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Nazarbayev University Repository


Academic year: 2023

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I hereby declare that this manuscript, entitled “3D PRINTING OF BIOCOMPATIBLE CRYOGELS FOR BONE TISSUE ENGINEERING”, is the result of my own work except for quotations and citations duly acknowledged. Natural biopolymers are highly regarded and commonly used in tissue engineering to create scaffolds that support living cells. For the construction of high-accuracy hydrogel scaffolds via 3D printing, the shear thinning properties of the commonly used bioinks lead to morphological compromises such as smaller pore diameters.

This composite formulation makes it possible to create highly porous and biocompatible hydrogel scaffolds with extra large pore sizes (d > 100 µm) using a combination of 3D printing and cryoglation techniques. These scaffolds have the potential to serve as platforms for various tissue engineering applications, and their morphological properties and cell viability data can be tailored accordingly. Overall, our approach provides a simple and cost-effective method of building hydrogel scaffolds with high accuracy.

Alexander Trifonov, for their guidance and help, as well as for the opportunity to work under their supervision. Figure 3.6 Using NIH/3T3 fibroblast cells, 3D printed 2.86% Gel/OxAlg cryogels were characterized in vitro. A) Illustrations of cryogels after cell seeding at days 1, 7, 14 and 21 using live/dead staining.


  • Human Bone
    • The function of bone in the body
    • Bone development process
    • Bone structure and composition
  • Bone Tissue Engineering (BTE)
    • Scaffolds in Bone Tissue Engineering (BTE)
    • Biomaterials used for Scaffolds Fabrication
    • Crosslinking in the 3D Printed Scaffolds
    • Morphology of scaffoldsand its effect on tissue development
    • Cryogelation
    • Stability and integrity of scaffold
    • Scaffold fabrication techniques
  • Thesis statement
  • Aims and Objectives
    • Aim
    • Objectives

Immature osteogenic cells can be found both in the marrow and in the deep layers of the periosteum. They can be found on the surface of the bone, as well as in parts of the bone that are redundant, diseased, or ancient. These factors can be defective, leading to osteopetrosis, osteopetrosis incomplete, and Paget's disease of bone, among other disorders [7].

The extracellular matrix of the bone communicates topographical and chemical information to the cells in bone ultra-porous geometry [8,9]. Due to the high level of complexity and data variance, mechanical properties in bone microstructure cannot be accurately predicted by mathematical models [12,13]. One of the central areas of regenerative medicine is bone tissue engineering (BTE).

By creating hierarchical structures through ice templating, the porosity of the scaffolds can be controlled. One naturally occurring substance, chitosan, shares the same structural features as the glycosaminoglycans that make up most of the ECM. Another abundant natural element is HAp, which is also similar to bone tissue and an organic component of the hierarchical bone structure.

The porosity and pore dispersion of the scaffold allows inappropriate infiltration and ingrowth of cells. From the point of view of micropores, a small pore size reduced the permeability of the scaffold and cell migration. There is a correlation between the scaffold's porosity, pore size, shape, orientation and mechanical properties.

These results indicate that the porosity of the framework should exceed an average pore size of 150 μm [17,18]. First, most existing techniques only allow the fabrication of scaffolds with predetermined, fixed porosity. During the degradation process, the internal structural bonds of the polymer frameworks were broken, resulting in a decrease in molecular weight.

A wide range of applications in tissue engineering and regenerative medicine tailored to the needs of individual patients have made additive manufacturing, often known as 3D printing, one of the most revolutionary technologies in the modern world. Furthermore, we speculated that the mechanical stability, as well as the degradation rate of the 3D macroporous structure, could be controlled by the concentration of the polymer content in the ink, while maintaining the same level of low cytotoxicity.

Figure 1.1 Bone cells types (Osteocyte, Osteoblast, Osteogenic cell, and Osteoclast) [3]
Figure 1.1 Bone cells types (Osteocyte, Osteoblast, Osteogenic cell, and Osteoclast) [3]


  • Chemicals
  • Synthesis of biopolymer and formulation of ink
  • Shear thinning properties
  • Mechanical and morphological description
    • Morphological studies
    • Swelling Test
    • Deterioration Anaylsis
    • Accuracy (%)
    • Stress/strain testing
  • Cell culture
    • Scaffolds preparation for cell viability
  • Statistical analysis

Scaffolds were fabricated using a BIOX 3D printer manufactured by CELLINK in Sweden, maintaining temperature control via a temperature-controlled print bed and printhead. The scaffolds were printed at a uniform speed of 5 mm.s-1 and an extrusion pressure of 60–75 kPa to ensure uniform hydrogel fibers, with the pressure varying according to the ink composition. The carriers were then rinsed with water and lyophilized for 24 hours before long-term storage at 4 oC.

To perform morphological assessments of the 3D printed scaffolds, scanning electron microscopy was used with a JSM-IT200 (LA) model. To determine the swelling capacity, the lyophilized scaffolds were assessed by placing them in a 10 mM PBS solution and allowing them to stand at room temperature for 60 and 300 minutes. At specified intervals, the samples were removed from the water, dried to remove excess water on the surface, and weighed.

Four carriers (total N = 4) were selected from each composition, washed, lyophilized and reweighed after 1, 7, 14 and 21 days. To evaluate printing accuracy, the dimensional compliance of the computer-aided design (CAD) model and construction was examined. PA was defined as the ratio between the practical and theoretical areas (to avoid) voids.

