Rat MSCs seemed to differentiate slightly less, while human MSCs differentiated slightly more on the heat-treated Ti64. This suggests that identical therapies can be implemented on the heat-treated Ti64 as on the untreated Ti64.
Scope and Limitations
Plan of development
This will lead to the mechanical deterioration of the joint, reduced cell viability, proliferation and differentiation, as well as increased osteolysis leading to joint loosening (Allen et al., 1996; Anderson, 2001; Brodbeck & Anderson, 2009; Haleem-Smith et al. al., 2012). This chapter will initially introduce biomaterials used for implantation in section 2.1, followed by an overview of the biology of bone in section 2.2.
Biocompatible materials
Ti oxide film and biocompatibility
Ti is a reactive metal, in the sense that it undergoes a reaction with oxygen molecules from the surrounding environment (water, air, extracellular matrix); but in doing so it immediately creates an electrically and chemically inert film of titanium oxide (TiO2) about 4 nm thick on the surface of the metal (Schindler, 1984). The exposed oxygen molecules are hydroxylated in the presence of water molecules (water, atmospheric moisture or tissue fluids) as shown in Figure 2.1 B, leaving no Ti ion present on the surface and in contact with the environment (Steinemann, 1998). ).
Ti64 microstructure and mechanical compatibility
Both α and β phases (shown in Figure 2.2) have priority; Ti in α phase has increased tensile strength, creep strength and higher elastic modulus while Ti in β phase is more ductile. The addition of α and β stabilizers to commercially pure Ti will result in an α-β region (seen in Figure 2.3 A and B) in which the characteristics of both phases will be seen in the metal (Frosch & Stürmer, 2006; Pohler , 2000 Steinemann, 1998).
Thermal oxidisation
Bone Development
- Types of bone
- Bone cells
- Bone formation
- Bone remodelling
- Bone repair
These cells are found in continuous supply within the bone marrow and surrounding periosteum for bone development (Karsenty, 2007; Komori, 2006). These cells are responsible for bone resorption during the process of bone remodeling through phagocytosis.
Implant Development and fixation
- Manufacturing methods for implants
- Cemented vs Cementless debate
- Surface modifications to cementless implants
- Key points for current study
This continuity could potentially prevent a defined weak point at the scaffold-surface interface, resulting in a stronger unit (Peltola et al., 2008). To improve the physical properties of the implant, prevent stress shielding and promote bone ingrowth, the introduction of porosity is important (Dabrowski et al., 2010). Bone ingrowth will depend on pore size, shape, amount and interconnections, but there is no consensus on what these should be (Li et al., 2007).
However, these techniques do not allow the microcontrol of pore sizes, but instead produce a pore size range (Karageorgiou & Kaplan, 2005; Li et al., 2007).
Seeding cells into Ti64 implants
Cells on Ti64
Esther van Heerden 26 | P a g e As previously mentioned, the structure and porosity of Ti64 play a role in the osseointegration of the implant. Their work shows that the average pore size affected the proliferation rate of the mouse preosteoblasts over nine days, with the smaller pore sizes encouraging proliferation. However, the rate of differentiation was unaffected, concluding that the porosity and finish of the Ti surface did not inhibit or encourage differentiation.
It can be concluded that scaffold geometries influence cell proliferation on Ti64, but differentiation occurs independently and can be enhanced by nanometric roughness.
Seeding of a RPD implant
Human osteosarcoma cell lines were seeded on the scaffolds and showed a seeding efficiency of 30–40% on the homogeneous scaffolds with a minimum pore size of 100 μm, 12 h after seeding. On the scaled scaffold with pore sizes ranging from 100 µm to 750 µm, with 100 µm pore size at the center of the scaffold, the seeding efficiency increased to 70% 12 h after seeding. 2011) points out, not only is the contact surface important, but the structure and size of the pores play an important role in cell migration and adhesion.
