Skip to main content
NCBI home page
Saudi Journal of Biological Sciences logoLink to Saudi Journal of Biological Sciences
. 2020 May 23;27(8):2143–2148. doi: 10.1016/j.sjbs.2020.05.029

Hydroxyapatite based biocomposite scaffold: A highly biocompatible material for bone regeneration

Ceng Li a, Weiguang Qin b, Sivalingam Lakshmanan c,⁎, Xiaohui Ma d, Xiaowei Sun e, Bo Xu f,⁎
PMCID: PMC7384365  PMID: 32742182

Abstract

The conventional approaches for treating bone defects such as autografts donor tissue shortages and allografts transmission of diseases pose many shortcomings. The objective of this study was to design a nano strontium/magnesium doped hydroxyapatite (Sr/Mg-HA) with chitosan (CTS) and multi-walled carbon nanotubes (MWCNT) (Sr/Mg-HA/MWCNT/CTS) biocomposite was created to support the growth of osteoblasts using a solvent evaporation method. To help the growth of osteoblasts, a solvent evaporation technique was used to design a nano strontium/magnesium doped hydroxyapatite with chitosan and multi-walled carbon nanotubes biocomposite. We studied the biocompatibility and efficiency in vitro of biocomposite following physicochemical analyzes. Tests of biocompatibility, cell proliferation, mineralization, and osteogenic differentiation have shown that in-vitro safety and effectiveness of biocomposite are good. The performance of biocomposite was more efficient in in-vitro as well as in vivo experiments than in Sr/Mg-HA nanoparticles. Briefly, the Sr/Mg-HA/MWCNT/CTS biocomposite is an ideal candidate for effective bone repair in clinics with excellent mechanical properties with durable multi-biofunctional antibacterial properties and osteoinductivity.

Keywords: Antibacterial activity, Bone, Biocompatibility, Hydroxyapatite, Osteoblast cell

1. Introduction

Skeletal disorders and trauma have become a global, functional problem and a clinical challenge for bones and fractures (Lems and Raterman, 2017, Xie et al., 2019). A bone graft autologously is known as the good scaffolds for fixing bone defects and fractures, which has an exceptional bone fusion therapeutic effect (Nandi et al., 2010). The drawbacks of autologous bone graft, for instance, the morbidities of the donor site, its restricted availability, trauma and pain, however, limit its broad use. Although the allograft and xenograft could defeat the downsides of the autologous bone join, they may bring different worries up in the potential spreading of sickness and invulnerable dismissal (Oryan et al., 2014; Offner et al., 2019). The advancement of bone tissue engineering has proposed promising ways to bone regeneration in recent decades. Perfect bone fix composite should have comparative properties to regular bones, incorporating high porosity, magnificent cytocompatibility, great degradability, reasonable mechanical quality, and the upgraded capacities for mineralization and bone cell proliferation (Ibrahim et al., 2016, Chen et al., 2016).

Chitosan (CTS) is a natural polymer and has extensively been utilized as a platform in bone tissue engineering and other biomedical applications because of its cytocompatibility, degradability, pore development conduct, appropriateness for cell ingrowths and inborn bactericidal nature (Ramya et al., 2012, Escárcega-Galaz et al., 2018, Ahmad et al., 2020). Though, CTS based biocomposite materials have ideal mechanical quality and low interconnected porosity for cell connection, which are deprived to be improved further (Wang et al., 2005, Spinks et al., 2006). Poor mechanical characteristics nevertheless restrict the medical applications of CTS in hard tissue engineering (Souza et al., 2017, Sanchez et al., 2019). Different methods to boost the mechanical properties of CTS based biocomposite have been suggested (Javaid et al., 2019, Price et al., 2019).

A further strategy for the enhancement of the compressive strength of the CTS based implants is the nano-reinforcement (Li et al., 2013, Wang et al., 2019, Cao et al., 2020). The long-term effort is made to use MWCNTs notable electrical, thermal, mechanical and chemical characteristics, to improve the properties of polymer biocomposites. The introduction of MWCNT as refurbishing materials into the polymer medium leads to substantial changes in the mechanical characteristics resulting in better properties for biocomposite than pristine polymer (Limón-Martínez et al., 2019).

