Bone regeneration induced by local delivery of a modified PTH

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Tissue Engineering and Regenerative Medicine

Bone regeneration induced by local delivery of a modified PTH-derived peptide from nano-hydroxyapatite/chitosan coated true bone ceramics Liang Yang, Jinghuan Huang, Shuyi Yang, Wei Cui, Jianping Wang, Yinping Zhang, Jingfeng Li, and Xiaodong Guo ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00780 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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ACS Biomaterials Science & Engineering

Bone regeneration induced by local delivery of a modified PTH-derived peptide from nano-hydroxyapatite/chitosan coated true bone ceramics Liang Yang,#,1,2 Jinghuan Huang,#,3 Shuyi Yang,2 Wei Cui,2 Jianping Wang,1 Yinping Zhang,1 Jingfeng Li,*,1, Xiaodong Guo*,2 1

Department of Orthopedics, Zhongnan Hospital of Wuhan University, 169 Donghu

Road, Wuhan 430071, People’s Republic of China. 2

Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, People’s Republic of China. 3

Department of Orthopaedics, Shanghai Jiaotong University Affiliated Sixth People’s

Hospital, 600 Yishan Road, Shanghai 200233, People’s Republic of China. #

These two authors contributed equally to this work.

*Corresponding author: [email protected] (Jingfeng Li); and [email protected] (Xiaodong Guo)

Abstract A novel PTH-derived peptide, PTHdP, including a repetitive aspartic acid sequence at the C-terminal and phosphorylated serine at the N-terminal has been previously designed. To evaluate its potential as a bone growth factor for bone tissue engineering, true bone ceramics incorporated with nano-hydroxyapatite coating and chitosan

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(CH/TBC) was developed as a desired three-dimensional porous delivery carrier in this study. In vitro results showed that PTHdP could be high-efficiently loaded and subsequently released in a controlled and sustained manner from CH/TBC. Bioactivity of released PTHdP was retained and able to exert a significant effect on promoting or inhibiting osteogenesis actions when exposed intermittently or continuously, respectively for MC3T3-E1 cells culture. As evaluated in a critical size rabbit radial defect model by radiographic and histological examination, combination of CH/TBC scaffolds with PTHdP exhibited a remarkably stronger capacity to stimulate new bone formation than control and pure CH/TBC groups. These results indicated the novel PTHdP peptide achieved high affinity to bone mineral without interference in bioactivity, and local delivery of PTHdP from apatite materials could be a promising alternative for future bone tissue engineering. Keywords: Parathyroid hormone; bone-targeting; drug local delivery; true bone ceramics; bone regeneration

Introduction Bone grafting is a treatment frequently performed in trauma, orthopedic and craniomaxillofacial surgery for bone defects repair, and the acknowledged gold standard bone graft is autologous cancellous bone.1 Whereas, autologous bone grafts are associated with various complications risks such as chronic pain, hematomas, infections and blood loss. Furthermore, for some patients, there may be insufficient bone to be harvested.2 Consequently, the development of new and alternative

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approaches to bone healing has been extensively motivated. With progressive cognition of the bone regeneration process, new therapeutic strategies including bone graft substitutes, bioactive growth factors and stem cell-based methods are being investigated.3-5 Then, an effective strategy is established to develop sustained and controlled release of bioactive molecules from desirable bone graft biomaterials into sites of damaged bone.6,7 Thereinto, bioactive proteins and peptides have attracted extensive attention for their enhanced osteogenic properties to induce rapid regeneration of bone.8-10 Parathyroid hormone (PTH) is an 84 amino acid peptide hormone that plays an important physiological role in calcium regulation and bone remodeling.11 The PTH(1–34), comprising the first 34 amino acids of the N-terminus fragment of PTH, conserves most of the functions of PTH.12 It is acknowledged that PTH(1-34) can exhibit anabolic or catabolic effects, depending on the concentration and mode of administration. Generally, prolonged and high doses of exposure to PTH(1-34) leads to increased bone resorption, yet intermittent doses of higher amounts or infusion of low doses results in bone formation.13 In addition to its prevalent application in treating osteoporosis, intermittent administration of PTH(1-34) has been found to significantly stimulate bone defect regeneration,14,15 making it available to expand the application of PTH(1-34) to a bone graft procedure.16,17 However, the inconvenience of daily injection, low bioavailability with short lasting duration and even potential safety risk of systemic exposure severely hinder the broader application of PTH(1-34).18,19 To overcome these disadvantages, localized and controlled delivery

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of PTH(1-34) via high affinity immobilization on drug delivery vehicles, such as PLGA microspheres,20 fibrin polymer,21 biomimetic CaP coating,22 polyethylene glycol-based matrix,23 and atelocollagen membrane,24 have been explored. As these researches concluded, a proper local delivery system could be effective to maintain the bioactivity of PTH(1-34) and achieve local bone healing without the potential problems caused by systemic PTH exposure. Nevertheless, previous researches were almost aimed at designing specific delivery vehicles to realize local and controlled delivery of PTH(1-34), while there is relatively little work focusing on modification of the inherent properties of PTH(1-34). Recently, we have designed and synthesized a novel PTH-derived peptide (S

[PO4]

VSEI–QLMHN–LGKHL–NSMER–VEWLR–KKLQD–VHNF DDD), including a repetitive Asp (aspartic acid) sequence and phosphorylated Ser (serine), and this modified PTH(1-34) peptide, PTHdP, could maintain its full bioactivity and significantly improve the osteogenic differentiation level in vitro.25 Actually, the synthesis process of PTHdP was similar to a previous novel peptide P24, derived from residues of the knuckle epitope of BMP-2 using FMOC/tBu solid-phase method by our group.26 Related researches have shown that the P24 peptide revealed a controlled and sustained release profile from the surface mineralization-modified true bone ceramics (TBC) and nano-HA/collagen/PLLA containing chitosan microspheres scaffold, mainly due to its high affinity bound to HA components through the repetitive Asp and phosphorylated Ser functional groups.27,28 Based on these previous

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researches, the PTHdP was considered to possess parallel high affinity to bone mineral with P24 peptide. The objective of this study was to evaluate the potential of PTHdP to be applied as a bone growth factor for bone tissue engineering. Thus, we have developed a desired delivery scaffold by coating TBC substrates with nano-hydroxyapatite (nHA) and chitosan (CS). TBC was selected for its inherent natural trabecular structure with an organized crystal of bone minerals, which was considered highly biomimetic and able to facilitate the growth of vessels into the material to provide a suitable environment for bone regeneration.29,30 Considering the high affinity of PTHdP to apatite, nHA coating and chitosan were further applied on TBC surface to improve the bioactivity and peptide immobilization capability.31,32 The biological effects of PTHdP released from the composite scaffolds were examined both in vitro and in vivo to determine a promising alternative strategy for future bone tissue engineering.