Ti64 heat treatment effects on cells
In this study, it is hypothesized that seeding three-dimensional (3D) scaffold structures with cells that are able to differentiate and produce bone will improve the potential for osseointegration of implants. Thus, the aim of this study is to investigate the effect that the process of thermal oxidation or heat treatment will have on laser-sintered 3D Ti64 scaffolds on the adhesion, proliferation and differentiation potential of seeded mesenchymal stem cells in vitro. Evaluation of cell adhesion, growth and differentiation on untreated and heat-treated Ti64 scaffolds.
Comparison of the results of untreated and heat-treated Ti64 tests to assess the effects of the heat treatment on the capacity of the cells.
Ti64 Scaffolds
Cell culture preparation
Thermal Oxidisation
Tissue Culture
- Source of rat mesenchymal stem cells
- Source of human mesenchymal stem cells
- Source of human fibroblast cells
- Culturing of MSCs and hFibs
- Differentiation of MSCs
- Counting of MSCs
- Freezing of MSCs
Cells were thawed to culture and build up the number of cells for experiments. The thawed cells were maintained in a standard culture medium1 following the protocol outlined in Appendix B.2: Maintenance of MSCs in standard culture medium. Cells were counted according to section 4.2.6 and viewed using phase contrast microscopy to observe growth over 21 days.
Cells were counted according to section 4.2.6 and viewed using phase contrast microscopy throughout the differentiation process.
Tissue culture work on Ti64 scaffolds
- Seeding and culturing of MSCs on scaffolds
- Osteogenic differentiation of MSCs on scaffolds
- Passaging of cells from scaffolds
- Microscopy observation of cells in scaffolds
To observe cell proliferation on the scaffolds, cells were harvested from the scaffolds and counted in a hemocytometer as described in section 4.2.6. The scaffold was washed several times to obtain the maximum number of cells for counting. Hoechst LIVE staining medium4 was used to stain cell nuclei within the scaffold.
The protocol for cell staining is described in Appendix C.5: Staining of osteogenic differentiation on Ti64 scaffolds.
Design and heat treatment of Ti64 scaffolds
To visualize any differences in the topography of the untreated and heat-treated Ti64 substrates, stereoscopic images were taken. A and B show the surface topography of the untreated Ti64 and C and D show the heat treated Ti64. A and C show the base surface of the respective Ti64 substrates, where the struts are seen as bright and unfocused.
B and D show the surface of the pillars of the corresponding Ti64 substrates, where the base is seen out of focus.
Pilot study to test the seeding efficiency of cells on Ti64 scaffolds
This lower seeding efficiency shows that the sample scaffolds were able to retain more cells by encapsulating the scaffold structure. Using the method of seeding established for hFibs, an experiment was run to determine the seeding efficiency of rat mesenchymal stem cells (rMSCs) seeded onto the untreated and heat-treated experimental Ti64 scaffolds. The results in Table 5.2 show that for rMSCs (passage number 6) on untreated Ti64 scaffolds, the seeding efficiency was 40%, and for that on the heat-treated Ti64 scaffolds, it was approx. 36%.
These results indicate that the seeding efficiency of rat MSCs was slightly better on untreated Ti64 than on heat-treated, but not significantly.
Growth and proliferation of rat MSCs on Ti64 scaffolds
The cells on both the untreated and heat-treated Ti64 reached either a maximum or a plateau at day 16, yet continued to proliferate on the cell culture dishes. Esther van Heerden 44 | P a g e Particularly noticeable was that the number of cells on the struts was very variable. For example, Figure 5.5 B and D showed a greater number of cells adhering to the scaffold struts, whereas C and E showed almost no cells on the struts.
Growth on the Ti64 scaffolds was slightly slower, showing a three-day doubling time on both the untreated and heat-treated Ti64, compared to the two-day doubling time of the cells on the dishes.
Differentiation of rat MSCs into osteoblasts on Ti64 scaffolds
The results also show that there was a slightly higher number of cells on the heat-treated Ti64 than on the untreated one, but not as noticeable. For the time-course study performed with rMSCs on heat-treated Ti64 scaffolds, cells were recultured in differentiation medium for 21 days on 21 scaffolds. Esther van Heerden 49 | Page Results in Figure 5.6 show the progression of differentiation of rMSCs located on the bottom and abutments of untreated and heat-treated Ti64 scaffolds, as well as on tissue culture dishes.