The induction of osteoinduction is one of the most significant aspects of bone repair (Mehrotra et al., 2019). While CTS can heal bones, there can be no reconstruction of vast areas of bone defects. Therefore, a variety of bioceramics were used in hard tissue repair by combining CTS. Hydroxyapatite (HA) is one of BTE's most widely used bioceramics that is well biocompatible, has a high osteoinductivity and is capable of bonding to bone tissue. Numerous investigations have discovered that bone recovery can be fundamentally advanced by joining HA into a biopolymer.

The biological and physicochemical characteristics of hydroxyapatite may be further improved by doping HA with mono-mineral and multi-minerals. By adding, for example, a certain amount of strontium (Sr) or magnesium (Mg) the mechanical characteristics of brittle HA will greatly improve. Research has demonstrated that Sr and Mg can advance bone recovery and development by empowering osseous separation while lessening bone ingestion, by restraining osteoclasts arrangement (Jiang et al., 2019, Arcos and Vallet-Regí, 2020, Chen et al., 2020, Li et al., 2020).

For this research, Sr/Mg-HA and Sr/Mg-HA/MWCNT/CTS biocomposite were prepared for bone repair and rejuvenation. Such Sr/Mg-HA and biocomposite have been tested with their structural, physical and biological characteristics.

2. Materials and method

2.1. Preparation of minerals doped hydroxyapatite and biocomposite

To prepared minerals doped hydroxyapatite, 0.08 M of calcium chloride, 0.01 M of magnesium chloride, 0.01 M of strontium chloride and 0.06 M of phosphoric acid were joined in a 1:6 M proportion (natural bone ratio). Mineral (Ca + Mg + Sr) arrangement was sonicated in 50 mL of twofold refined water for 10 min until a homogeneous suspension was acquired. Phosphoric acid (diluted with 50 mL of twofold refined water) was added drop wise to this arrangement for more than 30 min, with nonstop blending, with the pH kept up over 10 by the option of sodium hydroxide arrangement, as important. The arrangement was sonicated for a further 1 h, and afterward permitted to stand medium-term, after which the item was sifted under suction and dried in encompassing conditions. The powders were warmed in a high-temperature heater. Tests were warmed from 100 − 1200 °C, holding at 900 °C for 5 h, trailed by cooling the items from 900 °C to 100 °C and afterward cooling to room temperature.

In the wake of sonicating MWCNT in the CH3COOH, Sr/Mg-HA nanoparticles were added to the blend and sonicated to scatter the nanoparticles. The homogenous blend was then placed into a CTS arrangement. The biocomposite scaffold were prepared after casting the obtained mixture into a Petri glass 60 mm in diameter and evaporating the solvent at 50 ◦C for 48 h in an oven.

3. Characterization of minerals doped hydroxyapatite and biocomposite

The application of XRD was used for the main phases of Sr/Mg-HA nanoparticles and biocomposite (X’Pert Pro, PANalytical, Netherlands). Sr/Mg-HA nanoparticles and biocomposite morphology were examined using TEM (JEOL, EM-2100F). A universal testing machine (Instron 5567) tested the compressive and mechanical strength of the Sr/Mg-HA nanoparticles and biocomposite (Han et al., 2019).

3.1. Antibacterial test

In-vitro antibacterial tests were used to determine the antibacterial characteristics of Sr/Mg-HA nanoparticles and biocomposite. On three sample groups including control and Sr/Mg-HA nanoparticles and biocomposite, E. coli and S. aureus were grown. The optical density of the microbial solution has been determined after one day of culture (Kim et al., 2011).

3.2. In vitro MTT assay

After three and seven days of culture, cell proliferation was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) analysis. The cultivation medium was first replaced with the solution MTT in the 96-well pot. The MTT solution was replaced by the DMSO after four hours of incubation and dissolved for 20 min at RT. 100 µl reagents have finally been carefully distributed to 96- well plates. The optics densities were tested with a microplate reader at 570 nm (Song et al., 2019).

3.3. In vitro Live/dead cell assay

Fluorescence stain has been used to determine the structure of osteoblast cells. The medium has been removed from the pools after three and seven days of rising, and phosphate buffer solution washed twice on the cells of Sr/Mg-HA and biocomposite. Also, the cells were recolored utilizing the Live/dead cell measure packs for four hours. The recolored tests were washed twice with phosphate buffer solution and were seen under a fluorescent magnifying instrument (Wu et al., 2017).