The texture analyzer (TA-XT2-Stable Micro Systems, U.K) was used to calculate the elastic modulus of the printed scaffolds. The stress-strain curve between 10% and 20% strain was analyzed and the slope of the linear part of the curve was calculated using Equation (4) to estimate the elastic modulus (E). Following the procedure described in [74], fibroblast cells of the NIH/3T3 line were seeded on sterilized, freeze-dried scaffolds and cultured in a CM at 37°C with 5% CO2.

On days 1, 7, 14 and 21, the viability of the cells in the scaffolds was assessed using live/dead staining. After the culture medium was aspirated, the scaffolds were stained for 40 minutes in an incubator at 37°C with 5% CO 2 using a staining medium containing 2 M calcein-AM, 4 μM ethidium homodimer and 5 g/ml Hoechst. The scaffolds were then cleaned (x3) with PBS and transferred to imaging chambers (ibidi, -Slide) filled with CM.


  • FTIR analysis of oxidized Alginate, formulations of bioink and their rheological properties
  • Printing and characterization of Gelatin/Oxidized Alginate cryogel scaffolds
  • Scanning electron microscope and mechanical results of the selected 2.86% w/v
  • Cell seeding efficiency on Gelatin/Oxidized Alginate scaffold (2.86% w/v)

The mass of the resulting scaffolds of different compositions was found to be less than 5%. Swelling and degradation kinetics of the scaffolds were studied by soaking them in PBS for 1 and 5 h. The water retention of the scaffolds was consistent over two weeks, with only a slight downward trend for the lower concentration scaffolds.

The lower swelling capacity of the 2.50% and 2.67% w/v scaffolds can be attributed to their disordered morphology and reduced total pore volume. The SC of the scaffolds was calculated after one and five hours, as well as their temporal water absorption and degradation rate in PBS at 37 oC (*#$ shows significant changes related to the 5 hour measurement . with p<0.05). A visual representation in the form of a web map summarizes the correlation between the ink concentration, PA, and the mechanical and morphological characteristics of the 3D printed scaffolds, as stated in (F).

SEM analysis was used to investigate the configuration and shape of the selected scaffold focusing on 2.86% w/v. In addition, the analysis of the pore size distribution in Figure 3.5 (E) showed that the majority of the pore diameters were between 160 and 200 µm. The experimental research showed that a concentration of 4 µM of the cross-linking agent had a significant impact on the long-term stability without affecting the morphology of the scaffolds or the shear-thinning properties of the ink.

The SEM micrographs were taken after cryoglation and include images of the full-size optical impression (A), a top view (B), a cross-section (C), and a magnified image of a representative area (D). The successful colonization and seeding of scaffolds by cells is crucial for the formation of the extracellular matrix and integration of the scaffold into the tissue [79,80]. The physical properties of the scaffold can influence cell behavior, and previous studies have shown that porosity design can improve cell long-term viability and establish a 3D cellular network within printed scaffolds [81,82 ].

The images show an overlay of live/dead staining with nuclear staining, providing information about the morphology of the cells within the printed scaffolds. Cell morphology was elongated on days 7 and 14, but on day 21, most cells dispersed and formed a network of interconnected cells. With the Zen light program, color enhancement was done for improved visibility (maximum intensity). B) Quantitative study of cell viability.

Figure 3.1  FTIR measurements of alginate(-) and oxidized alginate(-). The oxidation reaction-introduced aldehyde  groups
Figure 3.1 FTIR measurements of alginate(-) and oxidized alginate(-). The oxidation reaction-introduced aldehyde groups' vibrational band is shown by the arrow


Mechanical properties of optimized diamond lattice structure for bone scaffolds fabricated via selective laser melting. Chemical gelation of hydrogel-based biological macromolecules for tissue engineering: Photo- and enzyme-crosslinking methods. Fabrication of a poly (ɛ-caprolactone)/starch nanocomposite scaffold with a solvent casting/salt leaching technique for bone tissue engineering applications.

Pore ​​architecture effects on the chondrogenic potential of patient-specific 3-dimensionally printed porous tissue bioscaffolds for auricular tissue engineering. Selection of the optimal 3D printed pore and the surface modification techniques for in vivo reconstruction of the tracheal scaffold via tissue engineering. Synthesis of silver-plated bioactive nanocomposite scaffolds based on grafted beta-glucan/hydroxyapatite via freeze-drying method: antimicrobial and biocompatibility evaluation for bone tissue engineering.

A review on the use of computational methods to characterize, design and optimize tissue engineering scaffolds with a potential in 3D printing fabrication. Wang, 3D bioplotting of gelatin/alginate scaffolds for tissue engineering: Influence of cross-linking degree and pore architecture on physicochemical properties, J. Kim, Three-dimensional collagen/alginate hybrid scaffolds functionalized with a drug delivery system (DDS) for bone tissue regeneration, Chem.


Figure 1.1 Bone cells types (Osteocyte, Osteoblast, Osteogenic cell, and Osteoclast) [3]
Figure 1.2  Human bone hierarchical structure [8]
Figure 1.3 Components used in bone tissue engineering. [14]
Table 1.1 : Different techniques for fabrication of scaffolds with benefits and limitations [19, 58]

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