Nano-Hydroxyapatite Bone Substitute Functionalized with Bone Active

Subscriber access provided by University of Colorado Boulder

Article

Nano-Hydroxyapatite Bone Substitute Functionalized with Bone Active Molecules for Enhanced Cranial Bone Regeneration Arun Kumar Teotia, Deepak Bushan Raina, Chandan Singh, Neeraj Sinha, Hanna Isaksson, Magnus Tagil, Lars Lidgren, and Ashok Kumar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14782 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 12, 2017

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 free 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 accessible to all readers and 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.

ACS Applied Materials & Interfaces 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 47

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

ACS Applied Materials & Interfaces

Nano-Hydroxyapatite Bone Substitute Functionalized with Bone Active Molecules for Enhanced Cranial Bone Regeneration Arun Kumar Teotia1, Deepak Bushan Raina1,2, Chandan Singh3, Neeraj Sinha3, Hanna Isaksson2,4, Magnus Tägil2, Lars Lidgren2, Ashok Kumar1,* 1

Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur- 208016, India 2

Department of Orthopedics, Clinical Sciences, Lund, Lund University, Lund-221 85, Sweden 3 4

Center for Biomedical Research, SGPGIMS Campus, Lucknow- 226014, India

Department of Biomedical Engineering, Lund University, Lund-221 00, Sweden

*Corresponding Author: Prof. Ashok Kumar, Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur (IITK), Kanpur, UP-208016, India Email: [email protected] Phone: +91-512-2594051

KEYWORDS Bisphosphonates, bone morphogenic proteins, cranial model, nano-hydroxyapatite, osteoinductive, solid state NMR

1 ACS Paragon Plus Environment


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

Abstract The aim of this study was to synthesize and characterize a nano-hydroxyapatite (nHAP) and calcium sulphate bone substitute (NC) for cranioplasty. The NC was functionalized with low concentrations of bone morphogenetic protein-2 (BMP-2) and zoledronic acid (ZA) and characterized both in vitro and in vivo. In vitro studies included MTT, ALP assays and fluorescent staining of Saos-2 (human osteoblasts) and MC3T3-E1 (murine pre-osteoblasts) cells cultured on NC. An in vivo study divided twenty male Wistar rats into four groups: control (defect only), NC, NC+ZA and NC+ZA+rhBMP-2. The materials were implanted in an 8.5 mm critical size defect in the calvarium for 12 weeks. Micro-CT quantitative analysis was carried out in vivo at 8 weeks and ex vivo after 12 weeks. Mineralization was highest in NC+ZA+rhBMP-2 group (13.0 ±2.8 mm3) compared to NC+ZA group (9.0 ±3.2 mm3), NC group (6.4±1.9 mm3), and control group (3.4 ±1.0 mm3) after 12 weeks. Histological and spectroscopic analysis of the defect site provided a qualitative confirmation of neo-bone, which was in agreement with the micro-CT results. In conclusion, NC can be used as a carrier for bioactive molecules and functionalization with rhBMP-2 and ZA in low doses enhances bone regeneration.


Page 2 of 47

Page 3 of 47

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


1.0 Introduction Bone defects in the calvarium can arise due to various reasons such as surgical craniotomy, or due to traumatic events. Cranioplasty is the technique used to repair such defects,1 both for cosmetic reasons and to protect the underlying brain.2 Bone xenografts and allografts3,4 have been used for cranioplasty, but autografts still remains the gold standard.5,4 However, graft harvest with potential donor site morbidity6,7 limits the use. In addition, metal, plastic (methyl methacrylate), ceramic or other support materials are in clinical use.7 Apart from being non-bioactive, the acrylate cements poses challenges with exothermic setting reaction leading to necrosis,8,9 inflammatory reaction, monomer leaching and ischemia.10–12 A synthetic bone graft substitute should in addition to being biocompatible, ideally be both osteoconductive and osteoinductive, and have a mechanical strength similar to that of bone. Hydroxyapatite (calcium phosphate) and calcium sulphate (CS) based materials are used as bone substitutes and bone fillers.13,14 Hydroxyapatite (HAP) is a natural component of bone, has excellent biocompatibility, and a slow turnover of several years. A porous structure is achieved by mixing HAP with CS, with the HAP remaining intact and CS being gradually released within months.15– 18