Materials and methods Synthesis and characterization of CS/nHA/TBC scaffolds TBC was synthesized as previously reported.27 nHA coatings were prepared by the acknowledged sol–gel dip-coating method. Briefly, Ca(NO3)2·4H2O (Sigma-Aldrich, Italy) was dissolved and stirred in distilled water at room temperature to obtain the Ca precursor. Then, (NH4)2·HPO4 solution (Sigma-Aldrich, Italy) was added by dropwise to form CaP ultrasonic dispersed solutions with a constant Ca/P molar ratio at 5/3. NH4OH was simultaneously added dropwise to control the pH approximately at 10.

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Subsequently, CaP solutions were put in an incubator to obtain dipping sols. Prepared TBC substrates were immersed into dipping sols and slowly withdrawn, then dried and sintered at 700 °C. Such dipping–drying–sintering process was repeated thrice to ensure coatings deposited adequately. Afterwards, chitosan (CS) was dissolved in the acetic acid solution at a concentration of 3% (w/v), meanwhile polyethylene glycol was added with centrifugation to make the chitosan solution homogeneous without bubbles. Finally, the obtained chitosan solution was irrigated dropwise to the prepared nHA/TBC scaffolds, then being freeze-dried to achieve the CS/nHA/TBC scaffolds (denoted as CH/TBC throughout this manuscript). CH/TBC scaffolds were randomly selected and characterized by field scanning electron microscopy (SEM, Quanta 200, Holland) to investigate the surface morphology. The samples were sprayed with a golden layer about 20 nm thick , then characterized at 20 keV at various magnifications. X-ray spectroscopy (EDS) was applied to quantify the energy-dispersive atomic concentrations of elements in the samples. For statistical analysis, each sample was investigated by five random areas to obtain the mean and standard deviation for each element calculated. PTHdP loading on CH/TBC scaffolds The PTHdP peptide was synthesized by FMOC/tBu solid-phase method as noted above. 10 or 100 ug PTHdP was dissolved in 1mL phosphate buffered saline (PBS) solution respectively at 0 ºC with gentle rotation. Each CH/TBC scaffold (10 mm × 2 mm, diameter × height) was soaked in the solution for 12 h and subsequently washed with PBS. The PTHdP/CH/TBC scaffolds were refrigerated at -20 °C overnight and

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then placed in a freeze-dryer to be frozen at -55 ºC for 48 h. Finally, all materials were sterilized and frozen at -20 °C until used. Loading efficiency of PTHdP was quantified by measuring the amount of PTHdP left in the PBS solution since the initial amount of PTHdP added to the solution was predetermined. The concentration of PTHdP left in PBS solution was measured by a PTH(1–34) ELISA kit (Santa Cruz) using the manufacturer’s instructions. The optical density read at 450 nm by a microtiter plate reader was used to calculate the PTHdP concentrations. The method to quantify loading efficiency of Bovine serum albumin (BSA) was substantially identical to the PTHdP, which was just performed by a BSA protein assay kit (Pierce, Rockford, IL) instead. All samples were assayed in triplicate. In vitro release study The PTHdP/CH/TBC samples were dipped in 5 mL PBS solution and kept in a shaking incubator (40 rpm) at 37 ºC for a period of 30 days. At designated time intervals, the release solution was collected by centrifugation and then completely replaced with equal amount of fresh PBS medium. The concentration of PTHdP in the PBS solution was measured by the PTH(1–34) ELISA kit noted above. Absorbance read at 450 nm by a microtiter plate reader was used to calculate the PTHdP concentrations with a standard curve. Similarly, In vitro release profiles of BSA were investigated by a BSA protein assay kit. All samples were assayed in triplicate. In vitro cell culture MC3T3-E1 cells were purchased from China Center for Type Culture Collection (Wuhan University) and cultured in the basic medium α (αMEM, Gibco) containing

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1% penicillin streptomycin (P/S, Gibco) and 10% fetal bovine serum under 37 ºC, 5% CO2 and 95% relative humidity. To perform osteogenic differentiation researches, 50 µM ascorbic acid, 100 nM dexamethasone and 10 mM β-glycerophosphate were respectively supplemented to the MC3T3-E1 culture medium. MC3T3-E1 cells were seeded at a density of 5 × 104 cells per well in 24-well plates and the culture medium was changed every other day for the in vitro experiments. In vitro bioactivity of released PTHdP To investigate the bioactivity of released PTHdP from CH/TBC scaffolds, both intermittent and continuous exposure of PTHdP in vitro were performed. MC3T3-E1 cells were prior cultured for 16 h and then divided into five groups: control (blank), CH/TBC, PTHdP-10/CH/TBC, PTHdP-100/CH/TBC, and 100 ng/mL PTHdP group. For the intermittent exposure experiments, extracts obtained by immersing each sample in the αMEM medium for every first 6 h in 48-h incubation cycle were added to MC3T3-E1 cells culture at which time the medium was replaced. Thus, MC3T3-E1 cells were exposed to PTHdP for the first 6 h of each 48-h incubation cycle, and then cultured in the absence of PTHdP during the subsequent time.33 The PTHdP directly added to cell culture at a concentration of 100 ng/mL was used as positive control.25 For the continuous exposure experiments, MC3T3-E1 cells were seeded at a density of 5 × 104 cells ml−1 on each sample (2 mm × 10 mm, height × diameter), cultured continuously and finally harvested at the designated time to be tested. The cell viability of MC3T3-E1 cells grown in each group was evaluated by the live/dead staining approach cultured for 1 day. Briefly, the culture medium was

