Biomaterials (v.28, #7)
Modelling of fracture behaviour in biomaterials
by I. Ichim; Q. Li; W. Li; M.V. Swain; J. Kieser (pp. 1317-1326).
One of the most frequent causes of degradation and failure of quasi-brittle biomaterials is fracture. Mechanical breakdown, even when not catastrophic, is of particular importance in the area of biomaterials, as there are many clinical situations where it opens the path for biologically mediated failures. Over the past few decades the materials/biomaterials community has developed a number of numerical models, but only with limited incorporation of brittle failure phenomena. This article investigates the ability of a non-linear elastic fracture mechanics (NLEFM) model to reliably predict failure of biomaterials with a specific focus on the clinical settings of restorative dentistry. The approach enables one to predict fracture initiation and propagation in a complex biomechanical status based on the intrinsic material properties of the components. In this paper, we consider five examples illustrating the versatility of the present approach, which range from the failure of natural biomaterials, namely dentine and enamel, to a restored tooth, a three unit all ceramic bridge structure and contact-induced damage in the restorative layered materials systems. It is anticipated that this approach will have ramifications not only to model fracture events but also for the design and optimisation of the mechanical properties of biomaterials for specific clinically determined requirements.
Keywords: Fracture; Strain softening; Brittle; Dental biomaterials; Finite element analysis
Physical and biological properties of barium cross-linked alginate membranes
by Heiko Zimmermann; Wahlisch Felix Wählisch; Claudia Baier; Markus Westhoff; Randolph Reuss; Dirk Zimmermann; Marcus Behringer; Friederike Ehrhart; Alisa Katsen-Globa; Christoph Giese; Uwe Marx; Vladimir L. Sukhorukov; Vasquez Julio A. Vásquez; Peter Jakob; Stephen G. Shirley; Ulrich Zimmermann (pp. 1327-1345).
We describe the manufacture of highly stable and elastic alginate membranes with good cell adhesivity and adjustable permeability. Clinical grade, ultra-high viscosity alginate is gelled by diffusion of Ba2+ followed by use of the “crystal gun” [Zimmermann H. et al., Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging. Biomaterials 2003;24:2083–96]. Burst pressure of well-hydrated membranes is between 34 and 325kPa depending on manufacture and storage details. Water flows induced by sorbitol and raffinose (probably diffusional) are lower than those caused by PEG 6000, which may be related to a Hagen–Poiseuille flow. Hydraulic conductivity, Lp, from PEG-induced flows ranges between 2.4×10−12 and 6.5×10−12mPa−1s−1. Hydraulic conductivity measured with hydrostatic pressure up to 6kPa is 2–3 orders of magnitude higher and decreases with increasing pressure to about 3×10−10mPa−1s−1 at 4kPa. Mechanical introduction of 200μm-diameter pores increases hydraulic conductivity dramatically without loss of mechanical stability or flexibility. NMR imaging with Cu2+ as contrast agent shows a layered structure in membranes cross-linked for 2h. Phase contrast and atomic force microscopy in liquid environment reveals surface protrusions and cavities correlating with steps of the production process. Murine L929 cells adhere strongly to the rough surface of crystal-bombarded membranes. NaCl-mediated membrane swelling can be prevented by partial replacement of salt with sorbitol allowing cell culture on the membranes.
Keywords: Hydrogels; Hydraulic conductivity; NMR-imaging; AFM; Topography; Cell culture
Blood plasma contact activation on silicon, titanium and aluminium
by Sara Arvidsson; Agneta Askendal; Pentti Tengvall (pp. 1346-1354).