More differentiation was seen in the untreated scaffolds than in the heat-treated scaffolds; as well as more on the base than on the supports of scaffolding.
Seeding and growth of human MSCs on Ti64 scaffold
Using passage 6 hMSCs, the seeding efficiency test was repeated on the untreated and heat-treated Ti64 scaffolds following the same method as previously described. The results shown in Table 5.4 showed a seeding efficiency of 30% for the cells on the untreated Ti64 scaffolds and 40% for those on the heat-treated Ti64 scaffolds. Esther van Heerden 53 | Page The next set of tests was to determine the growth and proliferation potential of the younger hMSCs on the untreated and the heat-treated Ti64 scaffolds.
The results of the growth of hMSCs on the untreated and heated treated Ti64 scaffolds and in culture dishes are shown in Figure 5.9.
Differentiation of human MSCs into osteoblasts on Ti64 scaffolds
The results also show that there were slightly more cells on the untreated Ti64 than on the heat treated one. Fluorescence microscopy results of untreated and heat-treated Ti64 frameworks, shown in Figure 5.11, show the progression of differentiation on the frameworks. Figure 5.11 also shows that the extent of differentiation at the struts is much smaller than that at the base.
Differentiation was slightly more abundant in heat-treated Ti64 scaffolds than in untreated scaffolds.
Untreated vs heat treated Ti64 cell work
This was shown by assessing cell adhesion, growth and differentiation on untreated Ti64 compared to that on the heat-treated Ti64. For all three markers, the expression or activity levels were higher on the heat-treated Ti64 than the untreated Ti64. The visual extent of mineralized nodules is also greater in the heat-treated Ti64.
This would explain the greater saturation of bone matrix seen on the heat-treated Ti64 in the human MSC tests in this study, as well as the improved biocompatibility of heat-treated Ti64 reported by Saldaña et al.
Location of cells on the scaffolds
As the pore size of the scaffold did not restrict cell movement, most cells were able to settle on the Ti64 substrate. Cytoskeletal reorganization at contact interfaces during attachment results in morphological and behavioral changes in the cell that have lasting effects on cell growth and differentiation capacity (Gwynn, 1994). In order to achieve better cell seeding on the scaffold supports, we can consider cell seeding in a rolling culture environment.
This situation would avoid the gravitational settling of cells through the pores on the base, allowing more cells to settle on the struts.
Technical difficulties
Finally, Stenderup, Justesen, Clausen, and Kassem (2003) emphasized that not only in vitro cell aging is important when considering effects on cell proliferation, but also patient age. Although patient age combined with time in culture had a significant effect on the proliferative capacity of the cells, it did not appear to affect the capacity to differentiate. In summary, the proliferative abilities of MSCs are strongly influenced by patient age and cell senescence during in vitro culture.
The tissue source of MSCs showed differentiation potential into any of the mesenchymal lineages; however, MSCs were more likely to differentiate into cells associated with the tissue from which they were derived.
Heat treatment of Ti64 does not adversely affect the cells capabilities
Ti64 may be considered an osteoconducive material
Cell proliferation and differentiation are sensitive to cell source and age of
Cell differentiation is also dependent on cell’s location in the scaffolds
Research methods to consistently seed struts of scaffolds
MSC versus osteoblast adherence and success on scaffolds
In vivo tests of the osseointegration of the scaffolds
On Monday and Friday, all medium was aspirated from the cells and filled with a full volume of standard medium. Resuspend cells to a density of ±10,000 cells/cm2 in standard culture medium (see standard culture medium recipe). Aspirate standard culture medium and pipette an equal volume of osteogenic medium (see Osteogenic Medium Recipe).
Aspirate the supernatant and resuspend the cells in standard culture medium (see recipe for standard culture medium) to a concentration of 2x10 6 cells/ml.
