Preparation of Diatom-Doped Bio-Nanocomposite Materials for Bone Tissue Scaffolds

Abstract

Naturally sourced materials have an important place in bone tissue engineering due to their biocompatibility and biodegradability. Non-diatom, diatom-doped chitosan/hydroxyapatite (CS/HAp) and collagen/chitosan/hydroxyapatite (Col/CS/HAp) as three-dimensional tissue scaffolds were produced by freeze drying technique. It was determined by SEM analysis that CS/HAp, CS/HAp/Di, Col/CS/HAp, Col/CS/HAp/Di scaffolds have 160 μm, 130 μm, 390 μm and 340 μm pores, respectively. The diatoms in the structure have approximately 9-16 μm in length, 8-20 μm in diameter and nanopore sizes of 260-330 nm. Cell culture studies were performed using the 3T3 cell line to study the non-toxic nature of biocomposite scaffolds that support cell attachment and proliferation. The cells in the scaffolds without diatom proliferate in a reticulated manner, whereas in the scaffolds containing diatom the cells were wrapped around the scaffold like a cover. The suggested scaffolds have the potential to meet the basic requirements in biocompatibility, cytocompatibility and interconnected pore structure.

Keywords:
Bio-nanocomposite; Bone-tissue; Diatom; Scaffold; Tissue engineering scaffold

1. Introduction

Large numbers of people suffer from bone defects caused by accident, injury, trauma, tumors, or bone-related diseases¹. Bone is a living and porous tissue that protects the body's organs and soft tissues. It functions as blood production and mineral reserves². As is known, biologically produced bone forms have self-healing properties. In addition, large bone defects do not heal spontaneously and require surgical intervention for treatment³. Clinically used bone grafts can be divided into two main types, biological and synthetic, according to their origin.

Disadvantages of biological grafts may include immune rejection and biocompatibility issues⁴. In order to solve these problems, suitable synthetic materials are being researched more and more every day. Bone tissue engineering, which works towards the production of synthetic grafts that includes intradisciplinary sciences, plays an important role in achieving this goal^5,6.

Bone tissue engineering aims to simulate the tissues in biological systems in the best way by using bone tissue scaffolds, cells and growth factors alone or together to repair bone damages or reconstruct damaged tissue⁷. Polymers and their derivatives have been investigated in many areas since 1990, because of their environment-friendly nature and practical applications in biomedical, pharmaceutical, and cosmetic fields from micro to nanoscale engineering^8,9. In doing so, it uses biocompatible polymers that can mimic the extracellular matrix and tries to regenerate or repair damaged or dysfunctional tissue skeletons in the human body¹⁰. The most important feature of tissue scaffolds to be used in bone tissue engineering is that they are osteoinductive and/or osteoconductive. Osteoinductive tissue skeletons allow osteoprogenitor cells to attach to the scaffold, migrate to the scaffold, differentiate and ultimately form new bone. Osteoconductive tissue scaffolds enable the formation of the 3-dimensional structure of the bone by supporting the formation of the capillary structure of the bone and the proliferation of bone cells by directing the cells from the main tissue to the material^11-13. Apart from these features, the tissue scaffolds to be used must be biocompatible, porous and biodegradable and at the same time have sufficient mechanical strength since they will be exposed to mechanical stress when implanted in the body.

Hydrogel, a natural 3D scaffold, can be prepared from synthetic or natural polymers. Compared to synthetic hydrogels, natural hydrogels tend to have greater inherent biocompatibility and desirable biodegradability^14-16. Hydrogels obtained from various materials emerge as an approach in biomedical in the field of tissue engineering. Recent research shows that it focuses on hydrogels carried out biological materials. These hydrogels can be prepared from protein structured polymers such as collagen, gelatin, and/or polysaccharide polymers such as chitosan, alginate^17,18. In addition, bio-ceramic hydroxyapatite and diatom with a bioactive structure in the composite hydrogel structure prepared to form the tissue scaffold can also be used¹⁹.

Diatoms are unicellular eukaryotic organisms that live in aqueous environments and are the largest source of biosilica formation. Diatoms of a wide variety of shapes form an amorphous silica shell with symmetrically dispersed nano-micropores with high mechanical stability^20,21. Diatom is a cheap and unlimited source of biogenic silica^22,23. Thanks to its unique porosity and morphology, it has been proposed for use in drug delivery systems, bio-fixing agents, molecular catalysis and photonics applications^24-26. As silicon plays an important role in bone formation, regeneration and mineralization, we think diatom, an inexpensive source of silica, will be a promising natural resource in bone tissue engineering. As a result of the research we have done, we see that our opinion is supported by encountering a very limited number of studies aimed at this purpose in recent years^20-22. In addition to inorganic materials such as hydroxyapatite, zirconia, glass ceramic, tri-calcium phosphate, the use of amorphous silica particles has been proposed to support mineralization in bone regeneration studies^22,27,28. In addition, silica has been used successfully with hydroxyapatite to increase osteo conductivity for bone regeneration^29,30.