In the present work, blood plasma protein deposition to spontaneously air oxidized silicon, titanium and aluminium was re-investigated in vitro. Immunological— and null ellipsometry methods were used to detect and quantitate adsorbed proteins, RIA methods to study the retention of preadsorbed125I-HSA upon exposure to buffer or blood plasma, and kallikrein-specific colorimetric substrate S-2302 to follow the surface generation of kallikrein.The results show that the contact activation of coagulation and complement systems are connected on Si and Ti, but not on Al, via coagulation factor XII. Preadsorbed125I-HSA was most readily displaced on silicon, followed by titanium and aluminium. The surfaces displayed different antibody binding patterns after short and long-time exposures to plasma. Titanium and silicon bound anti-HMWK after 1min in plasma, but aluminium did not. When the plasma incubation time was prolonged up to 2h the anti-HMWK binding disappeared totally on titanium and decreased on silicon. During the same time period, anti-C3c binding increased to the three types of surfaces. Also, the anti-C3c binding onto Si and Ti, but not Al, disappeared after incubation in Factor XII deficient plasma or when a specific coagulation factor XII (Factor XII) inhibitor, corn trypsin inhibitor (CTI) was added to normal plasma. The surface contacted plasmas cleaved the kallikrein-specific reagent S-2302 both after single surface contact, and after reincubation of surfaces in fresh plasma. The results show that C3b and Factor XIIa and their degradation products were retained at the surfaces.
Keywords: Adsorption; Contact activation; Complement; Silicon; Titanium; Aluminium
The inhibition of TNF- α-induced E-selectin expression in endothelial cells via the JNK/NF- κB pathways by highly N-acetylated chitooligosaccharides
by C.-W. Chia-Wen Lin; L.-J. Li-Jing Chen; P.-L. Pei-Ling Lee; C.-I. Chih-I Lee; J.-C. Jui-Che Lin; J.-J. Jeng-Jiann Chiu (pp. 1355-1366).
Chitooligosaccharides (COS) have been shown to regulate various cellular and biological functions. However, the effect of COS on inflammatory responses of the cells remains unclear. We investigated the regulatory effect of highly N-acetylated COS (NACOS) on tumor necrosis factor- α (TNF- α)-induced endothelial cell (EC) E-selectin expression, which is crucial for leukocyte recruitment. ECs were kept as controls or pre-treated with NACOS for different times, and then stimulated with TNF- α for 4h. The results show that pre-treating ECs with NACOS inhibited the TNF- α-induced E-selectin expression in a dose- and time-dependent manner. This NACOS-mediated inhibition in E-selectin expression was regulated at the transcriptional level, but not due to changes in mRNA stability. Stimulation of ECs with TNF- α-induced rapid increases in the phosphorylation of their mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated kinase (ERK), c-Jun-NH2-terminal kinase (JNK), and p38 MAPK]; the inhibitor for JNK (i.e., SP600125), but not those for ERK (i.e., PD98059) and p38 MAPK (i.e., SB203580), attenuated this TNF-α-induced E-selectin expression. Pre-treating ECs with NACOS inhibited the TNF- α-induced JNK activation, suggesting that JNK was involved in the inhibitory effect of NACOS on TNF- α-induced E-selectin expression. Pre-treating ECs with NACOS inhibited the TNF- α-induced p65 and p50 mRNA expressions. Gel shifting and chromatin immunoprecipitation assays showed that NACOS blocked the TNF- α-induced increases in the binding activity and in vivo promoter binding of nuclear factor- κB (NF- κB) in ECs. Our findings provide a molecular mechanism by which NACOS inhibit TNF- α-induced E-selectin expression in ECs, and a basis for using NACOS in pharmaceutical therapy against inflammation.
Keywords: Cell signaling; Chitin/chitosan; Endothelial cell; Gene expression; InflammationAbbreviations; MTT; 3; (4; 5-dimethylthiazol-2-yl)2; 5-diphenyl-tetrazolium bromide; PD98059; 2′-amino-3′-methoxyflavone; SB203580; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SP600125; athra(1; 9-cd)pyrazol-6(2; H; )-one
An in vitro pyrogen safety test for immune-stimulating components on surfaces
by Marina Hasiwa; Karin Kullmann; Sonja von Aulock; Christoph Klein; Thomas Hartung (pp. 1367-1375).
Due to the discovery of novel surgical techniques and new materials medical devices are increasingly used. Contact of these biomaterials with human tissue or blood commonly leads to inflammation of varying degrees, sometimes resulting in severe health problems. Possible causes are limited biocompatibility or pyrogenic contamination of the material.We adapted the recently validated in vitro pyrogen test (IPT), based on human whole blood cytokine release, to test the safety of biomaterials. Human whole blood is brought into direct contact with the surface of the test material and the release of the pro-inflammatory cytokine IL-1 β is measured. This procedure represents a human-relevant assay allowing the detection of pyrogens of different origins, e.g. Gram-negative (lipopolysaccharide, LPS) or Gram-positive (lipoteichoic acid, LTA), peptidoglycan (cell wall components of most bacteria) and fungal zymosan by direct material contact. The sensitivity of the test system allows a starting concentration of 10pg/ml for LPS, 10ng/ml for zymosan and 1μg/ml for LTA and peptidoglycan from different strains. Furthermore, we have shown that the test for solid materials can be carried out with cryo-preserved blood, which results in an even lower detection limit.
