Conclusions and Future Works
At each step in the development of a biodegradable scaffold for bone tissue engineering, it is important to answer certain critical questions:
Are the materials to which tissue is exposed biocompatible?
Does the material have satisfactory properties?
Will the cells function on the material as required for bone growth?
If not, what bioactive molecules must be present and how will they be delivered?
How do cellular function and material properties change when a different manufacturing technique or material formulation is used?
Already much progress has been made in scaffold development. The introduction of MWCNT in the scaffolds for bone tissue engineering seems to be promising in terms of cell viability and growth on the scaffolds. However, realizing 3-D PLLA/MWCNT composite scaffolds by PAM technique is very difficult because of lack of homogeneity in polymer dispersion extruded through PAM glass capillaries. So, this technique should be optimized in terms of capillary diameter and extrusion pressure and speed. Then, another problem to solve related to PAM technique is the control of scaffold micro- and nano-porosity since PAM can only control scaffold macro-porosity related to 2-D single layer geometry and 3-D total scaffold structure.
By the way, the results obtained from cell experiments revealed that hFOB 1.19 cells “feel” the composite scaffold so close to the bone extracellular matrix. This phenomenon, called durotaxis, that links the adhesion and growth of cells to the stiffness of the substrate is accomplished by use MWCNT in these experiments.
However, it is still not clear how 3-D geometry of the scaffolds affects cell growth and morphology in a time dependant way and which role MWCNT can play in this process. Maybe the role of MWCNT is just to provide high stiffness of the scaffolds in the durotaxis, but, after that, from a chronological point of view, it is supposed that they are not relevant in the 3D pattern recognition processes.
Moreover, another important point is the role of cell type (hFOB 1.19) chosen for experiments. These cells exhibit a particular behavior as function of temperature chosen for culture. In particular, when cultured at 37°C (temperature at which cell tests have been performed) hFOB 1.19 cells exhibit little or no division since they start to differentiate into mature osteoblastic phenotype. That’s why the index of cell density on both scaffolds (composite or not) and on both films (composite or not) was not relevant to measure if cells are viable or not on the structures. For this reason cell viability assays have been performed when cells were into a differentiation state and not into a proliferation one. Results of Cell Viability Assays revealed higher cell viability (recorded after 72 hours from cell seeding) on 3-D PLLA/MWCNT composite scaffolds in comparison to pure 3-D PLLA scaffolds and both (composite and not) 2-D films.
The limit of this work is related to choice of hFOB 1.19 cells and their particular temperature culture conditions. Normally, most of cell lines are cultured at 37°C at which most of cell tests could be performed while cell proliferation is regular. In this work instead, cell proliferation stopped at temperature of cell viability assays, and so, it has been much more difficult to create an index to indicate what kind of scaffold is more suitable for this kind of cells. In addition, It could be advantageous to test cell viability on a large time scale and not only until 72 hours from cell seeding.
In the future of bone tissue engineering, it could be interesting to realize nano-porous scaffolds to give cells more attachment sites to colonize them. In addition, the length of MWCNT should be optimized because this particular MWCNT length used in this work was determinant just from a macroscopic point of view (scaffold stiffness), but it is not relevant in providing bone cells growth directions. In particular, MWCNT length should be shorter and similar to cell dimension.
In this way, MWCNTs could be functionalized with growth factors or other drugs in order to help bone cells to growth and colonize scaffolds. That’s why, in this case, nano-porosity of scaffolds is necessary for drugs and growth factors release from a microscopic point of view.
However, in the design of an innovative scaffold for bone tissue engineering, there is always a trade-off to optimize between macroscopic mechanical properties (stiffness), and cell microscopic dimension (nano-porosity), since introduction of nano-pores into scaffolds could affect the stiffness ,and, on the contrary, the large dimension of MWCNTs could affect cell properties and diffusion patterns into scaffold.