Hydroxyapatite has similar components and structure to natural bone. It also has good biodegradability, biocompatibility and osteoconductivity. It also has the function of absorbing and accumulating calcium ions in body fluids and can support bone regeneration in polymer- based composites^31,32. There is evidence that HAp plays a role in biological processes such as angiogenesis, wound healing, ECM (Extra Cellular Matrix) organization, and inflammation³³. HAp derivatives are successfully used as scaffolding materials to treat vascular diseases due to their properties of bone and skin tissue regeneration, chondrocyte growth, biocompatibility and anti-inflammation³⁴. However, its use alone in tissue engineering applications is limited due to its non-biodegradable, poor mechanical properties, and processing difficulties³⁵. Therefore, hydroxyapatite is used by being included in composite polymer hydrogels.

Chitosan is the second most abundant bio-polysaccharide in the world, created by the deacetylation of chitin produced from shellfish, insects and fungi³⁶. Chitosan is currently a material of great interest in tissue engineering³⁷. The mechanical and physical properties of the hydrogel formed are directly related to the deacetylation degree and molecular weight of chitosan. Chitosan used in composite hydrogels has low cost, antibacterial, biodegradable, biocompatible and bioactive, easy to sterilize preparties. All these features can be controlled by changing the deacetylation level³⁸. Their disadvantages are that they are easily affected by parameters such as pH and temperature. It also shows poor mechanical properties³⁹. Therefore, it must be compounded with other materials such as hydroxyapatite, calcium phosphate, gelatin and alginate while forming a hydrogel⁴⁰.

Collagen is the most abundant ECM protein and provides an appropriate environment for cell adhesion and signaling molecules^41,42. Until now, collagen has been used in the repair and renewal of many tissues such as bones, skin and heart⁴³. Biologically, collagen has positive properties such as low inflammatory response and low antigenicity, biodegradability and biocompatibility⁴⁴. Collagen and collagen-based materials play an important role in maintaining the structural integrity and biological function of tissues. Therefore, it is widely used in tissue regeneration and tissue engineering studies⁴⁵. Unlike collagen in natural tissues, mechanical strength is insufficient in collagen-based biomaterials for the absence of covalent crosslinking. Because the crosslinking is performed by various methods to increase the mechanical performance of the tissue⁴⁶. Besides these, many studies have been done using various manufacturing techniques, including different synthetic materials and/or combinations with biomolecules⁴⁷.

In recent years, biopolymers have been used together with nanomaterials on bone tissue engineering studies. The freeze drying technique has been the subject of many research in terms of its ease and successful results. The scaffolds based on the cellulose-graft-polyacrylamide/nHA semi-IPN nanocomposite can bind to living bone through the formation of apatite layers on its surface can be used in bone tissue engineering⁴⁸. Chitosan/Alginate/Diatom scaffolds have been fabricated an alternative potential in the field of tissue engineering because of its high porous and non-toxic properties⁴⁹. Nanoclay particles were incorporated into polyvinyl alcohol–chitosan to improve the mechanical properties and bioactivity for bone tissue replacement applications⁵⁰. Novel biocompatible nanocomposite scaffolds have been prepared by freeze drying method using TiO₂ doped in grafted chitosan/hydroxyapatite for bone tissue engineering applications⁵¹. Alginate and hyaluronic acid hydrogel polymers reinforcing with titanium oxide nanoparticles has been developed for orthopedic field. This nanocomposite was prepared using freeze drying technique⁵². Hydroxyapatite and polymethylmethacrylate was fabricated to obtain porous polymeric-ceramic material⁵³.

Designing biomaterials for bone tissue engineering applications is still a challenge regarding the natural complex structure of hard tissues. In this study, in addition to chitosan/hydroxyapatite and gelatin/chitosan/hydroxyapatite hydrogels, diatom doped forms of these hydrogels were prepared. The liquid part of these four different bio-composite hydrogels, which were created to take advantage of the best properties of each material, was completely removed by freeze drying, and as a result, porous, 3-dimensional scaffolds were formed. Scaffolds produced in this combination for the first time were examined by SEM, FT-IR and cell culture studies.