Keywords: In vitro test; Human whole blood; Lipopolysaccharide; Lipoteichoic acid; Peptidoglycan; Zymosan
Engineering of bone using bone marrow stromal cells and a silicon-stabilized tricalcium phosphate bioceramic: Evidence for a coupling between bone formation and scaffold resorption
by M. Mastrogiacomo; A. Papadimitropoulos; A. Cedola; F. Peyrin; P. Giannoni; S.G. Pearce; M. Alini; C. Giannini; A. Guagliardi; R. Cancedda (pp. 1376-1384).
Resorbable porous ceramic constructs, based on silicon-stabilized tricalcium phosphate, were implanted in critical-size defects of sheep tibias, either alone or after seeding with bone marrow stromal cells (BMSC). Only BMSC-loaded ceramics displayed a progressive scaffold resorption, coincident with new bone deposition. To investigate the coupled mechanisms of bone formation and scaffold resorption, X-ray computed microtomography (μCT) with synchrotron radiation was performed on BMSC-seeded ceramic cubes. These were analyzed before and after implantation in immunodeficient mice for 2 or 6 months. With increasing implantation time, scaffold thickness significantly decreased while bone thickness increased. The μCT data evidenced that all scaffolds showed a uniform density distribution before implantation. Areas of different segregated densities were instead observed, in the same scaffolds, once seeded with cells and implanted in vivo. A detailed μX-ray diffraction analysis revealed that only in the contact areas between deposited bone and scaffold, the TCP component of the biomaterial decreased much faster than the HA component. This event did not occur at areas away from the bone surface, highlighting coupling and cell-dependency of the resorption and matrix deposition mechanisms. Moreover, in scaffolds implanted without cells, both the ceramic density and the TCP:HA ratio remained unchanged with respect to the pre-implantation analysis.
Keywords: Bioceramic; Bone tissue engineering; Animal model; MicroCT; X rays microdiffraction; Synchrotron radiation
Coculture of endothelial and smooth muscle cells on a collagen membrane in the development of a small-diameter vascular graft
by H.-C. Hsi-Chin Wu; T.-W. Tzu-Wei Wang; P.-L. Pei-Leun Kang; Y.-H. Yang-Hwei Tsuang; J.-S. Jui-Sheng Sun; F.-H. Feng-Huei Lin (pp. 1385-1392).
In this study, we have evaluated the feasibility of developing a biodegradable collagenous small diameter vascular graft of 2mm diameter and 1cm length. In brief, bi-layer type I collagen membrane was fabricated under vacuum suction and lyophilization methods. The smooth muscle cells were inoculated into the lower side of the porous membrane, while endothelial cells were seeded onto upper smooth side of the membrane. After cultured for 7 days, the vascular substitute was either harvested for in vitro examination or in vivo implanted in the subcutaneous layer for biocompatibility test.The tubular vascular prosthesis was then used as a temporary absorbable guide that served as an in vivo vascular graft to promote the complete regeneration of rat inferior vena cava. After implantation for 12 weeks, a thin continuous layer of endothelial cells and smooth muscle cells were lined with the vascular lumen and tunic media, respectively. Histology results showed that there were no signs of significant thrombogeneity and intima hyperplasia. This tissue engineered vascular substitute not only had enough tensile strength and good biocompatibility, but also advanced vascular regeneration. In the future, we suggest that this biodegradable vascular substitute will provide with the possibility in application on small diameter prosthetic grafts in artificial blood vessels.
Keywords: Biomimetic material; Collagen; Co-culture; Vascular grafts
Influence of calcium phosphate crystal assemblies on the proliferation and osteogenic gene expression of rat bone marrow stromal cells
by Yang Liu; Paul R. Cooper; Jake E. Barralet; Richard M. Shelton (pp. 1393-1403).