4. Results and discussion

4.1. X-ray diffraction analysis

The X-ray diffraction patterns of Sr/Mg-HA nanoparticles and biocomposite are shown in Fig. 1. The XRD peaks of Sr/Mg-HA nanoparticles could be listed to hexagon-staged pure hydroxyapatite with impressive planes at 002, 211, 112, 300, 130 and 213 (JCPDS card no. 09-0432) (Venkatesan and Kim, 2010, Fahami and Nasiri-Tabrizi, 2013). The diffraction patterns of Sr/Mg-HA nanoparticles relative to those of biocomposite are significantly different, suggesting that the Sr/Mg-HA nanoparticles were successfully incorporated in the MWCNT-CTS matrices. Nevertheless, it is worth pointing out that the MWCNT diffraction patterns were hard to detect in the biocomposite patterns, due to the convergence of the main peaks of MWCNT and hydroxyapatite by 26° (Hooshmand et al., 2014, Rodrigues et al., 2016). In addition, the characteristic plane of MWCNT cannot be identified with diffraction patterns of the biocomposite even though MWCNT was wrapped directly in the prepared hydroxyapatite. This also may explain the active biocomposite fabricated. Furthermore, the broad diffraction planes indicate a high biocomposite consistency.

Fig. 1.

Fig. 1

Open in a new tab

XRD patterns of Sr/Mg-HA nanoparticles (A), and biocomposite (B).

4.2. Morphological analysis

The structure and the morphology of the prepared samples were seen under TEM (Fig. 2). The pictures with comparing chosen zone electron diffraction examples and high-goals TEM pictures of the prepared samples were recorded. The prepared Sr/Mg-HA nanoparticles show a rod-like surface. Fig. 2 (A, B) showed the morphology of biocomposite: an interlaced system of MWCNT covered with Sr/Mg-HA, the particles were tied down onto the out mass of the MWCNT (Park et al., 2019). The SAED images of composite demonstrate the trademark ring example of rod structure, and they affirm the higher level of crystallinity of nanoparticles.

Fig. 2.

Fig. 2

Open in a new tab

Morphological characterization of (A) Sr/Mg-HA nanoparticles and (B) biocomposite.

4.3. Mechanical test

Carbon nanotubes can promptly be added as building squares to accomplish mechanically stable bony platform networks (Eivazzadeh-Keihan et al., 2019). The impact of CNTs stacking on the mechanical characteristics of certain polymers utilized in bone tissue engineering. For recovery of bone deformities with composite configurations and perplexing shapes, fruitful reconciliation is basic (Pei et al., 2019). Bone tissue engineering framework substances must keep up the most suitable auxiliary geometry as the procedure of recovery continues. In addition, the mechanical test outcomes appear (Fig. 3) that both Sr/Mg-HA and MWCNT could improve the young's modulus of the frameworks. It has been demonstrated that MWCNT and Sr/Mg-HA nanoparticles can improve the mechanical properties of different frameworks. Through the fuse of MWCNT and Sr/Mg-HA nanoparticles in the delicate composite framework, we can strengthen the CTS platform (Fig. 3).

Fig. 3.

Fig. 3

Open in a new tab

(A) compressive strength and (B) tensile strength of prepared samples.

4.4. Antibacterial test

The aftereffects of the antibacterial tests (Fig. 4) demonstrated that the Sr/Mg-HA nanoparticles with the MWCNT-CTS (Sr/Mg-HA/MWCNT-CTS) biocomposite had antibacterial exercises against E. coli and S. epidermidis. As appeared in Fig. 4A and B, after one day of culture, the antibacterial proportions of the Sr/Mg-HA nanoparticles and biocomposite bunches were fundamentally higher than those of the microbial arrangement on the Sr/Mg-HA nanoparticles (Tsai et al., 2018). Every one of these outcomes showed that the fuse of Sr and Mg-doped HA nanoparticles improved the antibacterial action of the biocomposite against the development of E. coli and S. epidermidis microscopic organisms (Kolmas et al., 2014). Besides, it was nothing unexpected that the biocomposite additionally had the certain bactericidal capacity, since the CTS and MWCNT itself previously had a specific level of antibacterial action (Goy et al., 2009, Kassem et al., 2019).

Fig. 4.