Bone naturally is a bio-composite of collagen as the organic part19 and nano-hydroxyapatite

(nHAP) as the inorganic part.20 Using nHAP in synthetic grafts with crystals resembling natural bone, the bioactivity is enhanced compared to when using larger size HAP particles21,22 Additionally, use of nHAP has a positive effect on protein adhesion,23 cell adhesion, proliferation24 and integration.25,26 HAP in combination with CS can locally deliver drugs,27,28 antibiotics,29 and bioactive molecules (e.g. bone morphogenetic protein’s (BMP’s)) to the defect site in a controlled manner.30



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

The nHAP-CS ceramic materials are biocompatible, resobable31, osteoconductive and have good mechanical properties, but these materials have limited osteoinductive potential.32 CSHAP based materials when implanted in vivo are reported to show no adverse effects or physiological changes at the local implant site.16 The osteoinductive properties of nHAP-CS materials can be enhanced by incorporating suitable anabolic agents (e.g. BMPs),33–35 and bioactive molecules e.g. gelatin.27 BMPs alone or in combination with ceramic and polymeric materials have been used to replace autografts to repair craniofacial, spinal fusion and non-union defects in clinics, without evidence of being superior to autograft.36–38 It can be attributed to the fact that BMPs not only affect osteoblast activity but also influences the osteoclast activity.39,40 Bisphosphonates specifically decrease the osteoclast activity by inducing apoptosis via farnesyl pyrophosphate synthase (FPPS) blockage. If the osteoclast mediated bone resorption is restricted, a net increase in bone formation is achieved. Most of current clinically used bisphosphonates are administered parenterally or orally. However, drawbacks such as osteonecrosis and renal toxicity at high systemic levels41 pave a way for local delivery. In a previous study, we showed that zoledronic acid (ZA), positively influenced bone formation at very low molecular concentrations.27 BMPs and bisphosphonates have been delivered using collagen gels, inorganic-organic composites, polymer matrices etc. but most of them have issues related to controlled release, stability and administration.42–44 A HAP-CS matrix overcomes many of these issues, BMPs interact with HAP similar to how it interacts with native HAP in bone,45 providing a controlled release. Bisphosphonates with a high affinity towards calcium ions bind to HAP,46 and decreases the osteoclast mediated resorption. CS slowly leaches creating a porous matrix for cells to infiltrate and differentiate into osteoblasts.27


Page 4 of 47

Page 5 of 47

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


In this study, we aimed to synthesize nHAP-CS cement (NC) with the hypothesis that nHAP will mimic the nanotopography of bone resulting in better integration of nHAP with mineralizing new bone, BMP and ZA incorporation was done to provide osteoinductive properties to the NC. Moreover, physicochemical analysis was performed to evaluate material properties and cell culture studies were performed to establish the biocompatibility of the NC. The final aim of the study was to investigate if the developed material can act as a carrier for rhBMP-2 and ZA, to enhance the regeneration of the bone and to evaluate bone formation in an in vivo rat critical cranioplasty model.

2.0 Materials and Methods Materials Calcium sulphate dihydrate (CSD), calcium nitrate and di-ammonium hydrogen phosphate were purchased form S.D. Fine-Chem, India. rhBMP-2 was purchased from Medtronic (Medtronic, Infuse® Bone Graft), zoledronic acid (ZA) (Novartis) was purchased from local pharmacy. Dulbecco’s modified Eagle’s medium (DMEM), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), β-glycerophosphate, ascorbic acid, SIGMAFASTTM p-Nitrophenyl phosphate (pNpp), Trypsin-EDTA, Masson’s trichrome staining kit, Alizarin RedS (alizarin) stain were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) (US origin), phenol red free α-MEM were purchased from Gibco® (MA, USA). Antibiotic cocktail (penicillin and streptomycin) was purchased from Hi-Media (India). Minimum Essential Medium Eagle-alpha modification (α-MEM) was purchased from Thermo scientific (MA, USA). All other chemicals used were of analytical grade. Wistar rats (males)



