composites represented as a function of the amount of aldehyde groups. pNC (5 wt%) prepared with 4.0 equivalent of AL was used.


15


Page 16 of 28

Preparation of pNC-β-TCP composite To develop biofunctional bone substitutes, β-TCP was employed as an inorganic component. It is known that, in contrast to α-TCP paste, paste of poorly water-soluble β-TCP does not undergo self-setting and easily diffuses due to its low mechanical property. It is known that bisphosphonate groups not only interact with calcium ions but also replace orthophosphate ions from apatite surfaces.38 Therefore, bisphosphonate groups on pNC strongly interact with calcium phosphate to form pNC-β-TCP composite with relatively high viscoelastic property. pNC dispersion was mixed with β-TCP particles (approximately 3 μm in diameter) to form pNC-βTCP composites (Figure 3a). Induration of composites immediately occurred after mixing (few seconds), and the shear modulus value increased during the incubation period and reached a plateau after 5 min (Figure S7). The viscoelastic property of the composites was tuned by the concentration of β-TCP and pNC. The combination of 20 wt% β-TCP and 5 wt% pNC showed high shear modulus, low loss tangent, and stability in water, while the composite composed of 50 wt% β-TCP and 5 wt% pNC dissolved in water although self-setting in air was observed (Figure 3b). Over 7 wt% pNC impaired viscoelastic property due to ineffective crosslinking (Figure 3c). A combination of pNC and β-TCP allows for fabricating an injectable β-TCP bone substitute. Moreover, we examined the thixotropic property of pNC-β-TCP composites. Thixotropy refers to viscous property that decreases under shear stress and recovers soon after the stress is removed, which is useful for single syringe injection of materials. pNC dispersion itself exhibited thixotropic property due to the entanglement of nanofibers with high aspect ratio (Figure S8). When periodic shear strain of 1 and 100% was applied to pNC-β-TCP composites, the G’ value decreased to one hundredth at 100% shear strain due to breakage of the network


16

Page 17 of 28 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

Biomacromolecules

structure, and it reverted to almost original value within few tens of seconds (Figure 3d). The entanglement of pNC and reversible, physical crosslinking between bisphosphonate groups and calcium ions may reconstruct network structures and restore mechanical property. This result suggests that pNC-β-TCP composites possess thixotropic property and can be injected using a single syringe.

Bone adhesiveness of pNC-β-TCP composites Implanted bone substitute often fails to integrate with the host bone, which impairs osteoconductivity. Bone adhesiveness of the implanted material depends on its physical interaction with the bone or biochemical interaction based on cell adhesion. To evaluate the physical adhesive property of the composite to hard tissues, adhesion strength to dentin matrix (ivory plate) was measured by sandwiching pNC-β-TCP composites with two pieces of ivory plates. pNC-β-TCP composites exhibited higher adhesion strength to ivory plates compared to βTCP composite (Figure 3e). Bisphosphonate groups may attach to the dentin matrix and enhance adhesion strength, although adhesion strength shown here was weak because only physical interaction worked between dentin and composites. Bone-adhesive pNC-β-TCP composites may be fixed at defect sites in the bone after injection. To investigate whether this composite biochemically interacts with the bone, apatite formation on pNC-casted films was evaluated using SBF. SBF is widely used to predict apatite formation by materials in vitro.39 Bioactive implanted materials spontaneously form bone-like apatite on their surfaces after incubation in SBF. After 2 weeks of incubation of pNC-casted films in SBF at 37 °C, huge amounts of agglomerates of apatite particles were formed on the surface of pNC films, but not on the glass substrate (Figure 3f). Since negatively charged functional groups, such as phosphate, carboxy,


17

Biomacromolecules

and sulfonic groups act as a core for apatite formation,39 the bisphosphonate groups on pNC accumulated calcium and phosphate ions to form bone-like apatite on their surfaces. Apatite formation was also observed on pNC-α-TCP composites, but not on α-TCP paste (Figure S9). Bisphosphonate groups on pNC promoted biomineralization, which contributes to enhancing osteoconductivity and integration with bones.

c

1

1000

100 0.1 10

1

0.01 0

20

40

1000

1

100 0.1 10

1

0.01 2

60

4

β-TCP (wt%) 1% 100% 1% 100% 1% 100% 1% 100% 1%

Shear modulus (kPa)

100

e

100

10

10

1 1 0.1

6

8

10

pNC (wt%)

Adhesion strength (Pa)

d

tanδ

tanδ

Shear modulus (kPa)

pNC-β-TCP

Shear modulus (kPa)

b

a

1000 750 500 250 0

tanδ

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

Page 18 of 28

*

b-TCP

β-TCP

f

Glass

b-TCP+pCNF

pNC-β-TCP pNC

0.1

0.01 G'

tand tanδ

G"

