Frontiers in biofabrication of ligament and bone-ligament interface scaffolds

Ligaments are the responsible of joint stability and their injuries represent a common disease with a very high incidence both in general population and athletes. The ligament natural healing process is often inadequate to restore its structural, mechanical and biological properties and, in most cases, it results in the formation of scar tissue with inferior mechanical properties. Two different strategies were proposed to reach a complete and long-term functional repair of the damaged tissue: reconstructive and regenerative approaches. The reconstructive approach includes conservative and reconstructive treatments and can be considered a valid and relatively fast solution for ligament injuries. However, currently used therapies are mainly limited to pain control and/or tissue replacement, without fully restoring tissue functionality and influencing the long-term functional repair. The regenerative approach represents a new strategy based on Tissue Engineering (TE) that aims at fabricating an immunological tolerant tissue substitute to permanently restore the functionality of the damaged one, without the need for supplementary therapies. Nevertheless, important challenges must be solved to obtain complete ligament repair that will lead to a clinically effective and commercially successful application. The main aim of the present research is to investigate both approaches in order to restore the ligaments function to improve the patient health and quality of life. The first step of this research project was the mechanical analysis of the ligament tissue to obtain the initial guidelines to choose, on one side, the adequate reconstruction technique and, on the other side, the biomaterials and microfabrication techniques for the fabrication of a scaffold that support the tissue regeneration. Using the human Medial Patello-femoral Ligament (MPFL) as subject of this study, tensile and stress relaxation analyses were performed to provide an assessment of the mechanical and viscoelastic properties of the MPFL tissue substance and of the structural properties of the bone-ligament-bone (femur-MPFL-patella) complex. The mechanical analysis helped in understanding MPFL physiological function and its contribution as stabilizer for the correct selection of repair and reconstruction methods. In particular, in this thesis, two different surgical techniques were compared to reconstruct the MPFL. Although the results demonstrated the inadequacy of both techniques, the through tunnel tendon and the double converging tunnel, to restore the mechanical and structural properties of the native MPFL, these surgical treatments are currently used in clinical practice. However, ambiguous long-term results with postsurgical complications including wear and degradation of the reconstruction represent high risk factors for the treatment success. A possible solution to restore the ligament function, emerged in this work, was the design of scaffolds able to mimic the mechanical, topographical and biological properties of the native tissue. Moving in this direction, the attention of this research was focused on the design, fabrication and biological validation of a soft-molecular imprinted (soft-MI) electrospun scaffold for ligament TE applications and of a 3D fiber deposited-electrospun scaffold (3DF-ESP) triphasic scaffold for the regeneration of the bone-ligament interface. The integration of ESP and soft-MI represented a powerful and promising technique for the fabrication of scaffolds for TE applications, because it allowed to mimic the structural environment through the fiber microtopographies and the biological one through the selective bindings of specific endogenous proteins and growth factors (GF). The imprinted scaffolds demonstrated affinity and selectivity for their template molecule. The soft-MI imprinted ESP scaffolds showed an interconnected porous structure characterized by a fibrous nanotopography and by a 3D striped microtopography that can influence cell behavior. In the scaffolds, cells were aligned along the macrostructure after 2 days of culture demonstrating that the microtopography can influence orientation, alignment, adhesion, morphology and proliferation. The high porosity and permeability observed are excellent features to promote nutrient supply and distribution. Furthermore, the scaffold surface properties observed, such as wettability and roughness, can promote cell adhesion and growth. The imprinting of ESP with several GFs resulted in a significant effect on cell behavior. FGF-2 promoted cell proliferation and FGF-2 imprinted scaffolds showed a statistically higher metabolic activity over the time and a higher number of cells after 7 days. BMP-2 and TGF-β3 imprinted scaffolds showed a statistically higher luciferase signal that indirectly demonstrate the presence and the bioactivity of the GFs. These results demonstrate for the first time the possibility to imprint nanofibers for creating bioactive ESP scaffolds for TE applications. The optimal surface microtopography was investigated developing a novel, flexible, scalable and reproducible microfabrication method for the production of micro-patterned electrospun scaffolds. Results indicated that the use of defined patterns could induce specific morphological variations in the hMSCs cytoskeletal organization that could be related to a differential activity of signaling pathways. Engineering interface tissues requests a complex strategy that includes the use of specific biomaterials and fabrication techniques to recreate the adequate multi-tissue transition but also specific cell types and culture conditions to promote growth and differentiation. Using structural features as leading criteria to mimic the bone-ligament interface, a 3DF-ESP triphasic scaffold with a linear gradient of materials, mechanical and structural properties was fabricated combining ESP and 3DF techniques. PCL and PLGA based scaffolds with a graded variation of their physicochemical and mechanical properties were fabricated in a two-step process to mimic the multi-tissue organization of the native ligament-to-bone insertion site. The use of different biomaterials processed by two different techniques allowed the fabrication of scaffolds with interconnected porous regions and with different morphologies and porosities. The 3DF-ESP triphasic scaffold showed an increase of the metabolic activity within culturing time. The partition metabolic activity analysis on the different areas showed a similar trend after 7 days in differentiation medium. A higher number of cells was detected comparing the triphasic scaffold to mixed control in basic (BM) and mineralization media (MM) while a similar trend was detected with 3DF control in ligament medium (LM). Total alkaline phosphatase (ALP) activity analysis showed a statistically higher activity of the triphasic scaffold in MM compared to the controls and, comparing the different areas, a trend in ALP activity in case of MM was detected. ALP seemed higher in the 3DF part and it decreased in the mixed and in the ESP regions. A different GAG amount between the triphasic scaffold and its controls was detected and, in addition to a similar trend in ALP activity, suggested that the triphasic configuration seems to influence hMSCs behaviour in vitro. The integration of ESP and 3DF represents a promising technique for the manufacturing of interface scaffolds able to come a step closer in mimicking the structural biological environment through the combination of different biomaterials at different scales. Finally, as alternative, an electrospun scaffold with increased three-dimensionality for ligament TE applications was designed and fabricated by tailoring crimp patterns on electrospun fibers by using thermal shrinkage. Results showed that the shrinkage of the scaffolds changed their topography from a flat surface into a wavy one and a correlation between the periodicity of the pattern and the cell shape was found. The mechanical analysis showed a trend of mechanical properties related to the percentage of shrinkage. Cellular migration analysis showed that wavy scaffolds enabled a more uniform distribution of cells across the scaffold and better cellular infiltration was found in wavy electrospun scaffolds compared to the flat ones. All the presented evidences suggested that the wavy scaffolds can improve cellular migration and are promising candidates for ligament regeneration.