2. Experimental Section

2.1. Materials

Chitosan (medium molecular weight) was purchased from Sigma-Aldrich (product of Iceland). Hydroxyapatite (nano-powder), Collagen Type-I (from calfskin) and diatomaceous earth (suitable for most filtrations) were purchased from Sigma-Aldrich (USA). Acetic Acid (Glacial, %100 Anhydrous), a solvent for chitosan and collagen, was obtained from ISOLAB chemicals (Wertheim, Germany). Glutaraldehyde used in crosslinking hydrogels was purchased from Merck (USA). DMEM (Dulbecco's Modified Eagle Medium), which was used as a medium for cell culture studies, was purchased from Sigma–Aldrich (USA). 3T3 cell line was purchased from the European Validated Cell Culture Collection (ECACC). All chemicals used in this study were at the analytical level.

2.2. Preparation of scaffolds

Chitosan and Collagen solution; Chitosan (3%, w/v) was dissolved in 1% acetic acid solution, which was stirred for 24 hours at 50 °C. Collagen (2.5%, w/v) was prepared by stirring in 1% acetic acid for 12 hours at 40 °C. With the preparation of chitosan and collagen solutions, biocomposite hydrogels with four different contents were prepared. The term BTS is encoded as the abbreviation of Bone Tissue Scaffold. In our study, four different mixtures have been called BTS-1 (CS/HAp), BTS-2 (CS/HAp/Di), BTS-3 (Col/CS/HAp) and BTS-4 (Col/CS/HAp/Di). The preparation of these four mixtures is given below, respectively.

BTS-1: 2 g of hydroxyapatite was added to 20 ml of chitosan solution and mixed until homogeneous.

BTS-2: 0.1 g of diatom was added to 20 ml of BTS-1 solution and mixed until homogeneous.

BTS-3: 10 ml of chitosan and 10 ml of collagen solution was mixed and 2 g of hydroxyapatite was added to this solution and mixed until homogeneous.

BTS-4: 0.1 g of diatom was added to 20 ml of BTS-3 solution and mixed until homogeneous.

In summary, Table 1 provides information on the materials contained in each composite scaffold.

Table 1
Bio-composite scaffolds and their ingredients.

	CS	HAp	Col	Di
BTS-1	✓	✓
BTS-2	✓	✓		✓
BTS-3	✓	✓	✓
BTS-4	✓	✓	✓	✓

These four hydrogels were kept overnight at -20 ^oC after crosslinking with glutaraldehyde (2.5%, v/v). It was then lyophilized (freeze dried) for 48 hours. In Figure 1, the visual of the scaffolds formed after the freeze drying (lyophilization) (Labconco FreeZone -105 ⁰C, USA) process is given.

Figure 1
Bone tissue scaffolds formed after freeze drying: (a) BTS-1, (b) BTS-2, (c) BTS-3 and (d) BTS-4.

2.3. Characterization studies

Scanning electron microscopy (SEM) (Zeiss Supra 40VP, Germany) was used to view the morphology of composite scaffolds before and after cell culture⁵⁴. The chemical bonds and functional groups of biocomposite tissue scaffolds were examined using Fourier Transform Infrared Spectroscopy (FT-IR) (Thermo Scientific Nicolet iS50, Germany) at a resolution of 0.5 cm^-1 and a frequency of 400-4000 cm^-1.

2.4. Cell culture studies

2.4.1. Cell line preparation and maintenance

Studies of cell culture were performed with the 3T3 mouse embryonic fibroblastic cell line (ECACC, UK). Cells were cultured in Petri dishes using Dulbecco’s modified Eagle’s medium (DMEM; Sigma, Germany) containing 10% FBS and 1% penicillin-streptomycin. Cells were subcultured every two days by keeping them in an incubator (EC-160, Nüve, Turkey) with 37°C, 95% humidity and 5% CO₂ environment prior to seeding.

2.4.2. Cell seeding into tissue scaffolds

Cell culture was carried out in sterile well culture dishes. Before cell seeding, the bottom of the culture dishes was covered with parafilm and washed with alcohol. Thus, the bottom of the cell culture dish was made hydrophobic, preventing the cells from migrating from the tissue scaffold to the surface of the culture dish. On the other hand, biocomposite bone tissue scaffolds were washed with 70% alcohol and left to dry. Then scaffolds and culture dishes were sterilized under UV light for 45 minutes. Before cell seeding, scaffolds were placed in culture dishes and kept in DMEM for 24 hours to interact with serum proteins. At the end of 24 hours, 1x10⁴ cells were seeded in each medium containing a scaffold. During 8 days⁵⁵ of cell culture, the culture medium was renewed every 2-3 days.

2.4.3. Morphological analysis

The culture medium on the tissue scaffolds was removed and the scaffolds were washed twice with PBS (Biowest, France). The cells were fixed by soaking the tissue scaffolds in 2.5% (v/v) glutaraldehyde solution for 30 minutes. The scaffolds were kept in 30%, 50%, 70%, 90% and 100% (v/v) ethanol solutions for 2 minutes, respectively, and dehydration was performed⁵⁶. It was then kept in hexamethyldisilazane (HMDS; BRB, Netherland) for 5 minutes and allowed to dry at room temperature. Scaffolds were made conductive by coating with gold-palladium for 400 seconds for SEM analysis.