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

weighing between 250 and 300 g were sourced from Indian Institute of Toxicology Research, Lucknow, India. 2.1 Synthesis of calcium deficient nano-hydroxyapatite (nHAP) nHAP was synthesized using wet chemical method. Briefly, an alkaline solution (pH 10.0) of calcium nitrate tetrahydrate Ca(NO3)2.4H2O (0.96 M) maintained at 90-100 °C under constant stirring was mixed with aqueous solution of di-ammonium hydrogen ortho-phosphate (NH4)2HPO4 (0.6 M) at a controlled rate. The pH of the system was constantly monitored and maintained at pH 10.0 by adding NH4OH solution. The nHAP precipitated out of the solution as white crystals. After completion of the reaction, the crystals were maintained in the mother liquor for maturation for 48 h at room temperature in alkaline conditions. After maturation the crystals were filtered out of the solution and washed thoroughly with milli-Q® Type-I water (DIH2O). The crystals were then dried at 120 °C. 2.2 Sintering of nHAP The synthesized nHAP was subjected to thermal treatment to enhance its crystallinity, density and phase purity. To investigate the effects of temperature and duration of thermal treatment on mentioned properties of nHAP, the material was subjected to different temperature conditions ranging from 500 °C upto 1000 °C with stepwise increment of 100 °C, with hold time from 1 hour to 4 h at a specific temperature. The optimal thermal treatment conditions and parameters were evaluated to get highly crystalline, high density material with pure phase, without calcium phosphate degradation products which gets generated on high temperature thermal treatment of HAP. 2.3 Synthesis of calcium sulphate α-hemihydrate (α-CSH) 6 ACS Paragon Plus Environment

Page 6 of 47

Page 7 of 47

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


Calcium sulphate α-hemihydrate (α-CSH) (CaSO4.0.5H2O) was synthesized from calcium sulphate di-hydrate (CSD) (CaSO4.2H2O) using a modification of the autoclave method. Briefly the CSD powder was dispersed in aqueous solution of H2SO4 (0.1 M). The dispersion was stirred for one hour followed by filtration. The crystals were then washed with Milli-Q® water until neutral pH. The crystals were then dispersed in DI-H2O at CSD: water ratio of 1:2 and the mixture was then placed in an autoclave at 121-125 °C for 4 h for conversion of CSD to α-CSH. After completion of the autoclave cycle α-CSH was removed and filtered while maintaining at 100 °C. The crystals were given wash 3× with boiling DI-H2O and dried in hot air oven at 95 °C for 12 h. 2.4 Synthesis of nHAP-CS bone substitute The synthesized nHAP and α-CSH were grinded to generate free flowing powder. 40% nHAP and 60% α-CSH were mixed to generate biphasic nano-cement (NC). To each gram of NC 600 µL of sterile saline was added and mixed thoroughly, which sets into a solid mass within 15 min. 2.5 Physicochemical characterization of the synthesized materials The phase conformation of the materials was carried out using X-ray diffraction (XRD) (Miniflex, Rigaku, Japan). FTIR (Tensor 27, Bruker, Germany) and Raman analysis (Olympus, USA) was used to study the molecular composition of the material and determine its purity. The microstructure, crystal morphology, crystal architecture and particle size were analyzed by SEM (Zeiss, Germany) analysis, Transmission electron microscope (TEM) (Tecnai G2, FEI) and DLS (Malvern, UK). TEM analysis was performed to check nHAP crystal size, shape and