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removed and washed with PBS thrice. Then, a mixture of propidium iodide (2 × 10-3 mmol/L) and calcein-AM (2 × 10-3 mmol/L) (Sigma) in 2.0 mL PBS was applied to make cells stained in an incubator for 15 min and subsequently washed with PBS. The cell growth profile was observed by confocal laser scanning microscopy. The intracellular cAMP content in MC3T3-E1 cells was measured by a cAMP ELISA kit (Endogen/Pierce, Rockford, IL) using the manufacturer’s instructions. After 6 h of incubation, the cells in each group were lysed directly in the medium by adding 0.1 N HCl and 0.5 mM isobutylmethylxanthine to protect the produced cAMP. The cAMP measurements were also normalized by the total amount of DNA by a determined Hoechst assay.34 Briefly, lysates and serial dilutions of calf thymus DNA were reacted with the Hoechst 33258 (0.5 mg/mL, Sigma) for 10 min kept in dark before measuring fluorescence. Measurements were corrected according to basic cAMP/DNA levels in control (blank) cultures. Considering the differences about the amounts of PTHdP loaded in each scaffold, bioactivity was expressed as units (nmol cAMP/mg DNA) per mg of peptide. The cell proliferation profile was investigated by MTT assay. After incubated for 1, 3, and 7 days, the cell cultures were completely removed and washed with PBS twice at the designated time. MTT (100 µL, 5 mg/mL) was added to each specimen and incubated for another 4 h at 37 °C. The mixed solution was removed and dimethyl sulfoxide (300 µL) was added to dissolve the product. The absorbance of solutions read at 490 nm by a microplate reader (Thermo lab systems, America) was used to calculate the cell amounts.

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The alkaline phosphatase (ALP) activity was evaluated after cultured for 7 and 14 days. Culture medium was removed and washed with PBS thrice at the designated time, then lysed in 0.2% Triton X-100. Cell suspension was sonicated for 1 min and then centrifuged for 10 min at 4 °C. Aliquots of supernatants were subjected to the working solution according to the manufacturer's protocol (Beyotime). The absorbance was recorded using a microplate reader at 405 nm. Data were expressed as µm produced p-nitrophenol per min per mg protein and normalized to total amounts of intracellular protein measured by the BCA assay kit. Additionally, ALP and Alizarin Red staining were carried out after cultured for 14 days as previously described.35 The expression level of runt-related transcription factor 2 (Runx2) was measured by the qRT-PCR analysis. After cultured for 7 and 14 days, total RNA of harvested MC3T3-E1 cells was extracted by Trizol reagent (Invitrogen Pty Ltd, Australia). Then, 1 µg of RNA of each specimen was completely reversed transcribed into cDNA by PrimeScrip RT reagent kit and the qRT-PCR analysis was performed on the Bio-Rad qRT-PCR system (Bio-Rad, MyiQ™, USA). Relative expression of the Runx2 target genes was normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. All samples were assayed in triplicate. In vivo animal experiments Animal experiments were approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology. Forty-eight New Zealand white rabbits (6 months old, 2.5 ± 0.4 kg)

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purchased from the Animal Experimental Center of Tongji Medical College, Huazhong University of Science and Technology were divided to four groups: control, CH/TBC, PTHdP-10/CH/TBC, and PTHdP-100/CH/TBC group. Each rabbit was anesthetized by an intramuscular injection of xylazine (8 mg/kg) and sodium pentobarbital (25 mg/kg). Then, the foreleg was sterilized by povidone–iodine and the mid-diaphysis of the radius was exposed through a longitudinal extensile incision. A burr drill was applied to make the bilateral 15 mm critical size radial defects.36 The sterilized scaffolds were subsequently implanted, then soft tissues and skin were closed. Radiographic examination Rabbits were anesthetized absolutely by an intramuscular injection of xylazine (8 mg/kg) and sodium pentobarbital (25 mg/kg) at 6 and 12 weeks postsurgery, then examined using X ray and 3D-CT reconstruction analysis (GE Lightspeed Ultra 16, Milwaukee, WI). New bone formation was evaluated by the gray scale according to the modified scoring system,36,37 in which the connection of broken ends was determined by evaluating the increased density shadow radiographically. In addition, the connecting degree of new bone callus in defect sites was further determined. In each group mean grades were calculated to determine the bone regeneration profile. Histological examination At 6 and 12 weeks postsurgery, rabbits were sacrificed to obtain the excised radial specimens along with the adjacent ulna. Then, specimens were fixed in 10% formaldehyde and decalcified in 10% ethylene diamine tetraacetic acid .

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Blinded histomorphometrical analysis of the bone area ratio (BAR) (including peri-implant and inside the implants) and residual material area (RMA) were performed according to a modified scoring system.38 Briefly, after complete decalcification, samples were hemisectioned through the center of the defect site, creating two separate samples for each implant. A series of 5 um thickness sections was made axially (parallel to the radius) through one implant sample to evaluate the peri-implant bone formation and transversely (vertical to the radius) through the second implant sample to evaluate guided bone regeneration inside implants. Sections were then stained with hematoxylin-eosin (HE). At specific implantation time, eight pieces of histological sections in each sample were randomly selected. At least eight images were randomly obtained in one section under light microscope at 40× magnification. BAR was expressed as the percentage of newly formed bone area in the available pore space and RMA was expressed as the percentage of scaffold area in the total defect area using image analytical software Image-ProPlus (Media Cybernetics, USA). Immunohistochemical analysis of RANKL expression Immunohistochemical analysis was performed by the immunoperoxidase method using the biotinylated antibodies against RANKL (Santa Cruz Biotechnology). Briefly, sections were washed with PBS containing 0.3% Tween 20 for 1 h and incubated with anti-RANKL antibodies, then followed by horseradish peroxidase-labeled secondary antibody (DAB, Dako) according to the manufacturer’s instructions. As a negative control, primary antibody was omitted and the sections were incubated with 1% PBS

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to assess background staining while the lower cortex of the radius was used as a positive control to check the effectiveness of the antibodies. The quantification of RANKL expression was performed by analyzing the digital images in an optical microscopy by a calibrated examiner (Leica microsystem GmbH, Wetzlar, Germany) at 200× magnification. To avoid an edge amplification frequently occured in the immunohistochemical staining, quantification of RANKL expression was evaluated in the central region of the sections by an acknowledged quantitative analysis based on scores (hyperpositive +++, super-positive ++, positive +, and negative-) according to the published studies.39 Statistical analysis ANOVA was used to test for statistical significance with GraphPad Prism 6 (GraphPad Software, USA). All quantitative data were presented as arithmetic means ± standard deviation. Student’s t-test was applied to determine the statistical significance between all the groups. A value of p < 0.05 was considered statistically significant.