2.4.4. Cytotoxicity studies

Cytotoxicity tests were performed to determine whether bone scaffolds have cytotoxic potential. Mouse embryonic fibroblast cells (3T3) were used for this purpose. This cell line was used for many purposes including biomaterial science⁵⁷. Briefly, 2x10³ cells were seeded in each wells of 96-well plate in DMEM containing 10% FBS and 1% penicillin/streptomycin mixture with the humidified atmosphere (95% air with 5% CO2). The medium was removed after 24 h and medium containing different amounts of BTS samples extracted by the methods of Lin et al. (2013)⁵⁸ with slight modifications. After 24 hours of treatment, cell viability was determined by MTT method as described by Konus et al. (2020)⁵⁹.

2.4.5. Statistical analysis

All experiments were run in triplicates. Statistical analyses were performed using Student’s ttest for multiple comparisons (Minitab Software). Data are expressed as that mean value (±SD*P<0.05) is considered significant

3. Results and Discussion

3.1. SEM analysis

As seen in Figure 2, SEM images of non-diatom BTS-1 and BTS-3 scaffolds are given at different magnifications. Here, it is seen that BTS-1 and BTS-3 scaffolds have approximately 160 μm and 390 μm macroporosities, respectively. It has been observed through SEM that the BTS-3 scaffold, unlike BTS-1, has larger pores due to the collagen it contains.

Figure 2
Morphological images of non-diatom BTS-1 scaffold (a) 250x, (b) 500x, (c) 1000x and BTS-3 scaffold (d) 250x, (e) 500x, (f) 1000x.

In Figure 3, SEM images of diatom containing BTS-2 and BTS-4 scaffolds are given at different magnifications. Unlike BTS-1 and BTS-3 scaffolds that do not contain diatom, it has been observed that these scaffolds have macro and micro pores as well as nanopores originating from diatom. It has been observed that BTS-2 and BTS-4 scaffolds have pores from macro to nano at different magnifications. Here, it is seen that BTS-2 and BTS-4 scaffolds have approximately

Figure 3
Morphological images of diatom BTS-2 scaffold (a) 250x, (b) 2500x, (c) 10000x and BTS-4 scaffold (d) 250x, (e) 2500x, (f) 10000x.

130 μm and 340 μm macroporosities, respectively. It has been shown through SEM that the BTS-4 scaffold, unlike BTS-2, has larger pores due to the collagen it contains. It has been seen that the same type of diatoms found in these scaffolds are approximately 9-16 μm in length and 8-20 μm in diameter. It has also been observed that these diatoms have nanopore sizes of 260330 nm at regular intervals.

3.2. FT-IR analysis

FT-IR result graph of bone tissue scaffolds is given in Figure 4. The FT-IR data of the structures forming the scaffolds are given separately. It was determined that the OH groups of HAp in the

Figure 4
FT-IR graph of biocomposite scaffolds: (a) BTS-1, (b) BTS-2, (c) BTS-3 and (d) BTS-4.

BTS-1, BTS-2, BTS-3 and BTS-4 scaffolds were located in the asymmetric stretch band at 3288, 3290, 3291, 3284 cm^-1 levels, and in the asymmetric bending band at 1647, 1652, 1652, 1647 cm^-1 levels, respectively. It was determined that the PO₄^-3 groups of HAp were located in the asymmetric stretch band at the levels of 559, 558, 558,559 cm^-1 and in the asymmetric bending band at the levels of 1016, 1012, 1015, 1016 cm^-1, respectively^60-62.

The 892-1152 cm^-1 characteristic peaks of chitosan are located in the C-O-C asymmetric stretch band. In addition, it was observed that C=O stretching (amide I) at the 1633 cm^-1 level and NH bending (amide II) band at the 1537 cm^-1 level. The collagen specific 1630, 3324 cm^-1 levels were located in the N-H stretching (amide I) band, 1543 cm^-1 level was located in the C-N stretching and N-H bending (amide II) band. The peak in the range of 1076-1100 cm-1 of the diatom shows the vibrations in the asymmetric Si-O Si bonds, and the peak in the range of 750850 cm^-1 shows the vibrations in the symmetrical Si-O-Si bonds. The peak at 600 cm^-1 levels is due to the crystal structure of the diatom^60-64.

FT-IR results show that the prepared compounds are rich with in functional groups such as carboxylic, amino and amide groups. Characteristic bands of both hydroxyapatite and chitosan compounds were present in the material. Depending on the chemical bonds between chitosan and calcium/phosphate ions, phosphate ions and calcium can be homogeneously retained in the polymerized precursor on a molecular scale.