Fig. 4

Open in a new tab

(A) Antibacterial ratio of S. epidermidis and (B) antibacterial ratio of E. coli suspension growth on the Sr/Mg-HA and biocomposite for one day.

4.5. Biocompatibility assay

The BMSCs cell proliferation results utilizing MTT assay appear in Fig. 5A. There is no measurably huge distinction between each group on day one. Though, with the expansion of incubation time, the multiplication of cells demonstrates a kept developing pattern for prepared samples and the control sample. Although the multiplication on the control samples and Sr/Mg-HA nanoparticles is higher than those biocomposites on day four, the expansion of biocomposite is increasingly quick from day seven. The outcome shows that the biocomposite with higher Sr/Mg-HA nanoparticles substance can display better cell attachment and expansion, which ought to be specially picked for future in vivo examination and application.

Fig. 5.

Fig. 5

Open in a new tab

(A) Proliferation of bone marrow stromal cells after 1, 4 and 7 days of culture on prepared samples. (B) Fluorescence images of bone marrow stromal cells after 4 days of culture prepared samples.

The biocompatibility of cells is commonly envisioned utilizing a fluorescent live/dead test (Fig. 5B). On day four, live cells layer can be seen from all culture groups, which shows that the biocomposite and Sr/Mg-HA nanoparticles have no negative impact on biocompatibility. Additionally, the fluorescence pictures likewise display numerous filopodia, exhibiting admirable cells connection on the biocomposite (Mitran et al., 2018).

4.5.1. In vitro osteogenic differentiation

In vitro osteogenic properties of bone marrow stromal cells seeded on the Sr/Mg-HA nanoparticles and biocomposite was assessed by the estimation of alkaline phosphatase activity, since alkaline phosphatase was viewed as a beginning time marker of osteogenic properties. As appeared in Fig. 6A, the alkaline phosphatase activity was expanded with the expanding incubation occasion. Following seven days of incubation, the alkaline phosphatase activity in Sr/Mg-HA nanoparticles and biocomposite were essentially higher than that in control. Bone marrow stromal cells seeded on Sr/Mg-HA nanoparticles and biocomposite showed altogether higher alkaline phosphatase activity contrasted with those incubated on chitosan (control). Nonetheless, it was plainly that the biocomposite indicated fundamentally higher alkaline phosphatase activity than that of the Sr/Mg-HA nanoparticles.

Fig. 6.

Fig. 6

Open in a new tab

(A) Alkaline phosphatase activity assay of cells after culturing for 7 days. (B) mineralization formation evaluated by AR staining quantification.

Associatively, the mineralized scaffold created by the cells was resolved on day 7. Fig. 6B demonstrates that mineral creation was enormously delivered in cells incubated on Sr/Mg-HA nanoparticles and biocomposite scaffold, and the measure of the mineralized scaffold was fundamentally expanded contrasted with cells on biocomposite. The addition of Sr/Mg-HA nanoparticles in MWCNT-CTS went about as the osteoinductive factor can initiate osteogenic of bone marrow stromal cells without outside expansion. These in vitro assessments recommend that the biocomposite could surprisingly advance osteogenic differentiation, bone cell multiplication, andadhesion.

5. Conclusion

In short, the biocomposite displayed impressive mechanical characteristics, antibacterial inductivity, and osteoinductivity. The reinforcement effects of MWCNT also enhanced the biocomposite mechanical strength. The biological characteristics of the biocomposite were improved by dual-ionic doping. Strontium and magnesium were doped into the hydroxyapatite, which can stay away from cytotoxicity, yet can likewise accomplish osteoinductive and antibacterial characteristics for a significant time. Beside, dual-ion doping can successfully eliminate many issues caused by the use, like high prices, growth factors or antibiotics. The in vitro cell proliferation and live/dead experimentations showed that the biocomposite enhanced cell activity and encouraged bone regeneration. In brief, the biocomposite is suitable for potential bone regeneration, with its long-term active multi-biofunction of the osteoinductivity and its antibacterial characteristics.

Footnotes

Peer review under responsibility of King Saud University.