Calcium phosphates (CaPs) have been investigated as substrates to promote bone formation both in vitro and in vivo. The aim of this study was to examine the proliferation and differentiation of rat bone marrow stromal cells (BMSCs) cultured on three-dimensional (3D) octacalcium phosphate (OCP) crystal assemblies. The cytotoxicity of OCP crystal assemblies was evaluated by measuring the lactate dehydrogenase (LDH) release from BMSCs during 10h of incubation with OCP crystal assemblies. The proliferation of BMSCs on OCP crystal assemblies in medium with or without osteogenic supplements was also investigated using the MTT assay with tissue culture treated plastic (TP) as the control. The tissues formed by BMSCs cultured on OCP crystal assemblies for 24 days were examined following staining with haematoxylin and eosin (H&E), alkaline phosphatase (ALP) and Van Gieson's techniques. The influence of OCP crystal assemblies on mRNA expression of α chain of collagen type I (Coll-Ia), ALP and osteocalcin (OC), osteonectin (ON), osteopontin (OP), lumican, Cbfa1, EST317 and EST350 by the BMSCs were also investigated using semi-quantitative RT-PCR.Although OCP crystals were relatively cytotoxic compared with TP, proliferation of BMSCs occurred when seeded onto OCP crystal assemblies. BMSCs cultured on OCP demonstrated similar proliferation rates as found on the control and no significant difference ( P<0.05) in the number of cells cultured in medium supplemented with or without osteogenic additives on TP and OCP. The deposition of collagen and ALP were detected in tissue synthesised by BMSCs cultured on OCP crystals assemblies. OCP crystal assemblies down-regulated basal bone ECM proteins, including Coll-Ia, ON and lumican, in the first week of culture, whilst up-regulation of the same genes was observed after 24 days of culture. The observed down-regulation of Cbfa1 on OCP substrates was consistent with the negative effect of OCP crystal assemblies on the genes encoding bone ECM proteins. The up-regulation of OC mRNA expression by OCP crystal assemblies could be related to the requirement for synthesis of more OC proteins to control the concentration of calcium ions in culture medium.
Keywords: Bone tissue engineering; Bone marrow; Calcium phosphate; mRNA; Osteoblast
Non-destructive quantitative 3D analysis for the optimisation of tissue scaffolds
by Julian R. Jones; Gowsihan Poologasundarampillai; Robert C. Atwood; Dominique Bernard; Peter D. Lee (pp. 1404-1413).
In tissue engineering, porous scaffolds are often used as three-dimensional (3D) supports for tissue growth. In scaffold design, it is imperative to be able to quantify the pore sizes and more importantly the interconnects between the pores. X-ray micro-computed tomography ( μCT) has become a popular tool for obtaining 3D images of scaffold biomaterials, however images are only qualitative. In this work, methods were developed for obtaining pore size distributions for both the macropores and their interconnects. Scaffolds have been developed, by foaming sol–gel derived bioactive glasses, which have the potential to fulfil the criteria for an ideal scaffold for bone tissue engineering. μCT images were obtained from scaffolds with different pore structures. The images were thresholded and three algorithms were applied in 3D to identify pores and interconnects and to obtain pore size distributions. The results were validated against mercury intrusion porosimetry and manual 3D image analysis. The μCT data were then meshed such that predictions of permeability as a function of changes in the pore network could be made. Such predictions will be useful for optimising bioreactor conditions for tissue engineering applications. These techniques would be suitable for many other types of scaffolds.
Keywords: Scaffold; Image analysis; Porosity; Bioactive glass
Preparation of hemoglobin-loaded nano-sized particles with porous structure as oxygen carriers
by Jian Zhao; C.-S. Chang-Sheng Liu; Yuan Yuan; X.-Y. Xin-Yi Tao; X.-Q. Xiao-Qian Shan; Yan Sheng; Fan Wu (pp. 1414-1422).