3.3. Results of cell culture studies

In this study, cell culture studies were carried out under stagnant conditions for 8 days with the 3T3 cell line using the tissue scaffolds synthesized. Viability, attachment and morphology of 3T3 cells on the prepared scaffolds were investigated by the analyzes made during the culture and the effect of diatom-doped scaffolds on differentiation was evaluated by comparing them with other scaffolds.

The morphology of day 8 of 3T3 cells cultured on the produced tissue scaffolds is given in Figure 5. It is clear that cells attach to the scaffold, spread out and migrate into intrinsically linked pores. It was observed that the cells in the scaffolds without diatom proliferate in a reticulated manner, whereas in the scaffolds containing diatom the cells were wrapped around the scaffold like a cover. It is understood that diatom supports more cell proliferation thanks to its bioactive property.

Figure 5
SEM images of diatom-free BTS-1 (a) and BTS-3 (c) scaffolds and diatom-doped BTS-2 (b) and BTS-4 (d) scaffolds with 3T3 cells after 8 days of cell culture.

3.4. Results of cytotoxicity studies

As a result of the studies obtained, effective doses were found for the 3T3 cell line with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, as seen in Figure 6. The results are the mean values of the triplicate measurement of two different cytotoxicity assays.

Figure 6
Effect of scaffolds at different concentrations on 3T3 cell viability.

It was determined that BTS scaffolds used at different doses did not cause suspicious toxic effects on cells. The viability of cells was decreased slightly at higher doses but they were not found statistically significant (Figure 6).

4. Conclusions

This work aimed to investigate the properties of a biocomposite based on blends of biopolymers and hydroxyapatite with the addition of diatom to develop potential scaffold material. The use of biocomposite structures, such as collagen, chitosan, hydroxyapatite and diatom for scaffold preparation by freeze drying is beneficial because it can increase the biocompatibility of the material, physical and chemical properties. Looking at the morphological observation by SEM of the scaffolds, it was observed that the macropores of the scaffolds containing diatoms were slightly smaller than the scaffolds without diatoms and diatom-specific nanopores were observed. It was determined that the macropores of the collagen-containing scaffolds were larger. These pores are critical in cell attachment, differentiation and ECM formation. In addition, diatom-specific extra nanopores will further support these effects. In addition, the FTIR analysis that the asymmetric tension and bending bands of the structural bonds in the tissue scaffolds were inconsistent peaks.

As a result of cell culture studies, while cells proliferated in a reticulate manner in diatom-free scaffolds, cells in diatom-containing scaffolds completely covered the scaffolds like a cover. It is thought that the bioactive feature of the diatom promotes cell adhesion and proliferation more. Ultimately, it was determined that the scaffolds were highly biocompatible and have ideal pores as the cells proliferated by adhering to the scaffolds.

As a result of the cytotoxicity studies, it was concluded that BTS-1, BTS-2, BTS-3 and BTS-4 scaffolds did not cause toxic effects on 3T3 cells at any of the applied dose levels. With these results, the scaffolds make a positive contribution to the literature with the materials they contain. Diatom can be used as a source of bioactive silica with its unique nanopores in order to improve the osteoinductive properties of tissue scaffolds in bone tissue engineering studies.

The developed biocomposite scaffolds have promising potential for bone tissue engineering.

5. Acknowledgments

The authors would like to thank the scientific research council of Pamukkale University, Turkey, for research grant 2019FEBE052. The authors thanks to Prof. Dr. Cem GÖK and Molecular Biochemistry Laboratory of Biology Department of Pamukkale University for providing device usage and supports.