0.001

0.01 0

1

2

3

4

5

6

7

8

9

Time (min)

Figure 3. Fabrication of pNC-β-TCP composites and evaluation of their bone adhesive property. a) Photograph of pNC-β-TCP composite. b,c) The relationship between shear modulus and concentration of β-TCP and pNC. pNC (5 wt%) and β-TCP (20 wt%) were used for the measurements. Red squares denote the composite with high viscoelastic property and high stability in water. The measurement was performed at a fixed strain and frequency (1%, 1 Hz). d) Thixotropic property of pNC-β-TCP composite (5 wt% pNC and 20 wt% β-TCP). The strain was varied at 1 and 100% every 1 min at a fixed frequency (1%). e) Adhesion strength of β-TCP and pNC-β-TCP composites to dentin matrix (ivory plate). pNC (5 wt%) and β-TCP (20 wt%) were used for the measurements. *P < 0.05, Student’s t-test (n=3). f) SEM images of the glass substrate and pNC films immersed in SBF for 4 weeks at 37 °C. Apatite particles were observed on the surface of the pNC film.


18

Page 19 of 28 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

Biomacromolecules

Suppression of osteoclast formation by pNC In addition to the physical characteristics of pNC (physical crosslinking, thixotropy, and bone adhesiveness), pNC may serve as a pharmacologically active component in the bone resorption process. Bone resorption is a multistep process comprising proliferation of precursor cells, differentiation to osteoclasts (osteoclastogenesis), and degradation of organic/inorganic structures by mature resorptive cells.40 The bone matrix is degraded by secretion of HCl from osteoclasts through ion transporting events, which drops the pH to ~4.5.40 Since bisphosphonate groups on pNC form ion-crosslinking with calcium ions, the decrease of pH induces protonation of bisphosphate groups (AL: pK1=2.5, pK2=6.5). At pH=4.5, half of the P-OH groups in AL were pronated, which attenuate crosslinking and collapse the structure and release pNC. Actually, the weight of pNC-β-TCP composites decreased after incubation in PBS with low pH (2 and 5) (Figure 4a). This result indicates that pNC-β-TCP composites degrade under osteoclast microenvironment by protonation of bisphosphonate groups. Therefore, pNC bearing pharmacologically active bisphosphonates will be released in response to a pH decrease elicited by active osteoclasts, called osteoclast-responsive. To examine the pharmacological activity of pNC, mouse macrophage-like cells (RAW264.7, osteoclast precursor cells) were differentiated into osteoclasts in the presence of pNC. Osteoclasts are multinucleated cells formed by the fusion of monocyte/macrophage progenitor cells through activation factors, such as macrophage colony-stimulating factor (MCSF), RANKL, and a tumor necrosis factor-family cytokine.18 RAW264.7 cells were cultured in the presence of RANKL for 4 days, and tartrate-resistant acid phosphatase (TRAP) staining showed the formation of multinucleated osteoclasts (Figure 4b). Conversely, exposure of RAW264.7 cells to 2.5 mg mL–1 of pNC during osteoclast differentiation in the presence of


19


Page 20 of 28

RANKL drastically suppressed osteoclast formation. This behavior was dose-dependent and corresponded to the inhibition level induced by AL exposure (Figure 4c). pNC dispersion did not exert any cytotoxic effect on RAW264.7 cells even at 5 mg mL–1 (Figure S10). Consequently, pNC suppressed osteoclast formation without cytotoxicity. To assess the activity of osteoclasts, pit formation was evaluated using an apatite plate. As a result, pit formation was suppressed in pNC-exposed samples, while differentiated osteoclasts formed large pits (Figure 4d). These results indicate that pNC released from pNC-β-TCP composites may be taken up by osteoclast precursor cells and may suppress osteoclast formation through the inhibition of farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthase in the mevalonate pathway.18 Bisphosphonate groups function not only as crosslinking points but also as drugs that impair bone resorption by osteoclasts. The released pNC might be favorable for uptake by cells due to its size and has potential for use as a carrier in drug delivery systems. Since injectable bones, especially α/β-TCP, require slow degradation property, suppression of osteoclast activity contributes to controlling the degradation rate of composites. On the other hand, due to the difficulty of the long-term culture of osteoclasts (>5 days), local release of pNC and uptake of pNC by osteoclasts remain unclear. The use of primary osteoclasts and animal models may give detailed mechanisms on the release profile and uptake of pNC.