References

  1. Ahmad N., Ahmad R., Ahmad F.J., Ahmad W., Alam M.A., Amir M., Ali A. Poloxamer-chitosan-based Naringenin nanoformulation used in brain targeting for the treatment of cerebral ischemia. Saudi J. Biolog. Sci. 2020;27:500–517. doi: 10.1016/j.sjbs.2019.11.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arcos D., Vallet-Regí M. Substituted hydroxyapatite coatings of bone implants. J. Mater. Chem. B. 2020;8(9):1781–1800. doi: 10.1039/c9tb02710f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen Z.X., Lei Q., He R.L., Zhang Z.F., Chowdhury A.J.K. Review on antibacterial biocomposites of structural laminated veneer lumber. Saudi J. Biolog. Sci. 2016;23:S142–S147. doi: 10.1016/j.sjbs.2015.09.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cao S., Ryan P.M., Salehisahlabadi A., Abdulazeem H.M., Karam G., Černevičiūtė R., Antuševas A., Rahmani J., Zhang Y. Effect of Probiotic and Synbiotic Formulations on Anthropometrics and Adiponectin in Overweight and Obese Participants: A Systematic Review and Meta-analysis of Randomized Controlled Trials. J. King Saud Univ.-Sci. 2020;32:1738–1748. [Google Scholar]
  5. Chen M., Zhang H., Shan S., Li Y., Li X., Peng D. Fabrication of multiwalled carbon nanotubes/carrageenan-chitosan@ Ce and Sr substituted hydroxyapatite biocomposite coating on titanium: In vivo bone formation evaluations. J. King Saud Univ.-Sci. 2020;32:1175–1181. [Google Scholar]
  6. Escárcega-Galaz A.A., De La Cruz-Mercado J.L., López-Cervantes J., Sánchez-Machado D.I., Brito-Zurita O.R., Ornelas-Aguirre J.M. Chitosan treatment for skin ulcers associated with diabetes. Saudi J. Biolog. Sci. 2018;25:130–135. doi: 10.1016/j.sjbs.2017.03.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eivazzadeh-Keihan R., Maleki A., De La Guardia M., Bani M.S., Chenab K.K., Pashazadeh- Panahi P., Baradaran B., Mokhtarzadeh A., Hamblin M.R. Carbon based nanomaterials for tissue engineering of bone: Building new bone on small black scaffolds: A review. J. Adv. Res. 2019;18:185–201. doi: 10.1016/j.jare.2019.03.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fahami A., Nasiri-Tabrizi B. Characterization of mechanothermal-synthesized hydroxyapatite-magnesium titanate composite nanopowders. J. Adv. Ceram. 2013;2:63–70. [Google Scholar]
  9. Goy R.C., Britto D.D., Assis O.B. A review of the antimicrobial activity of chitosan. Polímeros. 2009;19:241–247. [Google Scholar]
  10. Hooshmand T., Abrishamchian A., Najafi F., Mohammadi M., Najafi H., Tahriri M. Development of sol-gel-derived multi-wall carbon nanotube/hydroxyapatite nanocomposite powders for bone substitution. J. Compos. Mater. 2014;48:483–489. [Google Scholar]
  11. Han L., Jiang Y., Lv C., Gan D., Wang K., Ge X., Lu X. Mussel-inspired hybrid coating functionalized porous hydroxyapatite scaffolds for bone tissue regeneration. Colloids Surf. B Biointerf. 2019;179:470–478. doi: 10.1016/j.colsurfb.2019.04.024. [DOI] [PubMed] [Google Scholar]
  12. Ibrahim S., Sabudin S., Sahid S., Marzuke M.A., Hussin Z.H., Bashah N.K., Jamuna- Thevi K. Bioactivity studies and adhesion of human osteoblast (hFOB) on silicon- biphasic calcium phosphate material. Saudi J. Biolog. Sci. 2016;23:S56–S63. doi: 10.1016/j.sjbs.2015.10.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Javaid M.A., Younas M., Zafar I., Khera R.A., Zia K.M., Jabeen S. Mathematical modeling and experimental study of mechanical properties of chitosan based polyurethanes: Effect of diisocyanate nature by mixture design approach. Int. J. Biol. Macromol. 2019;124:321–330. doi: 10.1016/j.ijbiomac.2018.11.183. [DOI] [PubMed] [Google Scholar]
  14. Jiang Y., Yuan Z., Huang J. Substituted hydroxyapatite: a recent development. Mater. Technol. 2019:1–12. [Google Scholar]
