3D screening device for the evaluation of cell response to different electrospun microtopographies.

Micro- and nano-topographies of scaffold surfaces play a pivotal role in tissue engineering applications, influencing cell behavior such as adhesion, orientation, alignment, morphology and proliferation. In this study, a novel microfabrication method based on the combination of soft-lithography and electrospinning for the production of micro-patterned electrospun scaffolds was proposed. Subsequently, a 3D screening device for electrospun meshes with different micro-topographies was designed, fabricated and biologically validated. Results indicated that the use of defined patterns could induce specific morphological variations in human mesenchymal stem cell cytoskeletal organization, which could be related to differential activity of signaling pathways. STATEMENT OF SIGNIFICANCE: We introduce a novel and time saving method to fabricate 3D micropatterns with controlled micro-architectures on electrospun meshes using a custom made collector and a PDMS mold with the desired topography. A possible application of this fabrication technique is represented by a 3D screening system for patterned electrospun meshes that allows the screening of different scaffold/electrospun parameters on cell activity. In addition, what we have developed in this study could be modularly applied to existing platforms. Considering the different patterned geometries, the cell morphological data indicated a change in the cytoskeletal organization with a close correspondence to the patterns, as shown by phenoplot and boxplot analysis, and might hint at the differential activity of cell signaling. The 3D screening system proposed in this study could be used to evaluate topographies favoring cell alignment, proliferation and functional performance, and has the potential to be upscaled for high-throughput.

Reconstruction of medial patello-femoral ligament: Comparison of two surgical techniques.

The medial patello-femoral ligament is considered the most important passive patellar stabilizer and its proper functionality is essential for the patello-femoral joint stability. In this work, 18 human knees were randomly divided into two groups and reconstructed through two different surgical techniques: the "Through tunnel tendon" and the "Double converging tunnel" reconstructions. Subsequently, the samples were mechanically tested to evaluate the structural properties of reconstructed femur-MPFL-Patella complex (rFMPC). Particular attention was given to maintain the anatomical orientation between the patella and the graft. Both procedures showed lower stiffness and higher ultimate strain and absorbed energy compared to the native MPFL, but the advantages of the double converging tunnel technique are related to the restoration of the native MPFL sail-shape, to a better stress distribution on the patella, to the use of a single interference screw as fixation device and to the simplicity, rapidity and cost-effectivity of the surgical procedure. The evaluation of the structural properties of rMPFL is fundamental to evaluate the adequacy of the different techniques to restore the physiological structural properties of the native MPFL.

Triphasic scaffolds for the regeneration of the bone-ligament interface.

A triphasic scaffold (TPS) for the regeneration of the bone-ligament interface was fabricated combining a 3D fiber deposited polycaprolactone structure and a polylactic co-glycolic acid electrospun. The scaffold presented a gradient of physical and mechanical properties which elicited different biological responses from human mesenchymal stem cells. Biological test were performed on the whole TPS and on scaffolds comprised of each single part of the TPS, considered as the controls. The TPS showed an increase of the metabolic activity with culturing time that seemed to be an average of the controls at each time point. The importance of differentiation media for bone and ligament regeneration was further investigated. Metabolic activity analysis on the different areas of the TPS showed a similar trend after 7 days in both differentiation media. Total alkaline phosphatase (ALP) activity analysis showed a statistically higher activity of the TPS in mineralization medium compared to the controls. A different glycosaminoglycans amount between the TPS and its controls was detected, displaying a similar trend with respect to ALP activity. Results clearly indicated that the integration of electrospinning and additive manufacturing represents a promising approach for the fabrication of scaffolds for the regeneration of tissue interfaces, such as the bone-to-ligament one, because it allows mimicking the structural environment combining different biomaterials at different scales.

Material and structural tensile properties of the human medial patello-femoral ligament.