6. References

• ¹
  Murugan R, Ramakrishna S. Bioresorbable composite bone paste using polysaccharide based nano hydroxyapatite. Biomaterials. 2004;25:3829-35.
• ²
  Preethi Soundarya S, Haritha Menon A, Viji Chandran S, Selvamurugan N. Bone tissue engineering: scaffold preparation using chitosan and other biomaterials with different design and fabrication techniques. Int J Biol Macromol. 2018;119:1228-39.
• ³
  Hollister SJ, Maddox RD, Taboas JM. Optimal design and fabrication of scaffolds to mimic tissue properties and satisfy biological constraints. Biomaterials. 2002;23:40954103.
• ⁴
  Flanagan TC, Wilkins B, Black A, Jockenhoevel S, Smith TJ, Pandit AS. A collagenglycosaminoglycan co-culture model for heart valve tissue engineering applications. Biomaterials. 2006;27(10):2233-46.
• ⁵
  Chen Q-Z, Bismarck A, Hansen U, Junaid S, Tran MQ, Harding SE, et al. Characterization of a soft elastomer poly (glycerol sebacate) designed to match the mechanical properties of myocardial tissue. Biomaterials. 2008;29(1):47-57.
• ⁶
  Karp JM, Langer R. Development and therapeutic applications of advanced biomaterials. Curr Opin Biotechnol. 2007;18(5):454-9.
• ⁷
  Ratner BD, Hoffman AS, Schoen FJ, Lemons JE. Biomaterials science: an introduction to materials in medicine. California: Elsevier Academic Press; 2013.
• ⁸
  Özcan Y, İde S, Jeng U, Bütün V, Lai YH, Su CH. Micellization behavior of tertiary amine-methacrylate-based block copolymers characterized by small-angle X-ray scattering and dynamic light scattering. Mater Chem Phys. 2013;138(2-3):559-64.
• ⁹
  Özcan Y, Orujalipoor I, Huang YC, Bütün V, Jeng US. Self-assembled and nanostructured copolymer aggregations of the tertiary amine methacrylate based triblock copolymers. Anal Lett. 2015;48(17):2693-707.
• ¹⁰
  Griffith LG, Naughton G. Tissue engineering-current challenges and expanding opportunities. Science. 2002;295:1009-14.
• ¹¹
  Wei G, Ma PX. Structure and properties of nano-hydroxyapatite/polymer composite scaffolds for bone tissue engineering. Biomaterials. 2004;25(19):4749-57.
• ¹²
  Albrektsson T, Johansson C. Osteoinduction, osteoconduction and osseointegration. Eur Spine J. 2001;10:S96-101.
• ¹³
  Burg KJL, Porter S, Kellam JF. Biomaterial developments for bone tissue engineering. Biomaterials. 2000;21:2347-59.
• ¹⁴
  Ahmed EM. Hydrogel: Preparation, characterization, and applications: a review. J Adv Res. 2015;6(2):105-21.
• ¹⁵
  Bedian L, Villalba-Rodríguez AM, Hernández-Vargas G, Parra-Saldivar R, Iqbal HMN. Bio-based materials with novel characteristics for tissue engineering applications–A review. Int J Biol Macromol. 2017;98:837-46.
• ¹⁶
  Catoira MC, Fusaro L, Di Francesco D, Ramella M, Boccafoschi F. Overview of natural hydrogels for regenerative medicine applications. J Mater Sci Mater Med. 2019;30:115-25.
• ¹⁷
  Francis L, Greco KV, Boccaccini AR, Roether JJ, English NR, Huang H, et al. Development of a novel hybrid bioactive hydrogel for future clinical applications. J Biomater Appl. 2018;33(3):447-65.
• ¹⁸
  Gok C, Aytas S. The role of colloidal systems in environmental protection. California: Elsevier Academic Press; 2014.
• ¹⁹
  Wang F, Li MS. A biomimetic method of hydroxyapatite powders synthesized in simulated body fluid. Key Eng Mater. 2005;297-300:1371-5.
• ²⁰
  Dalgic AD, Atila D, Karatas A, Tezcaner A, Keskin D. Diatom shell incorporated PHBV/PCL-pullulan co-electrospun scaffold for bone tissue engineering. Mater Sci Eng C. 2019;100:735-46.
• ²¹
  Dimas LS, Buehler MJ. Influence of geometry on mechanical properties of bio-inspired silica-based hierarchical materials. Bioinspir Biomim. 2012;7(3):036024.
• ²²
  Le TDH, Bonani W, Speranza G, Sglavo V, Ceccato R, Maniglio D, et al. Processing and characterization of diatom nanoparticles and microparticles as potential source of silicon for bone tissue engineering. Mater Sci Eng C. 2016;59:471-9.
• ²³
  Yusan S, Gok C, Erenturk S, Aytas S. Adsorptive removal of thorium (IV) using calcined and flux calcined diatomite from Turkey: evaluation of equilibrium, kinetic and thermodynamic data. Appl Clay Sci. 2012;67:106-16.