20

Page 21 of 28 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

Biomacromolecules

Figure 4. Suppression of osteoclast formation. a) Effect of pH on the weight of pNC-β-TCP composites. After 24 h of incubation of composites in PBS at different pH values, the composite weight decreased with decreasing pH due to protonation of bisphosphonate groups on pNC. *P < 0.05, **P < 0.01 when compared with the weight at pH=7.4, Student’s t-test (n=3). b) Bright field images of RAW264.7 cells exposed to media without and with 2.5 mg mL–1 of pNC. The cells were cultured in the presence of 50 ng mL–1 RANKL as an osteoclast formation factor. pNC dispersed in media formed loosely crosslinked crushed gels with calcium ions in media. Osteoclast formation was confirmed by TRAP staining. c) The area of osteoclasts exposed to pNC and AL. Osteoclast formation was suppressed in a dose-dependent manner after pNC exposure. Osteoclasts were stained with TRAP and the positively stained area was measured by ImageJ. * P< 0.05, **P < 0.01, Tukey’s multiple comparison test (n=5). d) Pit area formed on apatite plates where osteoclasts exposed to pNC dispersion were cultured. *P < 0.05, Tukey’s multiple comparison test (n=3). Scale bars represent 200 μm.


21


Page 22 of 28

Effect of pNC composite on osteoblast function Finally, we examined the effect of pNC composites on osteoblast activity. Regulation of not only osteoclast formation but also osteogenesis by osteoblasts may lead to regulation of bone remodeling toward bone regeneration. A mixture of α-TCP and β-TCP was used as a composite for osteoblast culture because β-TCP without pNC (control) did not undergo self-setting and collapsed in media. Human osteoblast-like cells (MG-63) were seeded onto α/β-TCP and pNCα/β-TCP composites and cultured for 3 weeks. Fluorescence analysis confirmed good adhesion of MG-63 cells on the surfaces of pNC-α/β-TCP composites (Figure 5a). The expression level of osteocalcin in the supernatants of cells cultured on the composites was quantified using an ELISA assay. Osteocalcin is a differentiation marker of osteoblasts and is secreted in the latter stages of differentiation.22 The production of osteocalcin from MG-63 cells cultured on pNC-α/βTCP composites was significantly higher compared to that from cells on α/β-TCP plates, indicating the promotion of osteoblast differentiation by pNC-α/β-TCP composites (Figure 5b). Since pNC induced apatite formation on their surfaces via bisphosphonate groups, the apatite surface on the composites formed during cultivation could provide suitable microenvironments for osteoblasts. Moreover, bisphosphonate itself may enhance osteoblast activity, as previously reported by several researchers.22,

41

However, the detailed mechanism has not yet been

elucidated. Further analysis of osteoblast functions such as mineralization ability needs to be examined. Enhancement of osteogenesis by pNC composites, in addition to suppression of bone resorption has a great impact on the treatment of bone defects and osteoporosis. Maintaining a balance between osteoblast and osteoclast activity is a crucial aspect for the design of nextgeneration bone substitutes.


22

Page 23 of 28

a

b 10

Actin Nuclei

Osteocalcin (ng/mL)

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

Biomacromolecules

*

8 6 4 2 0

α/β-TCP without

pNC-α/β-TCP pNC

Figure 5. Promotion of osteoblast differentiation. a) Fluorescence image of osteoblasts cultured on pNC-α/β-TCP composites for 3 weeks. α-TCP was used because β-TCP as a control did not undergo self-setting and was not available for cell culture. Composites were prepared by mixing 3 wt% pNC, 40 wt% α-TCP, and 40 wt% β-TCP. Actin filaments and nuclei were stained with phalloidin (red) and 4',6-diamidino-2-phenylindole (DAPI; blue), respectively. b) Osteocalcin production from MG-63 cells was upregulated in pNC-α/β-TCP composites. Osteocalcin levels in the supernatants were quantified using ELISA assay. *P < 0.05, Student’s t-test (n=3). Scale bar: 50 μm.


23


Page 24 of 28

CONCLUSIONS. In conclusion, we reported an injectable bone composed of biofunctionalized NC and β-TCP composite that regulates osteoclast/osteoblast activity for bone regeneration. pNC formed thixotropic, bone-mimicking composites through reversible, physical crosslinking with inorganic components, such as β-TCP. pNC-β-TCP composites possessed various characteristics, including tunable viscoelastic property, physical binding to bone, apatite formation, and pH responsiveness. Importantly, pNC composites collapsed in response to a pH drop (

An osteoclast-responsive, injectable bone of bisphosphonated

Recommend Documents