Results and discussions Characterization of CH/TBC scaffolds As the SEM results revealed, the nHA/TBC scaffolds maintained the constant three-dimensional trabecular structure with interconnected pores ranging from 200 to 850 µm. Meanwhile, an increased surface roughness due to the HA layer deposition was observed (Figure 1A). With incorporation of chitosan, cross-linked net structured

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chitosan was observed spreading throughout coated scaffolds surface or between the hole wall of scaffolds (Figure 1B, C). Nevertheless, the macrostructure of CH/TBC remained basically identical to the nHA/TBC. For higher magnifications observation at 5000×, the CH/TBC surface was found completely coated with a dense and uniform apatite layer (Figure 1D). Furthermore, the deposited apatite crystals revealed a hierarchical nanoscale with grain size less than 100 nm (Figure 1E). EDS analysis of scaffolds surface exhibited major peaks for Ca, P and O and all the obtained results of Ca/P ratios were corresponding to the HA crystal (Ca/P=1.67), indicating a successful deposition of nHA layer on substrates surface. To realize an effective application in bone tissue engineering, it is preferable to develop a bone graft biomaterial that can serve as not only a factor delivery vehicle but also a three-dimensional porous scaffold. In the present study, TBC was selected for its excellent osteoconductive capacity and biocompatibility, dependent on the inherent trabecular structure with an organized crystal of bone minerals.29,30 With incorporation of nHA coating and chitosan on TBC surface, few impacts were observed on the macrostructure. CH/TBC remained a constant three-dimensional porous structure with optimum pore size for bone ingrowth at a range of 100-800 µm.40 Besides, an increased microsurface roughness with deposition of hierarchical nanotopography HA coating was detected, which could probably resulted in greater surface areas and enhanced surface–protein interactions on CH/TBC scaffolds.41,42 The extensive distribution of chitosan inside the materials was considered beneficial to produce a hydrophilic surface for enhanced cellular responses and promote the drug

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loading capability.43 These outcomes suggested it considerable to apply the novel CH/TBC as a desired drug delivery scaffold in subsequent experiments.

Figure 1. SEM and EDS analysis of nHA-coated TBC and CH/TBC scaffolds: (A) nHA/TBC (35×), (B) CH/TBC (50×), (C) chitosan structure (500×), (D) CH/TBC (5,000×), (E) CH/TBC (50,000×), (F) EDS spectra of CH/TBC surface. In vitro PTHdP loading and release kinetics

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To investigate the drug delivery capability of CH/TBC scaffolds, BSA was applied as a control factor to be loaded and released. As shown in Figure 2A, the loading efficiency of PTHdP and BSA at a concentration of 10 ug/mL on CH/TBC was measured 96.5 ± 2.3% and 53.4 ± 6.9%, respectively. For higher peptide concentration at 100 ug/mL, the loading efficiency of PTHdP and BSA was correspondingly changed to 85.1 ± 4.8% and 58.2 ± 5.2%. The loading efficiency of PTHdP was significantly higher than that of BSA at each condition. Despite the loading efficiency decreased by approximately 11% with a raised peptide concentration, the total amount of PTHdP loaded into CH/TBC was much higher.

Figure 2. In vitro loading efficiency and release kinetics of PTHdP and BSA from CH/TBC scaffolds in PBS solution. *Significant statistical difference between groups (p < 0.05). The in vitro release profile of BSA and PTHdP was performed in both concentrations ( 10 and 100 ug/mL), respectively. As release profiles between different concentrations were observed basically identical, results were thus comprehensively analysed and integrated to be shown in Figure 2B. The in vitro release of BSA was in a burst manner and its total amount was almost released within

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5 days. In comparison, the release of PTHdP exhibited a biphasic release pattern, characterized by an initial rapid release approximately 58.1% within 5 days, then followed by a sustained release up to 30 days. During the whole period, the final amount of released PTHdP was about 65.6%. The drug loading efficiency was mainly mediated by the interaction between proteins and scaffolds. In this study, the hierarchical nano-structured HA coating on CH/TBC surface could provide greater surface areas for drug loading. Additionally, the incorporation of chitosan could further promote drug immobilization ability, in particular for those water soluble polyanionic species.31 As the isoelectric points (IEP) of Asp and phosphorylated Ser are both relatively low, PTHdP with these functional groups will possess an anionic charge in the PBS condition, thus resulting in some immobilization on chitosan by electrostatic interaction. Nevertheless, the main aspect for a successful immobilization of biomolecules onto scaffolds surfaces is determined essentially by the inherent properties of the biomolecule itself. As the repetitive Asp sequence is acknowledged for high affinity to bone mineral and has been extensively applied in bone-targeting drug delivery system,44,45 PTHdP could probably be driven and immobilized on CH/TBC by a high affinity bound to the nHA coating through its reactive repetitive Asp functional groups. Consequently, compared to BSA, which possessed no reactive functional groups and could just be immobilized by random physisorption or electrostatic interaction due to the low IEP, PTHdP revealed a significantly higher loading capability in each condition. Additionally, with the concentration of PTHdP raising from 10 to 100 µg/mL, a decreased loading efficiency

