Objectives

Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology.

In clinical orthopedics suitable materials that induce and restore biological functions together with the right mechanical properties are particularly needed for the regeneration of musculoskeletal tissue. An innovative solution to answer this need is represented by tissue engineering. This technique could overcome the limits of traditional approaches involving the use of homologous, autologous or allogenetic tissue (e.g. tissue availability, immune rejection and pathogen transfer). In this field, rapid prototyping techniques are emerging as the most promising tool to realize three-dimensional tissue constructs with highly complex geometries.

Based on CAD/CAM technology, rapid prototyping allows development of patient-specific 3D scaffolds from digital data obtained with latest generation imaging tools. These structures can be realized in different materials, tailoring their mechanical properties and architectural features. Most rapid prototyping techniques allow the creation of acellular 3D scaffolds, which must be subsequently seeded with cells. Conversely, 3D bioprinting can deposit bio-ink containing molecules/cells, providing desired spatial distribution of growth factors/cells within the scaffold. The need of printable materials suitable for processing with inkjet, dispensing, or laser-print technologies, forces the use of matrices within a specific range of viscosity. However, these materials have low mechanical features. To overcome this problem and to obtain a final construct with good mechanical properties, bioprinting tissue fabrication can rely on the alternate deposition of thermoplastic materials and cell-laden hydrogels. Since mechanical performance is determined not only by the material properties but also by the geometry (microarchitecture) of the structure, printing parameters can be modified to obtain the desired features.

The new 3D platform available at Rizzoli Orthopaedic Institute, consisting of a Computer Tomography (GE Medical Systems, Milano, Italia) and a 3D Bio-Printer (RegenHU, Villaz-St-Pierre, Switzerland) is used to address the above-mentioned issues. Preliminary results showed that it is possible to modify the microarchitecture of the printed structures adjusting their apparent density and stiffness in the range of the trabecular bone tissue. Additionally, it has been proven that the calcium phosphate based paste, used as bioink, allows cell attachment and proliferation. Therefore, the platform allows to print scaffolds with open and interconnected porosities and suitable mechanical properties. They can be filled with different components such as cells or soluble growth factors at specific locations.

Aim of study: The development of tissue engineering techniques evidenced that the healing of injured ligaments require the interactions of different cell types, local cellular environment and the use of devices. In order to gain new information on the complex interactions between mesenchymal stem cells (MSCs) and biodegradable scaffold, we analysed in vitro the proliferation, vitality and phenotype of MSCs grown onto a multilayered-woven-cylindric-array of Hyaff-11A8 fiber configured as ligament scaffold.

Methods: Sheep MSCs were isolated from bone marrow aspirates and grown at two different density (7,5x106/cm and 15x106/cm) in the scaffold. At different time points (2, 4, 6 days) cellular proliferation was analysed by MTT test and cellular viability by calcein-AM immunofluorescence dye and confocal microscopy analysis. Moreover, hyaluronic acid receptor (CD44) and typical matrix ligament proteins (collagen type I, III, laminin, fibronectin, actin) were evaluated by immunohistochemistry.

Results: MSCs growth was cell density-dependent and cells were uniformly distributed inside and along the scaffold. Confocal analysis showed that MSCs completely wrap the fibers at both cell concentrations analysed and were all viable both outside and inside the scaffolds only using the lower cell concentration. Moreover, MSCs expressed CD44, collagen type I, III, laminin, fibronectin and actin.

Conclusion: These data demonstrate that MSCs well survive in a hyaluronic acid-configured ligament scaffold expressing a protein important for scaffold interaction, like CD44, and proteins responsible of the functional characteristic of the ligaments.