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

architecture. Selected area electron diffraction (SAED) analysis was also performed to obtain the crystallographic data to analyze crystallinity and crystal structure of nHAP. 2.6 Mechanical analysis Compression analysis was performed to determine the mechanical properties of the NC in different CSH: nHAP ratios, using an INSTRON MTS mechanical analyzer. 1.6 cm × 1.6 cm × 1.6 cm dimension NC and CS monoliths were prepared 24 hours before the test by mixing different proportions of CSH and nHAP, the monoliths were allowed to set at 37 °C. The monoliths were subjected to compressive load with crosshead speed of 0.6 mm/min until failure. The yield force at fracture was recorded and compressive modulus (C) was calculated. 2.7 Nano-cement bed generation for 3D cell culture The NC bed was generated in the bottom of the 2D-TCP by pouring the NC mixed with saline (0.9% w/v) into the well plate and allowing to set. The NC bed was prepared so as to uniformly coat whole of the bed area with uniform thickness without leaving any gaps. The NC beds were pre-incubated with complete media 1 hour before seeding the cells to saturate cement with media. 2.7.1 Nano-cement functionalization with bioactive molecules The bioactive molecules were incorporate into the NC matrix for functionalization. The bioactive molecules (ZA, BMP-2) were mixed in water and then it was mixed with dry CSH: nHAP (6: 4) mixture using 600 µl water per gram of NC mixture. The material were casted into disc shape and then used for characterization using FTIR and XPS (PHI, VersaProbe III, USA). 2.8 Cell-material interactions


Page 8 of 47

Page 9 of 47

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


To investigate material biocompatibility and cell adhesion, human osteosarcoma, Saos-2, and MC3T3-E1 subclone-4, mouse pre-osteoblast cells were cultured on NC. The cell viability was assessed using MTT assay as described elsewhere.27 Saos-2 cells were cultured in DMEM media with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic cocktail (penicillin and streptomycin). For experiments the cells were trypsinized with trypsin-EDTA, (0.25% trypsin, 0.02% EDTA) checked for viable cell numbers and seeded on NC at a seeding density of 1 × 105 cells/scaffold. Treated two dimensional tissue culture plates (2D-TCP) were used as positive control. MC3T3-E1 preosteoblast cells were cultured in α-MEM media supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic cocktail (penicillin and streptomycin) (complete media), in a humidified incubator at 37°C with 5% CO2 environment. MC3T3-E1 cells were seeded (1 × 105 cells/well) on the NC coated TCP. The viability of the seeded cells was evaluated using MTT similar to the Saos-2 cells. Cell adhesion and spreading of both cell types seeded on the NC matrix was evaluated by SEM (Zeiss, Germany) and fluorescence imaging of the cells seeded on NC matrix, looking for cell appendages, and cell adhesion. For fluorescence imaging cells were stained with phalloidinfluorescein isothiocyanate (FITC) staining cytoskeleton green and propidium iodide (PI) nuclear stain (red) and imaged using confocal microscope (Leica, Germany). 2.8.1 Effect of bone active agents on MC3T3-E1 cells proliferation and functionality The MC3T3-E1 subclone 4 mouse pre-osteoblast cells were obtained from ATCC (Virginia, USA). These cells were used to evaluate the effect of bone anabolic and anticatabolic agents loaded into the NC on viability, proliferation, and differentiation of the cells. A total of 1.7 × 105 cells were cultured on NC containing either rhBMP-2/ZA alone or both or none of the bone active molecules. The cells were cultured in phenol red free α-MEM complete medium 9 ACS Paragon Plus Environment