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was detected. When the concentration was 10 µg/ml, there were conceivably sufficient scaffolds surface areas and abundant chitosan/nHA coating for peptide immobilization. In comparison, the surface area and chitosan/nHA coating of CH/TBC could be relatively decreased as the concentration increased 10-folds to 100 µg/mL. Furthermore, due to the anionic charge of PTHdP, there should be certain electrostatic repulsion existing between the PTHdP molecules. Consequently, the more PTHdP was immobilized on CH/TBC, the stronger a electrostatic repulsion would result. Since the total amount of PTHdP at 100 µg/ml was much higher than 10 µg/ml, some of the PTHdP (100 µg/ml) could be restricted access to CH/TBC scaffolds and thus loading efficacy dropped apparently. In the present study, BSA was immobilized mainly by random physisorption or electrostatic interaction. Whereas, previous studies have showed both these bonds were not strong due to the diffusion of peptide or dissolution of chitosan, and thus resulted in a burst release within a short time.28,46 Accordingly, the initial burst release of PTHdP could also be related to the diffusion of peptide absorbed on CH/TBC surface or chitosan disintegration. Despite the initial burst release, the release of PTHdP transformed to a controlled and sustained manner in the subsequent time. These results were consistent with previous researches, which demonstrated Asp-nHA conjunction could certainly account for a prolonged peptide retention and ensure a controlled release of peptide through the gradual hydrolysis of Asp-nHA bonds.27,47 In vitro bioactivity of released PTHdP

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As shown in Figure 3A, cells spontaneously attached in each group with no obvious dead cells (stained red) detected for 1 day culture, indicating all scaffolds were biocompatible and no cellular toxicity was caused by the initial burst release of PTHdP. Additionally, the distribution of cells growing on scaffolds demonstrated the three-dimensional porous structure of CH/TBC. As shown in Figure 3B, ALP activity was relatively low in control and CH/TBC groups, indicated by few black particles deposited in the cytoplasm of MC3T3-E1 cells. In contrast, significantly higher ALP activity was observed in the subsequent groups with increasing deposition of black particles. Particularly, the amounts of black particles per unit surface area in PTHdP-100/CH/TBC and PTHdP groups were numerous and significantly higher than the other groups. As shown in Figure 3C, the number of red mineralization nodules per unit surface area of PTHdP-100/CH/TBC and PTHdP groups was much higher than that of control, CH/TBC, and PTHdP-10/CH/TBC groups.

Figure 3. The proliferation and differentiation profile of MC3T3-E1 cells cultured in each condition. (A) Cell viability of MC3T3-E1 cells cultured in each sample for 1

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day, (B) ALP staining and (C) Alizarin Red staining of MC3T3-E1 cells after cultured with intermittent PTHdP exposure for 14 days .Scale bar = 100 µm. Biological effects of released PTHdP with intermittent exposure on MC3T3-E1 cells are shown in Figure 4. As shown in Figure 4A, the intracellular cAMP levels of PTHdP-10/CH/TBC, PTHdP-100/CH/TBC, and PTHdP groups were significantly higher than that of control and CH/TBC groups, but no significant difference was detected between CH/TBC and control groups. As shown in Figure 4B, Proliferation rates increased in each condition with prolonged culture time. But proliferation levels appeared indistinguishable among all the groups at each designated time. As shown in Figure 4C, the PTHdP-10/CH/TBC, PTHdP-100/CH/TBC, and PTHdP groups exhibited a significantly higher ALP activity than control and CH/TBC groups at each time. Furthermore, the ALP activity of PTHdP-100/CH/TBC and PTHdP groups were significantly higher than that of PTHdP-10/CH/TBC groups after culture for 14 days. No significant difference was detected between CH/TBC and control groups at each time. A similar profile was detected for the Runx2 gene expression after 7 and 14 days culture (Figure 4D).

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Figure 4. Intracellular cAMP level (A), proliferation (B), ALP activity (C), and osteogenic gene Runx2 expression (D) of MC3T3-E1 cells cultured with intermittent exposure to PTHdP in each condition.*Significant statistical difference compared to control (p < 0.05); #Significant statistical difference compared to CH/TBC (p < 0.05); ^Significant statistical difference compared to PTHdP-10/CH/TBC (p < 0.05). Biological effects of released PTHdP with continuous exposure on MC3T3-E1 cells are shown in Figure 5. As revealed in Figure 5A, the expression levels of intracellular cAMP in each condition revealed basically identical to Figure 4A as mentioned above. With prolonged culture time, proliferation rates increased in all conditions as shown in Figure 5B. At day 1, proliferation levels appeared indistinguishable among all the groups. At later times cultured for 3 and 7 days, the CH/TBC groups revealed a significantly higher proliferation level than the other groups, and the proliferation 21



level in PTHdP-10/CH/TBC, PTHdP-100/CH/TBC and PTHdP groups revealed significantly lower compared to control and CH/TBC groups. As shown in Figure 5C, CH/TBC groups revealed a significantly higher ALP level than other groups at each designated time. But ALP activity in PTHdP-10/CH/TBC; PTHdP-100/CH/TBC and PTHdP groups was significantly lower than control and CH/TBC groups at each time. Additionally, both PTHdP-100/CH/TBC and PTHdP groups exhibited a significantly lower ALP activity than the PTHdP-10/CH/TBC group after culture for 14 days. The Runx2 gene expression profile (Figure 5D) was observed similar to the ALP activity mentioned above.