The medial patellofemoral ligament (MPFL) is considered the most important passive patellar stabilizer and acts 50-60% of the force of the medial soft-tissue which restrains the lateralization of the patella between 0° and 30°. In this work, 24 human knees have been tested to evaluate the material properties of MPFL and to determine the structural behavior of femur-MPFL-Patella complex (FMPC). Particular attention was given to maintain the anatomical orientation between the patella and MPFL and to the evaluation of the elongation during the mechanical tests. The ultimate stress of the isolated ligament was 16±11MPa, the ultimate strain was 24.3±6.8%, the Young׳s Modulus was 116±95MPa and the strain energy density was 2.97±1.69MPa. The ultimate load of the whole structure, FMPC, was 145±68N, the ultimate elongation was 9.5±2.9mm, the linear stiffness was 42.5±10.2N/mm and the absorbed energy was 818.8±440.7Nmm. The evaluation of material and structural properties of MPFL is fundamental to understand its contribution as stabilizer and for the selection of repair and reconstruction methods.

Quasi-linear viscoelastic properties of the human medial patello-femoral ligament.

The evaluation of viscoelastic properties of human medial patello-femoral ligament is fundamental to understand its physiological function and contribution as stabilizer for the selection of the methods of repair and reconstruction and for the development of scaffolds with adequate mechanical properties. In this work, 12 human specimens were tested to evaluate the time- and history-dependent non linear viscoelastic properties of human medial patello-femoral ligament using the quasi-linear viscoelastic (QLV) theory formulated by Fung et al. (1972) and modified by Abramowitch and Woo (2004). The five constant of the QLV theory, used to describe the instantaneous elastic response and the reduced relaxation function on stress relaxation experiments, were successfully evaluated. It was found that the constant A was 1.21±0.96MPa and the dimensionless constant B was 26.03±4.16. The magnitude of viscous response, the constant C, was 0.11±0.02 and the initial and late relaxation time constants τ1 and τ2 were 6.32±1.76s and 903.47±504.73s respectively. The total stress relaxation was 32.7±4.7%. To validate our results, the obtained constants were used to evaluate peak stresses from a cyclic stress relaxation test on three different specimens. The theoretically predicted values fit the experimental ones demonstrating that the QLV theory could be used to evaluate the viscoelastic properties of the human medial patello-femoral ligament.

Shape and size of the medial patellofemoral ligament for the best surgical reconstruction: a human cadaveric study.

PURPOSE: The aim of this study was to investigate the shape and the attachments of the medial patellofemoral ligament (MPFL) in cadaver specimens to determine an anatomical basis for the best MPFL reconstruction. METHODS: Twenty fresh-frozen knees were used. Dissection protocol implied performing dissections from within the knee joint. We investigated the shape and the attachments between the MPFL and the quadriceps tendon, the patellar and femur insertions, and all the other relationships with the medial soft tissues of the knee. RESULTS: The distal fibers of MPFL were interdigitated with the deep layer of the medial retinaculum. All isolated ligament had a sail-like shape with the patellar side bigger than the femoral side. The femoral insertion, distinct both from medial epicondyle and adductor tubercle, was located at 9.5 mm (range 4-22) distal and anterior respect to adductor tubercle and proximal and posterior to epicondyle. The medial third of the thickness of patella was involved in the insertion. The proximal third of the patella is always involved in the MPFL attachment; in 45% of the cases, it was extended to the medial third and in one case, an extension at the distal third was found. Additionally in 35% (7 cases), it extended to the quadriceps tendon and it were inconstantly attached at the vastus medialis obliques (VMO) tendon and at the vastus intermedius (VI) tendon in an aponeurotic structure. CONCLUSIONS: The MPFL is a distinct structure that goes from patella to femur with a sail-like shape; its patellar insertion, that mostly occur via an aponeurosis tissue with VMO and VI, is at the proximal third of the patella but it may extend in some cases to the medial third patella or to the quadriceps tendon, or very rarely to the distal third of the patella. In the femoral side, the MPFL is inserted in its own site, in most cases distinct both from epicondyle and adductor tubercle, located on average at a 9.5 mm distance distally and anteriorly in respect to the adductor tubercle. Its lower margin was difficult to define. Given the importance of this structure, it must be reconstructed as anatomically as possible in its insertion and in its shape. Many attempts have been made to make functional reconstructions with less than excellent results.