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 10 of 47

(10% FBS) or in differentiation medium (α-MEM + 10% FBS + β-glycerophosphate (10 mM) + ascorbic acid (50 µg/mL)) and the media was changed after every 48 hours. The viability of the MC3T3-E1 cells was evaluated by MTT assay. The effect of the bone active agents loaded in NC was checked by analyzing the ALP activity of cells seeded on NC alone or NC loaded with bone active agents. The α-MEM complete medium and α-MEM differentiation medium containing the bone active agents were used as the control. 2.9 Rat cranial defect model and in vivo nano-cement implantation All the animal procedures were performed after approval from institute animal ethics committee (IAEC) and followed their guidelines (Reference no. IITK/IAEC/2014/1023). A total of 20 male Wistar rats weighing between 250 to 300 g were divided into 4 groups: group-1 empty (defect only), group-2 (nano-cement (NC) alone), group-3 (NC + ZA (10 µg/disc)), group4 (NC + rhBMP-2 (2 µg/ disc) + ZA (10 µg/ disc)) containing five rats in each group. 2.9.1 Surgical procedure for cranial defect Circular NC discs were prepared by pre-casting NC in molds with dimension 8.5 mm in diameter and 1.25 mm height. The surgery was performed under anesthesia, induced by intraperitoneal administration of mixture of ketamine hydrochloride (80 mg/kg) and xylazine hydrochloride (5 mg/kg). The rats were given antibiotic prophylaxis by intramuscular administration of ceftriaxone (40 mg/kg). The rats were placed in prone position and a midline incision extending from nasofrontal area upto external occipital protuberance was performed. The skin and underlying tissue was reflected laterally to expose the calvaria. The periosteum from the surrounding area was removed eliminating periosteal osteogenesis. In all groups, an 8.5 mm circular full thickness bone defect was created (using a circular trephine) bilaterally through


Page 11 of 47

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


the sagittal suture distal to the lambdoid suture and proximal to coronal suture in the cranium, by cutting at slow speed (~1500 rpm) under constant irrigation with sterile saline to prevent heat injuries. Subsequently, the bone piece was lifted using an elevator and defect site was cleaned for any bone fragments, which may lead to dura mater injury. The NC discs were placed into the cleaned defect site of the animal. In the control group no NC discs were implanted in the defect and the defect was left empty. After implanting the NC discs, the underlying tissue was sutured with 3-0 resorbable sutures and, the skin was sutured using 3-0 non-resorbable sutures. Antibiotic prophylaxis was given 3 days post-surgery. The animals were having free access to food and water ad libitum. 2.9.2 Blood sample collection and blood cell count Blood samples were collected in Na-EDTA coated tubes by retro-orbital puncture before surgery and first, third, sixth, 10th and 14th and 21st day post-surgery to check for total and differential blood cell count. 2.10 Radiological and micro-CT analysis of the defect site In vivo micro-CT analysis was performed of 3 animals/group at 8 weeks post-surgery (Skyscan 1076, Bruker, Belgium) using energy settings of 70 kV and 140 µA, with 300 ms exposure and 970 projections, resulting in a voxel size of 18.5 µm. Ex-vivo analysis was performed after sacrificing the animals and harvesting the cranium 12 weeks post-surgery. For radiological analysis imaging of excised bone was performed using in-vivo micro-CT analyser (nanoScan, Mediso Medical Imaging systems, Budapest, Hungary), the samples were aligned on the imaging bed and projections were taken at 65 kV, 1300 ms to get images for qualitative radiological analysis. The ex-vivo scanning was performed with energy settings of 65 kV, 1300 11 ACS Paragon Plus Environment


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

ms exposure and 720 projections (nanoScan, Mediso Medical Imaging systems, Budapest, Hungary). A RAMLAK filter was applied to the scanned images during post-reconstruction and an isotropic voxel size of 10 µm was achieved. In both in vivo and ex vivo images, the images were filtered (Gaussian-Blur, 2 pt. radius) and re-oriented to align all images in the same direction (Data viewer Skyscan, Bruker, Belgium). The aligned images were analyzed using CTAn (Skyscan, Bruker, Belgium). A circular ROI of 8.5 mm was analyzed to determine the mineralized volume (BV), and to visualize the resorption of the NC and NC-bone integration. Thresholding of mineralized volume determined by visual inspection and was kept constant. 2.11 Sample harvesting for ssNMR analysis for new bone generation at defect site Solid state NMR analysis was performed on the post sacrifice excised rat bone samples from the defect site. The bone samples were harvested and processed for ssNMR analysis as described elsewhere.47 Using a dental diamond cutting blade the bone from the periphery of the defect site was removed. The samples were immediately frozen at -20 °C until NMR analysis to avoid the variations due to preservation conditions as recommended in previously optimized study.47 The bone sample weights were measured, grinded and packed inside the 3.2 mm MAS rotor for recording solid-state nuclear magnetic resonance (ssNMR) experiments. 2.11.1 ssNMR spectroscopy All the ssNMR spectra were recorded on 600 MHz NMR spectrometer (Avance III, Bruker Biospin). Bruker’s 3.2 mm probe was used to record all the experiments. The operating frequencies were 600.156 MHz for 1H, 150.923 MHz for