Figure 5. Intracellular cAMP level (A), proliferation (B), ALP activity (C), and osteogenic gene Runx2 expression (D) of MC3T3-E1 cells cultured with continuous exposure to PTHdP in each condition.*Significant statistical difference compared to 22


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control (p < 0.05); #Significant statistical difference compared to CH/TBC (p < 0.05); ^Significant statistical difference compared to PTHdP-10/CH/TBC (p < 0.05). In principle the successful binding of proteins or biomolecules to delivery vehicle is simple, but as an effective therapeutic strategy the key procedure is ensuring the bioactivity of delivered drug for the subsequent interaction with target cell. In the present study, MC3T3-E1 osteoblasts revealed a significantly enhanced ALP activity, mineralization ability, and Runx2 expression than control and CH/TBC groups when exposed intermittently to released PTHdP, yet these positive effects were found dramatically decreased and even reversed to suppression of cell proliferation and differentiation as continuous PTHdP exposure was applied. These divergent results were considered fundamentally attributed to the specific effects of PTH on osteoblasts in vitro, depending on the mode of PTH treatment and drug concentration. As previous researches reported, intermittent/short-term exposure of PTH at adequate doses significantly supported osteogenesis actions in vitro, but with continuous exposure these osteogenesis actions could be strongly inhibited and the inhibitory effect increased with elevated PTH concentrations at a dose-dependent manner.33,48 In the present study, a continuous and high-dose exposure of released PTHdP could inevitably exist, especially for the first several culture days because MC3T3-E1 cells were cultured directly on each sample.and thus subjected to the initial rapid release of PTHdP from CH/TBC scaffolds. Consequently, a strong inhibitory effect of continuous PTHdP exposure was detected in each condition and the inhibition level significantly

decreased

in

PTHdP-10/CH/TBC

groups

23


as

compared

to


PTHdP-100/CH/TBC and PTHdP groups after culture for 14 days mainly due to a relative lower amount of released PTHdP. In principle, PTH exerts its pleiotropic effects on osteoblasts by initiating different signaling cascades ( cAMP/PKA, PLC/PKC, MAPKs and et al) and activates the subsequent gene and transcription factors expression.49 Thereinto, major effects of PTH on osteoblasts including inhibition of apoptosis and stimulation of differentiation were dependent on the cAMP/PKA signaling pathway, which could activate certain transcription factors and gene expression such as cAMP response element binding protein (CREB), activator protein-1 (AP-1) transcription factors and Runx2.50,51 In the present study, whether exposed intermittently or continuously, the released PTHdP could consistently activate a high intracellular cAMP expression in MC3T3-E1 cells and exert a significant effect to promote or inhibit osteogenesis actions. Compared to the positive control group (100 ng/mL PTHdP),25 the PTHdP released from PTHdP-100/CH/TBC groups revealed similar biological effects in each condition. These results supported the remained bioactivity of released PTHdP after being immobilized on CH/TBC scaffolds. Additionally, as MC3T3-E1 cells were directly cultured on each sample for the continuous exposure investigation, CH/TBC groups revealed significantly enhanced osteogenesis levels than the other groups, indicating an excellent biocompatibility and osteoinduction capability of pure CH/TBC scaffolds for its nanotopography HA coating and chitosan incorporation.41,42 Actually, unlike the situation in vivo, effects of PTH on osteoblasts in vitro are indeed complicated and variable due to different culture conditions, source of

24


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osteoblastic cells, cell differentiation stage and even cell density.48,52 Since our in vitro conditions could not reproduce the actual architectural and cellular complexity of bone tissues, the present in vitro results appeared insufficient to conclude the effects of released PTHdP in vivo. Nevertheless, present results could reliably demonstrate the retained bioactivity of immobilized PTHdP and provide valuable insights into the inhibitory effects associated with uncontrolled rapid PTHdP release. To further explore the mechanisms concerning the pleiotropic effects of PTHdP, more complicated conditions such as the osteoblasts/osteoclasts co-culture system may be preferred in the future research. General procedure of implantation All materials were successfully implanted to the critical deficient areas as shown in Figure 6. No apparent signs of infection were detected in implant areas during the 12 weeks postsurgery.

Figure 6. Segmental critical size rabbit radial defects about 1.5 cm were created by a surgery procedure and filled with prepared implants. Radiographic examination

25



X ray scanning performed at 6 and 12 weeks postsurgery is shown in Figure 7A. At 6 week postsurgery, a distinct segmental radial defect was present and no calluses formation was observed in the control group. In comparison, the defective sites were well occupied by implants and the boundary between implants and radius appeared obscure, but no significant bone calluses were detected in the CH/TBC group. The boundary became more obscure and plenty peri-implant bone formation were observed in PTHdP-10/CH/TBC and PTHdP-100/CH/TBC groups. With prolonged implantation period to 12 weeks, the defect area remained clear in the control group. Implants were integrated with host bone and the calluses around implants increased in CH/TBC groups. Contrastively, the boundary almost disappeared and bone-implants were closely united with shaped calluses formation in PTHdP-10/CH/TBC and PTHdP-100/CH/TBC groups. Particularly, bony calluses formation indicated by increased radiographic density was significantly higher in the PTHdP-100/CH/TBC group than the other groups. The 3D-CT reconstruction scanning ( Figure 7B) further demonstrated the connecting degree of new bone calluses in the defect site and revealed a similar profile to X ray.

26


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Figure 7. X ray scanning (A) , 3D-CT reconstruction scanning (B) and radiographic grades (C) in each group at 6 and 12 weeks post-implantation. *Significant statistical difference compared to control (p < 0.05); #Significant statistical difference compared to

CH/TBC

(p < 0.05); ^Significant

statistical difference compared to

PTHdP-10/CH/TBC (p < 0.05).

27



As shown in Figure 7C, the radiographic grades of CH/TBC, PTHdP-10/CH/TBC, and PTHdP-100/CH/TBC groups were significantly higher than that of control groups at each time. At 6 week postsurgery, the radiographic grades of PTHdP-10/CH/TBC and PTHdP-100/CH/TBC groups were significantly higher than the CH/TBC groups. With prolonged implantation period to 12 weeks, the radiographic grades in the PTHdP-10/CH/TBC group were similar to the CH/TBC group, and the PTHdP-100/CH/TBC group revealed a significantly higher radiographic grade than other groups. As a critical size bone defect model, the defects in animals should not naturally and spontaneously heal. In the present study, the application of 1.5 cm segmental rabbit radial defects was indeed critical size because few newly formed bones were detected in the control group within 12 weeks implantation. Thus, the animal experiment in vivo was rational and efficient to evaluate the bone regeneration ability of PTHdP/CH/TBC composite materials. As the results revealed, pure CH/TBC scaffolds could be naturally integrated with host bone at early implantation time, mainly due to its good biocompatibility and osteoconductive capacity. However, the PTHdP-10/CH/TBC and PTHdP-100/CH/TBC groups revealed significantly higher radiographic grades than the CH/TBC group at 6 week postsurgery. This result indicated the loaded PTHdP peptide could probably induce earlier new bone formation than pure CH/TBC scaffolds. With prolonged implantation time to 12 weeks, PTHdP-100/CH/TBC groups revealed a significantly higher radiographic grade than the other groups, suggesting a higher loaded amount of PTHdP was more