  15. Kim S.W., Baek Y.W., An Y.J. Assay-dependent effect of silver nanoparticles to Escherichia coli and Bacillus subtilis. Appl. Microbiol. Biotechnol. 2011;92:1045–1052. doi: 10.1007/s00253-011-3611-x. [DOI] [PubMed] [Google Scholar]
  16. Kassem A., Ayoub G.M., Malaeb L. Antibacterial activity of chitosan nano- composites and carbon nanotubes: a review. Sci. Total Environ. 2019;668:566–576. doi: 10.1016/j.scitotenv.2019.02.446. [DOI] [PubMed] [Google Scholar]
  17. Kolmas J., Groszyk E., Kwiatkowska-Różycka D. Substituted hydroxyapatites with antibacterial properties. BioMed Res. Int. 2014;2014:1–15. doi: 10.1155/2014/178123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li M., Liu Q., Jia Z., Xu X., Shi Y., Cheng Y., Zheng Y., Xi T., Wei S. Electrophoretic deposition and electrochemical behavior of novel graphene oxide- hyaluronic acid-hydroxyapatite nanocomposite coatings. Appl. Surf. Sci. 2013;284:804–810. [Google Scholar]
  19. Li Y., Clark C., Abdulazeeme H.M., Salehisahlabadi A., Rahmani J., Zhang Y. The effect of Brazil nuts on selenium levels, Glutathione peroxidase, and thyroid hormones: A systematic review and meta-analysis of randomized controlled trials. J. King Saud Univ.-Sci. 2020;32:1845–1852. [Google Scholar]
  20. Lems W.F., Raterman H.G. Critical issues and current challenges in osteoporosis and fracture prevention. An overview of unmet needs. Therapeutic Adv. Musculoskeletal Dis. 2017;9:299–316. doi: 10.1177/1759720X17732562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Limón-Martínez R.J., Olivas-Armendáriz I., Sosa-Rodarte E., Rodríguez-Rodríguez C.I., Hernández-Paz J.F., Acosta-Torres L.S., García-Contreras R., Santos-Rodríguez E., Martel-Estrada S.A. Evaluation of in vitro bioactivity and in vitro biocompatibility of Polycaprolactone/Hyaluronic acid/Multiwalled Carbon Nanotubes/Extract from Mimosa tenuiflora composites. Bio-Med. Mater. Eng. 2019;30:97–109. doi: 10.3233/BME-181036. [DOI] [PubMed] [Google Scholar]
  22. Mitran V., Ion R., Miculescu F., Necula M.G., Mocanu A.C., Stan G.E., Antoniac I.V., Cimpean A. Osteoblast Cell Response to Naturally Derived Calcium Phosphate- Based Materials. Materials. 2018;11:1097. doi: 10.3390/ma11071097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mehrotra S., Moses J.C., Bandyopadhyay A., Mandal B.B. 3D printing/bioprinting based tailoring of in vitro tissue models: Recent advances and challenges. ACS Appl. Bio Mater. 2019;2:1385–1405. doi: 10.1021/acsabm.9b00073. [DOI] [PubMed] [Google Scholar]
  24. Nandi S.K., Roy S., Mukherjee P., Kundu B., De D.K., Basu D. Orthopaedic applications of bone graft & graft substitutes: a review. Indian J. Med. Res. 2010;132:15–30. [PubMed] [Google Scholar]
  25. Oryan A., Alidadi S., Moshiri A., Maffulli N. Bone regenerative medicine: classic options, novel strategies, and future directions. J. Orthopaedic Surg. Res. 2014;9:18. doi: 10.1186/1749-799X-9-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Offner D., de Grado G.F., Meisels I., Pijnenburg L., Fioretti F., Benkirane-Jessel N., Musset A.M. Bone Grafts, Bone Substitutes and Regenerative Medicine Acceptance for the Management of Bone Defects Among French Population: Issues about Ethics, Religion or Fear? Cell Med. 2019;11 doi: 10.1177/2155179019857661. 2155179019857661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Price G.J., Bibi S., Imran Z., Nawaz M., Yasin T., Farooq A. Comparison of the effects of gamma or sonochemical irradiation of carbon nanotubes and the influence on the mechanical and dielectric properties of chitosan nanocomposites. Ultrasonics Sonochem. 2019;54:241–249. doi: 10.1016/j.ultsonch.2019.01.033. [DOI] [PubMed] [Google Scholar]