• ²⁴
  Aw MS, Simovic S, Yu Y, Addai-Mensah J, Losic D. Porous silica microshells from diatoms as biocarrier for drug delivery applications. Powder Technol. 2012;223:52-8.
• ²⁵
  Dolatabadi JEN, de la Guardia M. Applications of diatoms and silica nanotechnology in biosensing, drug and gene delivery, and formation of complex metal nanostructures. Trends Analyt Chem. 2011;30(9):1538-48.
• ²⁶
  Losic D, Mitchell JG, Voelcker NH. Diatomaceous lessons in nanotechnology and advanced materials. Adv Mater. 2009;21(29):2947-58.
• ²⁷
  Tautzenberger A, Kovtun A, Ignatius A. Nanoparticles and their potential for application in bone. Int J Nanomedicine. 2012;7:4545-57.
• ²⁸
  Zhang YF, Zheng YF, Qin L. A comprehensive biological evaluation of ceramic nanoparticles as wear debris. Nanomedicine. 2011;7(6):975-82.
• ²⁹
  Xu W, Ganz C, Weber U, Adam M, Wolter D, Frerich B, et al. Evaluation of injectable silica-embedded nanohydroxyapatite bone substitute in a rat tibia defect model. Int J Nanomedicine. 2011;6:1543-52.
• ³⁰
  Gibson IR, Best SM, Bonfield W. Chemical characterization of silicon-substituted hydroxyapatite. J Biomed Mater Res. 1999;44(4):422-8.
• ³¹
  Hu X, Shen H, Yang F, Liang X, Wang S, Wu D. Modified composite microspheres of hydroxyapatite and poly(lactide-co-glycolide) as an injectable scaffold. Appl Surf Sci. 2014;292:764-72.
• ³²
  Roh HS, Jung SC, Kook MS, Kim BH. In vitro study of 3D PLGA/n-HAp/β-TCP composite scaffolds with etched oxygen plasma surface modification in bone tissue engineering. Appl Surf Sci. 2016;388:321-30.
• ³³
  Pankajakshan D, Agrawal DK. Scaffolds in tissue engineering of blood vessels. Can J Physiol Pharmacol. 2010;88(9):855-73.
• ³⁴
  Burdick JA, Prestwich GD. Hyaluronic acid hydrogels for biomedical applications. Adv Mater. 2011;23(12):H41-56.
• ³⁵
  Mitra J, Tripathi G, Sharma A, Basu B. Mucoadhesion and drug permeability of free mixed films of pectin and chitosan: an in vitro and ex vivo study. Eur J Pharm Biopharm. 2009;71:325-31.
• ³⁶
  Hagesaether E, Hiorth M, Sande SA. Scaffolds for bone tissue engineering: role of surface patterning on osteoblast response. RSC Advances. 2013;3:11073-94.
• ³⁷
  Li X, Zhang L, Yin X. Microstructure and mechanical properties of three porous Si3N4 ceramics fabricated by different techniques. Mater Sci Eng A. 2012;549:43-9.
• ³⁸
  Huang Y, Onyeri S, Siewe M, Moshfeghian A, Madihally SV. In vitro characterization of chitosan–gelatin scaffolds for tissue engineering. Biomaterials. 2005;26(36):7616-27.
• ³⁹
  Vieira S, da Silva Morais A, Silva-Correia J, Oliveira JM, Reis RL. Natural-based hydrogels: from processing to applications, encyclopedia of polymer science and technology. New Jersey: John Wiley & Sons; 2017.
• ⁴⁰
  Kim SH, Yeon YK, Lee JM, Chao JR, Lee YJ, Seo YB, et al. Precisely printable and biocompatible silk fibroin bioink for digital light processing 3D printing. Nat Commun. 2018;9:1620-34.
• ⁴¹
  Lee A, Hudson AR, Shiwarski DJ, Tashman JW, Hinton TJ, Yerneni S, et al. 3D bioprinting of collagen to rebuild components of the human heart. Science. 2019;365(6452):482-7.
• ⁴²
  Toosi S, Naderi-Meshkin H, Kalalinia F, Peivandi MT, HosseinKhani H, Bahrami AR, et al. PGA-incorporated collagen: toward a biodegradable composite scaffold for bone-tissue engineering. J Biomed Mater Res A. 2016;104(8):2020-8.
• ⁴³
  Pal P, Dadhich P, Srivas PK, Das B, Maulik D, Dhara S. Bilayered nanofibrous 3D hierarchy as skin rudiment by emulsion electrospinning for burn wound management. Biomater Sci. 2017;5(9):1786-99.
• ⁴⁴
  Marelli B, Achilli M, Alessandrino A, Freddi G, Tanzi MC, Farè S, et al. Collagen-reinforced electrospun silk fibroin tubular construct as small calibre vascular graft. Macromol Biosci. 2012;12(11):1566-74.
• ⁴⁵
  Hamzah MSA, Ng C, Zulkarnain NIS, Majid HA, Razak SIA, Nayan NHM. Entrapment of collagen on polylactic acid 3D scaffold surface as a potential artificial bone replacement. Mater Today Proc. 2021;46(Pt 4):1668-73.
• ⁴⁶