28


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beneficial for prolonged bone defect repair. Due to the highly similar inorganic constituents of CH/TBC scaffolds to nature bone, scaffolds appeared visible for its high attenuation coefficient in particular for the 3D-CT examination, thus resulting in certain difficulty for observation. Therefore, it was essential to apply the histomorphometric analysis to make more accurate evaluation of the osteogenesis profile in each group. Histological and immunohistochemical examination As shown in Figure 8A, peri-implant bone formation profile was analyzed using the longitudinal section. At 6 week post-implantation, there was just a few newly formed bone around the broken ends detected in control groups. Limited new bone formation and numerous peri-implant fibrous tissues were detected in CH/TBC groups. Contrastively, large amounts of newly formed trabecular bones were found closely attached

to

the

margins

of

implants

in

the

PTHdP-10/CH/TBC

and

PTHdP-100/CH/TBC groups. With prolonged implantation period to 12 weeks, increased new bone formation around the broken ends was observed in control groups. In the CH/TBC group, peri-implant newly formed bone exhibited to be remodeled as bridge bony calluses, and a distinct boundary mainly composed of fibrous tissues was present in the callus–implant interface. No obvious bone formation was detected below the calluses. In comparison, bony calluses were integrated with implants with no boundary detected and several newly formed bones were observed growing penetratively from calluses into scaffolds interspace in PTHdP-10/CH/TBC groups. The callus-implant integration was more complete and greater amounts of new bones

29



were observed growing penetratively in the PTHdP-100/CH/TBC group.

Figure 8. Histological evaluation of the bone formation profile with HE staining at 6 and 12 week post-implantation. (A) Peri-implant new bone formation (40×, longitudinal section), (B) Center defect sites new bone formation (40×, cross section). Newly formed bones are stained red, the fiber tissues appear pinky and the residual scaffolds appear white. B indicates bone. F indicates fibrous tissues. The scale bar = 400 µm. As shown in Figure 8B, the bone formation profile in implants interspace was determined with cross-section at the center defect site. At 6 week post-implantation, there was nothing but irregular fibrous tissues observed in the control group. In the CH/TBC group, implants interspace was mostly occupied by abundant fibrous tissues but little new bone formation was observed. Contrastively, several newly formed 30


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osseous tissues were detected in the PTHdP-10/CH/TBC group although the interspace of implants remained filled predominantly with fibrous tissues. Furthermore, the quantity of newly formed osseous tissues was greater in PTHdP-100/CH/TBC implants interspace. With prolonged implantation period to 12 weeks, compared to minor osseous tissues in the control group, quite a few newly formed bones were observed in CH/TBC groups. The quantity of newly formed bone gradually increased in the subsequent PTHdP-10/CH/TBC and PTHdP-100/CH/TBC groups, along with the reduction of fibrous tissues and scaffolds trabeculae. Especially, as the results showed, defective sites were occupied almost by plenty of confluent

newly

formed

bones

with

few

fibrous

tissues

detected

in

PTHdP-100/CH/TBC groups. Meanwhile, the integrity of scaffolds trabecular structure was significantly decreased, indicating accomplished degradation of CH/TBC scaffolds.

Figure 9. Immunohistochemical staining of RANKL expression at center area of the

31



Page 32 of 48

defect site after 6 and 12 weeks post-implantation. Arrows point to positive cells with the brown precipitate. The scale bar = 100 µm. As shown in Figure 9, few RANKL-positive cells were detected in the CH/TBC group at 6 weeks post-implantation. In comparison, RANKL-positive cells were significantly more numerous in PTHdP-10/CH/TBC and PTHdP-100/CH/TBC groups. With prolonged implantation time to 12 weeks, the numbers of RANKL-positive cells in CH/TBC and PTHdP-10/CH/TBC groups were both relatively low. In the PTHdP-100/CH/TBC group, RANKL-positive cells remained numerous and were significantly higher than other groups. As revealed in Figure 10A, the bone area ratio (BAR) of CH/TBC, PTHdP-10/CH/TBC, and PTHdP-100/CH/TBC groups was significantly higher than control

groups

at

each

time.

The

BAR

of

PTHdP-10/CH/TBC

and

PTHdP-100/CH/TBC groups was also significantly higher than CH/TBC groups at both 6 and 12 weeks. Furthermore, the PTHdP-100/CH/TBC group revealed significantly higher BAR than the PTHdP-10/CH/TBC group at each time. As shown in Figure 10B, residual material area (RMA) was not significantly different among all the groups at 6 weeks. For prolonged implantation period to 12 weeks, RMA tinily changed in CH/TBC groups. In comparison, the RMA of PTHdP-10/CH/TBC and PTHdP-100/CH/TBC groups obviously decreased at 12 weeks and was significantly lower than that of CH/TBC groups. Particularly, the RMA of PTHdP-100/CH/TBC groups was also significantly lower than that of PTHdP-10/CH/TBC groups. As shown in Figure 10C, both the PTHdP-10/CH/TBC and PTHdP-100/CH/TBC group

32


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revealed a significantly higher RANKL expression level than the CH/TBC group at 6 weeks. With prolonged implantation time to 12 weeks, the RANKL expression level between CH/TBC and PTHdP-10/CH/TBC groups revealed not significantly different and both of them were significantly lower compared to the PTHdP-100/CH/TBC group.