13

C respectively. The ssNMR spectra


Page 12 of 47

Page 13 of 47

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


were recorded at the magic angle spinning (MAS) speed of 10 kHz. The MAS speed was controlled within accuracy of ± 2Hz by Bruker’s MAS pneumatic unit. 1

H-13C cross polarization (CP) experiment was recorded at MAS of 10 kHz for all the

samples. The

13

C cross polarization at magic angle spinning (CPMAS) spectra were recorded

with, relaxation delay of 5 s, 8 k transients, 1 ms contact time and 3.32 µs, 1H π/2 pulse length. The 1H decoupling field of 100 kHz was used during 13C acquisition. Line broadening of 30 Hz was used to process the 13C spectra. 2.12 Histological analysis of the bone samples The bone samples for histology were fixed in formalin for 24 hours at 4°C, and then cut transversely through the sagittal suture. One part of the specimen was decalcified with Na-EDTA (10%, pH 7.2) and embedded in paraffin, before being sectioned to 5 µm and stained with Masson’s trichrome stain. The other half was embedded in methyl-methacrylate (MMA) (Sigma) and was sectioned to 5µm and stained with Alizarin red S. 2.13 SEM and Raman analysis of undecalcified bone sections SEM and Raman analysis was performed on the bone sections to check de-novo bone formation and presence of HAP at defect site. The spectra were recorded at the margins of de novo formed bone using Raman microscope (Olympus, USA). The back scattered electron (BSE), SEM and EDX (JEOL, Japan) analysis of the undecalcified sections was used to check the composition of new tissue formed and to check the chemical composition and distribution of mineral content in the defect site. 2.14 Statistical analysis



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 14 of 47

All the in vitro experiments were carried out in triplicate keeping the minimum sample size of n = 3. For in vivo experiments at 8-weeks the sample size was kept at n = 3 and for the ex vivo micro-CT the sample size was n = 4 (except NC+rhBMP-2+ZA which has n = 5). Statistical differences between groups were determined using Student's t-test. 2.15 Ethics statement All the animal procedures were performed by following the guidelines of the institute animal ethics committee (IAEC) and using the approval number IITK/IAEC/2014/1023. Utmost care was taken to reduce the animal suffering during the experimentation.

3.0 Results 3.1 Synthesis and characterization of nano-HAP White colored free flowing nano-crystalline HAP powder was obtained by wet chemical synthesis route. The nHAP synthesized was given thermal treatment to enhance its crystalline properties and enhance its density. The nHAP was given different thermal treatment at different temperature and time durations to optimize sintering conditions. 3.1.1 X-ray diffraction (XRD) analysis The diffractograms of different nHAP samples given different thermal treatments showed effect of temperature treatment on the nHAP properties. When thermal treatment at 500 °C was given to nHAP, it showed no phase transformation, and there was no increase in the crystalline properties of the nHAP (Figure-S1a). When sintering was carried out at 600 °C for a period of 4 hours, phase transformation in the nHAP was observed. Upto a period of three hours there was no major changes in the diffraction pattern of nHAP observed, but when the thermal treatment


Page 15 of 47

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


was extended upto a period of 4 hours, occurrence of phase transformations was observed, which were more rapid and sharp at 700 °C as can be observed from the diffractograms (Figure S1c).