  Achilli M, Lagueux J, Mantovani D. On the effects of UV-C and pH on the mechanical behavior, molecular conformation and cell viability of collagen-based scaffold for vascular tissue engineering. Macromol Biosci. 2010;10(3):307-16.
• ⁴⁷
  Lin K, Zhang D, Macedo MH, Cui W, Sarmento B, Shen G. Advanced collagen-based biomaterials for regenerative biomedicine. Adv Funct Mater. 2019;29:1804943-59.
• ⁴⁸
  Saber-Samandari S, Saber-Samandari S, Kiyazar S, Aghazadeh J, Sadeghi A. In vitro evaluation for apatite-forming ability of cellulose-based nanocomposite scaffolds for bone tissue engineering. Int J Biol Macromol. 2016;86:434-42.
• ⁴⁹
  Özcan Y, Gönenmiş DE, Kızılhan E, Gök C. Highly porous biocomposite scaffolds fabricated by chitosan/alginate/diatom for tissue engineering. Polymer(Korea). 2022;46(4):455-62.
• ⁵⁰
  Zolghadri M, Saber-Samandari S, Ahmadi S, Alamara K. Synthesis and characterization of porous cytocompatible scaffolds from polyvinyl alcohol–chitosan. Bull Mater Sci. 2019;42(1):1-9.
• ⁵¹
  Abd-Khorsand S, Saber-Samandari S, Saber-Samandari S. Development of nanocomposite scaffolds based on TiO2 doped in grafted chitosan/hydroxyapatite by freeze drying method and evaluation of biocompatibility. Int J Biol Macromol. 2017;101:51-8.
• ⁵²
  Jamnezhad S, Asefnejad A, Motififard M, Yazdekhasti H, Kolooshani A, Saber-Samandari S, Khandan A. Development and investigation of novel alginate-hyaluronic acid bone fillers using freeze drying technique for orthopedic field. Nanomedicine Research Journal. 2020;5(4):306-15.
• ⁵³
  Mohammadzadeh Rad M, Saber-Samandari S, Sadighi M, Tayebi L, Mohammadi Aghdam M, Khandan AS. Macro-and micromechanical modelling of HA-Elastin scaffold fabricated using freeze drying technique. J Nanoanalysis. 2021;8(1):17-31.
• ⁵⁴
  Takanoglu D, Yılmaz K, Ozcan Y, Karabulut O. Structural, electrical and optical properties of thermally evaporated CdSe and In-doped CdSe thin films. Chalcogenide Lett. 2015;12(1):35-42.
• ⁵⁵
  Wissing TB, Bonito V, van Haaften EE, van Doeselaar M, Brugmans MMCP, Janssen HM, et al. Macrophage-driven biomaterial degradation depends on scaffold microarchitecture. Front Bioeng Biotechnol. 2019;7:87.
• ⁵⁶
  Mavis B, Demirtaş TT, Gümüşderelioğlu M, Gündüz G, Çolak Ü. Synthesis, characterization and osteoblastic activity of polycaprolactone nanofibers coated with biomimetic calcium phosphate. Acta Biomater. 2009;5(8):3098-111.
• ⁵⁷
  Bhatia SK, Yetter AB. Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements. Cell Biol Toxicol. 2008;24:315-9.
• ⁵⁸
  Lin WC, Lien CC, Yeh HJ, Yu CM, Hsu SH. Bacterial cellulose and bacterial cellulosechitosan membranes for wound dressing applications. Carbohydr Polym. 2013;94(1):603-11.
• ⁵⁹
  Konus M, Çetin D, Yılmaz C, Arslan S, Mutlu D, Kurt‐Kızıldoğan A, et al. Synthesis, biological evaluation and molecular docking of novel thiophene-based indole derivatives as potential antibacterial, GST inhibitor and apoptotic anticancer agents. ChemistrySelect. 2020;5(19):5809-14.
• ⁶⁰
  Mustafov SD, Sen F, Seydibeyoglu MO. Preparation and characterization of diatomite and hydroxyapatite reinforced porous polyurethane foam biocomposites. Sci Rep. 2020;10:13308-17.
• ⁶¹
  Öztürk Kiraz A, Kaya S, Gök C. Structural and electronic properties of nano hydroxyapatite. Acta Phys Pol A. 2020;137:1017-21.
• ⁶²
  Klinkaewnarong J, Swatsitang E, Maensiri S. Nanocrystalline hydroxyapatite powders by a chitosan–polymer complex solution route: synthesis and characterization. Solid State Sci. 2009;11(5):1023-7.
• ⁶³
  Shavandi A, El-Din Bekhit A, Ali MA, Sun Z. Bio-mimetic composite scaffold from mussel shells, squid pen and crab chitosan for bone tissue engineering. Int J Biol Macromol. 2015;80:445-54.
• ⁶⁴
  Hazar Yoruç AB, Karakaş A, Ayas E, Koyun A. Effect of precipitation method on properties of hydroxyapatite powders. Acta Phys Pol A. 2013;123:371-3.

Publication Dates

• Publication in this collection
  26 Sept 2022
• Date of issue
  2022

History

• Received
  10 May 2022
• Reviewed
  04 Aug 2022
• Accepted
  30 Aug 2022