Hb (hemoglobin)-loaded particles (HbP) encapsulated by a biodegradable polymer used as oxygen carrier were prepared. A modified double emulsion and solvent diffusion/evaporation method was adopted. All experiments were performed based on two types of biodegradable polymers, poly( ε-caprolactone) (PCL) and poly( ε-caprolactone-ethylene glycol) (PCL-PEG). The biodistribution and the survival time in blood of the particles were investigated in a mouse model. Encapsulation efficiency and pore-connecting efficiency were evaluated by a novel sulfocyanate potassium method. The influence of process parameters on the particle size and pore-connecting efficiency (PCE%) of nanoparticles have been discussed. The prepared conditions: solvent, external aqueous phase, pressure were discussed. The system utilizing dichloromethane (DCM)/ethyl acetate (EA) as a solvent with an unsaturated external aqueous phase yielded the highest encapsulation efficiency (87.35%) with a small mean particle size (153nm). The formation of porous channels was attributed to the diffusion of solvent. The PCE% was more sensitive to the rate of solvent diffusion that was obviously affected by the preparation temperature. The PCE% reached 87.47% when PCL-PEG was employed at 25°C. P50 of HbP was 27mmHg, which does not seem to be greatly affected by the encapsulation procedure. In vivo, following intravenous injection of 6-coumarin labeled HbP, the major organ accumulating Hb-loaded particles was the liver. The half-life of nano-sized PCL HbP was 3.1 times as long as the micro-sized PCL HbP. Also, Nano-sized as well as a PEGylated surface on HbP is beneficial for prolonged blood residence (7.2 fold increase).
Keywords: Nanoparticles; Hemoglobin; Encapsulation efficiency; Pore-connecting efficiency; Oxygen-carrying capacity; Circulation time
Bio-functional micelles self-assembled from a folate-conjugated block copolymer for targeted intracellular delivery of anticancer drugs
by S.-Q. Shao-Qiong Liu; Nikken Wiradharma; S.-J. Shu-Jun Gao; Yen Wah Tong; Y.-Y. Yi-Yan Yang (pp. 1423-1433).
In this study, a block copolymer, poly( N-isopropylacrylamide- co- N, N-dimethylacrylamide- co-2-aminoethyl methacrylate)- b-poly(10-undecenoic acid) (P(NIPAAm- co-DMAAm- co-AMA)- b-PUA) was synthesized, and folic acid was conjugated to the hydrophilic block through the amine group in AMA. This polymer was self-assembled into micelles, which exhibited pH-induced temperature sensitivity. They were smaller in size, and possessed a better-defined core–shell structure as well as more stable hydrophobic core than the random copolymer P(NIPAAm- co-DMAAm- co-UA), and provided a shell with folate molecules. An anti-cancer drug, doxorubicin (DOX) was encapsulated into the micelles. The mean diameter of the blank and DOX-loaded micelles was less than 100nm. DOX release was pH-dependent, being faster at low pH (endosomes/lysosomes). Therefore, DOX was readily released from the micelles into the nucleus after being taken up. More importantly, IC50 of DOX-loaded micelles with folate against folate receptor-expressing 4T1 and KB cells was much lower than that of the DOX-loaded micelles without folate (3.8 vs. 7.6mg/L for 4T1 cells and 1.2 vs. 3.0mg/L for KB cells). In vivo experiments conducted in a 4T1 mouse breast cancer model demonstrated that DOX-loaded micelles had a longer blood circulation time than free DOX ( t1/2: 30min and 140min, respectively). In addition, the micelles delivered an increased amount of DOX to the tumor when compared to free DOX. These bio-functional micelles may make a promising carrier to transport anticancer drugs specifically to tumor cells and release the drug molecules inside the cells to the cytosols for improved chemotherapy.
Keywords: pH- and temperature-sensitive; Micelles; Block copolymer; Doxorubicin; Biodistribution
Small interfering RNA delivery to the liver by intravenous administration of galactosylated cationic liposomes in mice
by Ayumi Sato; Motoki Takagi; Akira Shimamoto; Shigeru Kawakami; Mitsuru Hashida (pp. 1434-1442).