Figure 1: Physicochemical characterization of nHAP. a. XRD of nHAP unsintered and post sintering at 800 °C for 4 hours, b. FTIR spectra of n-HAP after thermal treatment at different temperatures, c. Raman spectra of n-HAP after thermal treatment at different temperatures, d.



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 16 of 47

DLS curve of n-HAP showing particle size distribution in nanoscale, e. TEM image of n-HAP (unsintered), f. SAED electron diffraction pattern of n-HAP (unsintered). When the thermal treatment was given at 800 °C there was marked phase transformation in the material showing high degree of single phase nanocrystalline HAP generation. The diffractogram of nHAP sintered at 800 °C for 4 hours (Figure 1a) when compared with the diffraction pattern for the ICDD standard for HAP (card no. 09-432) showed similar diffraction patterns showing thermal treatment leads to phase transformation and generation of highly crystalline, high density nHAP. The diffraction pattern of nHAP was also compared with ICDD files for calcium phosphate, and calcium oxide to check formation of these products as a result of heat treatment, which were not observed. 3.1.2 Fourier transform infrared (FTIR) analysis The FTIR vibrational spectra of nHAP after thermal treatment at different temperature conditions (Figure 1b) was analysed to check for any degradation products thus generated due to thermal treatment or any other type of impurities generated in the material. The spectra showed a peak at 960 cm-1 (nondegenerate symmetric stretching for P-O bond in PO4-), presence of 10001100 cm-1 broad peak characteristic for hydroxyapatite and at 1041 cm-1 representing asymmetric stretching for PO4- without peaks in other regions validates that nHAP is pure. The functionalization of NC with ZA was also analysed by FTIR analysis where characteristic peaks for PO4- , -CH stretch at 900 cm-1, peak for –CO 1000-1100 cm-1 peak for –C=N at 1635 cm-1 and a broad peak 3000-3300 cm-1 for –OH and –CH represents incorporation of ZA in NC (Figure S2).


Page 17 of 47

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


3.1.3 Dynamic light scattering (DLS) and TEM particle analysis Particle size analysis was carried out using dynamic light scattering (Malvern). A dispersion of nHAP particles was made in absolute alcohol, the particles were well dispersed by sonicating the particles for 15 minutes in a bath sonicator to generate a homogeneous dispersion before analysis. Most of the particles (Figure 1d) were having an average size of ~30 nm, with a size range distribution from 26 to 32 nm dimension range, clearly showing that the nHAP is having particle size in the nanometer range. The nHAP particle size and dimensions were further verified by TEM imaging (Figure 1e) showing particles in nano range. In selective area electron diffraction (SAED) analysis, diffraction rings characteristic for HAP were observed (Figure 1f). 3.1.4 Raman spectroscopy Raman spectra of the samples were recorded to analyze the composition, purity and effect of thermal treatment on the phase purity and degradation of nHAP to any other products (Figure 1c). The presence of the peak at 960 cm-1 (Figure S3a, b) that represents symmetric stretching (υ1) of the P-O bond in tetrahedral PO4- group is characteristic for the presence of HAP. Presence of other peaks at 580, 591, 608 cm-1 and peaks at 1028, ~1047 and ~1076 cm-1 (triply degenerate asymmetric stretching mode (υ4) and (υ3) of O-P-O bond in PO4- respectively) and at 446 cm-1 bending mode (υ2) represents HAP phase (Figure S3a, b). 3.1.5 X-ray photoelectron spectroscopy (XPS) analysis The XPS analysis was carried out of the NC functionalized with ZA and BMP-2. The presence of characteristic peaks for the specific elements at different binding energy levels (eV) were observed in different samples showing incorporation of the bioactive molecules in NC. Specific peak for calcium (Ca2p, 347.8 eV) presence of Ca2s peak confirms presence of calcium. 17 ACS Paragon Plus Environment