  28. Park J.E., Jang Y.S., Bae T.S., Lee M.H. Biocompatibility Characteristics of Titanium Coated with Multi Walled Carbon Nanotubes—Hydroxyapatite Nanocomposites. Materials. 2019;12:224. doi: 10.3390/ma12020224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pei B., Wang W., Dunne N., Li X. Applications of Carbon Nanotubes in Bone Tissue Regeneration and Engineering: Superiority, Concerns, Current Advancements, and Prospects. Nanomaterials. 2019;9:1501. doi: 10.3390/nano9101501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ramya R., Venkatesan J., Kim S.K., Sudha P.N. Biomedical applications of chitosan: an overview. J. Biomater. Tissue Eng. 2012;2:100–111. [Google Scholar]
  31. Rodrigues, B. V., Leite, N. C., das Neves Cavalcanti, B., da Silva, N. S., Marciano, F. R., Corat, E. J., Webster, T. J., Lobo, A. O. 2016. Graphene oxide/multi-walled carbon nanotubes as nanofeatured scaffolds for the assisted deposition of nanohydroxyapatite: characterization and biological evaluation. International journal of nanomedicine, 11, 2569. [DOI] [PMC free article] [PubMed]
  32. Spinks G.M., Shin S.R., Wallace G.G., Whitten P.G., Kim S.I., Kim S.J. Mechanical properties of chitosan/CNT microfibers obtained with improved dispersion. Sens. Actuat. B. 2006;115:678–684. [Google Scholar]
  33. Souza V.G.L., Fernando A.L., Pires J.R.A., Rodrigues P.F., Lopes A.A., Fernandes F.M.B. Physical properties of chitosan films incorporated with natural antioxidants. Ind. Crops Prod. 2017;107:565–572. [Google Scholar]
  34. Sánchez A.G., Prokhorov E., Luna-Barcenas G., Kovalenko Y., Rivera-Muñoz E.M., Raucci M.G., Buonocore G. Effect of Chemical Oxidation Routes on the Properties of Chitosan-MWCNT Nanocomposites. Current Nanosci. 2019;15(6):618–625. [Google Scholar]
  35. Song J.E., Tian J., Kook Y.J., Thangavelu M., Choi J.H., Khang G. A BMSCs-laden Quercetin/Duck's Feet Collagen/Hydroxyapatite Sponge for Enhanced Bone Regeneration. J. Biomed. Mater. Res. A. 2019;108:784–794. doi: 10.1002/jbm.a.36857. [DOI] [PubMed] [Google Scholar]
  36. Tsai S.W., Yu W.X., Hwang P.A., Huang S.S., Lin H.M., Hsu Y.W., Hsu F.Y. Fabrication and characterization of strontium-substituted hydroxyapatite-CaO- CaCO3 nanofibers with a mesoporous structure as drug delivery carriers. Pharm. 2018;10:179. doi: 10.3390/pharmaceutics10040179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Venkatesan J., Kim S.K. Effect of temperature on isolation and characterization of hydroxyapatite from tuna (Thunnus obesus) bone. Materials. 2010;3:4761–4772. doi: 10.3390/ma3104761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wang S.F., Shen L., Zhang W.D., Tong Y.J. Preparation and mechanical properties of chitosan/carbon nanotubes composites. Biomacromolecules. 2005;6:3067–3072. doi: 10.1021/bm050378v. [DOI] [PubMed] [Google Scholar]
  39. Wu J., Wu Z., Xue Z., Li H., Liu J. PHBV/bioglass composite scaffolds with co- cultures of endothelial cells and bone marrow stromal cells improve vascularization and osteogenesis for bone tissue engineering. RSC Adv. 2017;7:22197–22207. [Google Scholar]
  40. Wang D., Mu X., Cai W., Zhou X., Song L., Ma C., Hu Y. Nano-bridge effects of carbon nanotubes on the properties reinforcement of two-dimensional molybdenum disulfide/polymer composites. Compos. Part A: Appl. Sci. Manufact. 2019;121:36–44. [Google Scholar]
  41. Xie Y., Zhang L., Xiong Q., Gao Y., Ge W., Tang P. Bench-to-bedside strategies for osteoporotic fracture: From osteoimmunology to mechanosensation. Bone Res. 2019;7:1–13. doi: 10.1038/s41413-019-0066-7. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Saudi Journal of Biological Sciences are provided here courtesy of Elsevier

RESOURCES