Although small interfering RNA (siRNA) is a potentially useful therapeutic approach to silence the targeted gene of a particular disease, its use is limited by its stability in vivo. For the liver parenchymal cell (PC)-selective delivery of siRNA, siRNA was complexed with galactosylated cationic liposomes. Galactosylated liposomes/siRNA complex exhibited a higher stability than naked siRNA in plasma. After intravenous administration of a galactosylated liposomes/siRNA complex, the siRNA did not undergo nuclease digestion and urinary excretion and was delivered efficiently to the liver and was detected in PC rather than liver non-parenchymal cells (NPC). Endogenous gene (Ubc13 gene) expression in the liver was inhibited by 80% when Ubc13–siRNA complexed with galactosylated liposomes was administered to mice at a dose of 0.29nmol/g. In contrast, the bare cationic liposomes did not induce any silencing effect on Ubc13 gene expression. These results indicated that galactosylated liposomes/siRNA complex could induce gene silencing of endogenous hepatic gene expression. The interferon responses by galactosylated liposomes/siRNA complex were controlled by optimization of the sequence of siRNA. Also no liver toxicity due to galactosylated liposomes/siRNA complex was observed under any of the conditions tested. In conclusion, we demonstrated the hepatocyte-selective gene silencing by galactosylated liposomes following intravenous administration.
Keywords: Gene therapy; Liver; Liposome; Gene transfer
Self-assembled ternary complexes of plasmid DNA, low molecular weight polyethylenimine and targeting peptide for nonviral gene delivery into neurons
by Jieming Zeng; Xu Wang; Shu Wang (pp. 1443-1451).
Chemical conjugation of targeting ligands to polycation/plasmid DNA complexes has been widely used to improve the transfection efficiency of nonviral gene delivery vectors. However, conjugation reactions may reduce or even inactivate the biological activities of chemically sensitive moieties, such as proteins and peptides. Here we describe a new method for introducing targeting ligands into nonviral vectors, in which ternary complexes are formed via charge interactions among polyethylenimine (PEI) of 600Da, plasmid DNA and targeting peptides with positively charged DNA-binding sequence. Owing to the nerve growth factor (NGF) loop 4 hairpin motif in the targeting peptide, these ternary complexes are capable of mediating gene delivery efficiently and specifically into cells expressing the NGF receptor TrkA. In in vitro experiments, the complexes improved luciferase reporter gene expression by up to 1000-fold while comparing with that produced by complexes with nontargeting control peptide. In an in vivo experiment, the ternary complexes with the targeting peptide was 59-fold more efficient than the control ternary complexes in transfecting dorsal root ganglia (DRG), the peripheral nervous sites with TrkA-expressing neurons. In a cell viability study, the ternary complexes were remarkably different from DNA complexes by PEI of 25kDa, the gold standard for nonviral gene carriers, displaying no toxicity in tested neuronal cells. Thus, this study demonstrates an alternative method to construct nonviral delivery system for targeted gene transfer into neurons.
Keywords: Nonviral vectors; Targeted gene delivery; Neurons; TrkA receptor
Chemical composition, crystal size and lattice structural changes after incorporation of strontium into biomimetic apatite
by Z.Y. Li; W.M. Lam; C. Yang; B. Xu; G.X. Ni; S.A. Abbah; K.M.C. Cheung; K.D.K. Luk; W.W. Lu (pp. 1452-1460).
Recently, strontium (Sr) as ranelate compound has become increasingly popular in the treatment of osteoporosis. However, the lattice structure of bone crystal after Sr incorporation is yet to be extensively reported. In this study, we synthesized strontium-substituted hydroxyapatite (Sr-HA) with different Sr content (0.3%, 1.5% and 15% Sr-HA in mole ratio) to simulate bone crystals incorporated with Sr. The changes in chemical composition and lattice structure of apetite after synthetic incorporation of Sr were evaluated to gain insight into bone crystal changes after incorporation of Sr. X-ray diffraction (XRD) patterns revealed that 0.3% and 1.5% Sr-HA exhibited single phase spectrum, which was similar to that of HA. However, 15% Sr-HA induced the incorporation of HPO42− and more CO32−, the crystallinity reduced dramatically. Transmission electron microscopy (TEM) images showed that the crystal length and width of 0.3% and 1.5% Sr-HA increased slightly. Meanwhile, the length and width distribution were broadened and the aspect ratio decreased from 10.68±4.00 to 7.28±2.80. The crystal size and crystallinity of 15% Sr-HA dropped rapidly, which may suggest that the fundamental crystal structure is changed. The findings from this work indicate that current clinical dosage which usually results in Sr incorporation of below 1.5% may not change chemical composition and lattice structure of bone, while it will broaden the bone crystal size distribution and strengthen the bone.
Keywords: Bone crystal; Strontium-substituted; Hydroxyapatite; Nanocrystallite