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 47

Peaks for calcium were absent in ZA. Peak for oxygen (O1s, 531.8 eV) and for phosphorous (P2p3/2, 133.8 eV) were present in NC and nHAP. Presence of high intensity carbon peak (C1s, 284.8 eV) for C-C bond and weak nitrogen peak (N1s, 400 eV) for C-NH2 was present in ZA, NC+ZA and NC+BMP showing incorporation of organic content (Figure S4). 3.2 Mechanical analysis of the cement matrix The CS material showed mean compressive modulus of 424 ± 51 MPa. The NC material with 6:4 CSH: nHAP ratio showed mean compressive modulus of 328 ± 63 MPa (p-value >0.1) (Figure S5). 3.3 Cell-material interactions 3.3.1 Cellular attachment using microscopic studies For cell attachment analysis in vitro, cell culture studies were carried out on NC. For this Saos-2 (human osteosarcoma) (Figure 2a, c) and MC3T3-E1 (murine pre-osteoblasts) (Figure 2b, d) cells were cultured on NC matrix. Fluorescence (Figure 2a, b) and SEM (Figure 2c, d) imaging of the cells after 7 days of culture showed that cells are uniformly distributed over the surface of NC material. Cells showed extensions in all directions over the NC matrix with numerous filopodial extensions and anchoring appendages observed in cells growing in 3D environment.


Page 19 of 47

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


Figure 2: Cell proliferation study on NC. Fluorescent staining of a. Saos2 and b. MC3T3-E1 cells stained for cytoskeletal elements with FITC (green) and nucleus with PI (red) on NC. SEM image of c. SaoS2, d. MC3T3-E1. e. Graph representing cell proliferation analysis via MTT for MC3T3-E1 and Saos2 cell on NC matrix. (a, b magnification 40×). 3.3.2 Cell proliferation and functionality studies



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 20 of 47

The cells cultured for 15 days, showed upward trend in MTT values. It was observed that there was not significant increase in MTT values on day-1 and day-3 post seeding but subsequently the MTT values showed an increase and a doubling in the values was seen by day 10 (Figure 2e) representing that NC is biocompatible and supports cell proliferation. 3.3.3 Effect of bone active agents on MC3T3-E1 cells proliferation and functionality The MC3T3-E1 cell functionality was assessed when they were cultured on NC functionalized by incorporating either rhBMP-2 or ZA alone or both (Figure S2, S4). The 2D TCP without ZA in media was used as control. A marked and significant increase in MTT assay values was observed in all the groups (Figure 3a). 2D TCP showed the highest increase, but there were no significant differences among other groups (Figure 3a) implying that ZA was completely bound to the NC matrix and not freely available in the media. Free ZA mediated cytotoxicity was not observed even at day-6 post seeding on ZA loaded NC matrix.

Figure 3: Biocompatibility and bioactive molecule carrier study of NC. a. Comparative study of MC3T3-E1 cell proliferation on bioactive molecule (ZA and rhBMP-2) loaded NC matrix and 20 ACS Paragon Plus Environment

Page 21 of 47

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


effect on proliferation, b. Differentiation and functionality study of MC3T3-E1 cells cultured on NC matrix in presence of free or bound bioactive molecules (rhBMP-2 and ZA). The loading capacity and controlled release of bioactive molecules (ZA, rhBMP-2) in the NC matrix, was analysed by evaluatingdifferentiation (ALP activity) of MC3T3-E1 cells cultured on cement matrix with either rhBMP-2 loaded into the cement matrix or freely available in the media. It was seen that BMP caused differentiation of MC3T3-E1 cells even in absence of differentiating media (ascorbic acid and β-glycerophosphate) (Figure 3b) and higher levels of pNPP assay values were observed under rhBMP-2 presence at both time points in 2D conditions. When cells were cultured on 3D NC surface in α-MEM with 10% FBS (v/v) (NC+CM) or αMEM with 10% FBS (v/v) + ascorbic acid + β-glycerophosphate (NC+OM) there was significant increase in ALP levels in NC+OM (p-value

Nano-Hydroxyapatite Bone Substitute Functionalized with Bone Active

Recommend Documents