Figure 10. Histological analysis of bone area ratio (A), residual material area (B), and quantification of RANKL expression (C) for each group after implanted for 6 and 12 weeks. *Significant statistical difference compared to control (p < 0.05); #Significant statistical difference compared to CH/TBC (p < 0.05); ^Significant statistical difference compared to PTHdP-10/CH/TBC (p < 0.05). The in vivo results further demonstrated the excellent osteoconductive ability and biocompatibility of CH/TBC for the desired bone-implant integration and subsequent new bone ingrowth. This excellent property was considered substantially attributed to its three-dimensional natural trabecular structure with enhanced surface bioactivity for the deposition of nHA coating and chitosan incorporation. With PTHdP loaded on implants, the PTHdP-10/CH/TBC and PTHdP-100/CH/TBC groups both revealed a remarkably stronger ability to stimulate peri-implant new bone formation and induce guided bone regeneration into implants interspace for the implantation periods used.

33



These results were consistent with previous researches, which demonstrated enhanced osseointegration and bone defect regeneration was obtained by local application of PTH.21,23,24 In the present study, as PTHdP/CH/TBC implants were applied in vivo, the local concentration of released PTHdP for the first few days could be conceivably high due to the initial uncontrolled and rapid PTHdP release. As observed in vitro, this rapid release resulted in significant inhibitory effects on osteogenesis actions for MC3T3-E1 cells culture. Whereas, unlike the situation in vitro, the in vivo condition was indeed at relatively low extracellular pH and surrounded by hot tissues, thus the initial rapid release in vivo was expected to be accelerated and continued no more than 5 days according to the in vitro results.53 Furthermore, previous researches have demonstrated the lasting duration and half-life of bioactive PTH(1-34) were really short in vivo,18,19 suggesting the bioactivity of released PTHdP could just exist transitorily and then got eliminated soon by the in vivo condition. Therefore, given the present prolonged bone repair period up to 12 weeks, the potential catabolic effects caused by the initial rapid release in first few days appeared not dominant. Actually, after the rapid release, the remained PTHdP on CH/TBC was almost immobilized by the Asp-nHA conjunction, thus resulting in a controlled release of PTHdP during the prolonged implantation period as mentioned prior. Indeed, the Asp-nHA conjunction of PTHdP was a selective immobilization via its terminal repetitive Asp functional groups bonding to implants. This selective immobilization could ensure an oriented immobilization of PTHdP and attenuate the peptide activity with its active fragments blocked as previously reported.21,54,55 Consequently,

34


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immobilized PTHdP could regain its full bioactivity just after releasing from CH/TBC and thus avoid the possibility for the super-physiological local concentrations that could lead to bone resorption rather than bone formation. Nevertheless, both the PTHdP-10/CH/TBC and PTHdP-100/CH/TBC groups revealed significantly higher RANKL expression levels than CH/TBC groups. Since RANKL is a protein expressed on the osteoblast cell membrane and associated with osteoclastic activation,56 these results indicated the local released PTHdP was able to induce certain catabolic actions by stimulating osteoclasts generation. It is acknowledged that bone mass and turnover are maintained through the tightly coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts.57 As previous researches reported, intermittently administered PTH(1-34) could significantly increase bone turnover to exert its prominent anabolic effects.58,59 Thus, the elevated activation of RANKL was considered corresponding to the increased bone formation and could supply new osteoclasts for the desired bone remodeling during the bone repair process. Snice the increased RANKL could induce catabolic actions, the accelerated degradation of CH/TBC scaffolds occurred after 12 weeks implantation. But compared to the consistently increased new bone formation, the RANKL expression appeared to be limited to a certain level as it changed tinily between 6 and 12 weeks. This phenomenon suggested although the RANKL expression was stimulated concurrently with bone formation, the anabolic actions induced by released PTHdP were more dominant during the bone turnover balance. The RANKL expression in PTHdP-10/CH/TBC groups significantly

35



decreased at 12 weeks compared to 6 weeks, mainly duo to the dramatically decreased amount of released PTHdP which was insufficient to activate RANKL expression. Furthermore, PTHdP-100/CH/TBC groups exhibited significantly higher bone formation ability than PTHdP-10/CH/TBC groups, suggesting higher loaded amount of PTHdP (100 ug/mL) was probably a preferable strategy for a prolonged bone repair period. The sucessful combination of CH/TBC scaffolds with novel PTHdP was considered to be a foundational work that successfully demonstrate the great osteogenesis potential of PTHdP to be locally applied for bone tissue engineering. Nonetheless, the initial burst release caused by part unspecific immobilized PTHdP including random physisorption and electrostatic interaction could probably induce some potential catabolic effects within first few days, thus more desirable delivery devices should be developed and combined with the PTHdP to further investigate its actions by avoiding the potential catabolic effects. Additionally, the in-depth molecular mechanisms about the interaction between released PTHdP and the endogenous bone formation-related cells (MSCs and osteoblasts) and the underlying PTH-responsive signalling to regulate the specific anabolic/catabolic balance require further investigations.

Conclusions A desired delivery scaffold CH/TBC, true bone ceramics incorporated with nHA coating and chitosan, was successfully developed in this study to realize a local

36


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delivery of the novel PTHdP. High loading efficiency with controlled and sustained release of PTHdP from CH/TBC scaffolds was achieved in vitro. The released PTHdP retained its bioactivity and revealed a remarkably stronger ability to stimulate bone regeneration in the critical size rabbit radial defect model. Given its high affinity to bone mineral, local delivery of PTHdP from apatite materials could be a promising alternative for future bone tissue engineering.

Acknowledgments This research was supported by the National Natural Sciences Foundation of China (No: 81301538, 81672158, 81371939), the National Key Research and Development Program of China (2016YFC1100102), the Youth Science and Technology Morning Program of Wuhan (2014072704011256), High-level Personnel Program of Wuhan University (600400002), and Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund (cxpy2016035).

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For Table of Contents Use Only

Bone regeneration induced by local delivery of a modified PTH-derived peptide from nano-hydroxyapatite/chitosan coated true bone ceramics Liang Yang,#,1,2 Jinghuan Huang,#,3 Shuyi Yang,2 Wei Cui,2 Jianping Wang,1 Yinping Zhang,1 Jingfeng Li,*,1, Xiaodong Guo*,2

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Bone regeneration induced by local delivery of a modified PTH

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