Background: Synthetic grafts were developed to overcome problems related to autogenous grafts. The adhesive interactions of cells play a fundamental role in the healing process of ligament tissue engineering. One of the disadvantages of synthetic ligaments is the lack of biological cues for promoting cell adhesion and proliferation. The aim of this study is to evaluate the cell viability and adhesion to fibers of polyester implant for ligament tissue engineering and labrum reconstruction.
Methods: Mesenchymal stem cells were seeded (10x105) and cultured in an artificial prosthesis of polyester. Fragments were stained with calcein and photographs were taken at 24, 48, 72 and 120 hours, as well as later at two weeks in culture. The percentage of fluorescence was recorded using an Image J program.
Results: After two weeks of cell culture, the artificial prosthesis was covered by cells on average 98.57 ± 0.74% of the surface. The porous structure of the prosthesis was covered by a confluent layer of cells and extracellular matrix. Statistically significant differences were found between all the times analyzed (p=0.01).
Conclusion: Our results suggest that the cells seeded on the polyester prosthesis spread and proliferated until a confluent layer, showing that this has a good biocompatibility.
Keywords: Artificial prosthesis; Cell viability; Biocompatibility; Ligament reconstruction; Synthetic ligaments
It has been shown that the acetabular labrum has an important function in the normal biomechanics and stability of the joint. Labral tears are associated to a poor sealing of the joint fluid resulting in increased frictional forces and premature osteoarthritis [1,2]. The most common pathology in patients undergoing hip arthroscopy is labral tear . When labral repair is not possible, debridement or reconstruction are indicated. Some authors have found that arthroscopic labral reconstruction is superior to labral resection in patients with irreparable or mostly calcified labrum with positive clinical outcomes .Labrum reconstruction has recently become popular and multiple techniques using autografts or allografts reporting good outcomes at medium to long term have been described [5,6]. However, both grafts types are not exempt from complications. Autografts have the disadvantage of donor site morbidity, inadequate graft sizing or morphology, otherwise allografts require procurement from a tissue bank, presents a potential risk of immune reaction and infectious diseases transmission, and have shown a later integration [7-11]. Only one study reported the use of synthetic graft for hip labrum reconstruction, this synthetic prosthesis is a polyurethane meniscal substitute adapted for augmentation and reconstruction of segmental labral tissue loss or irreparable labral damage . Synthetic grafts were developed primary ligament reconstruction in the knee with the objective to overcome problems related to autologous and allogenic implants. Initially, some serious complications were reported with the use of those artificial prosthesis such as graft rupture, foreign-body, inflammation, and serious knee synovitis [13,14]. Although synovitis appears to be a rare complication, it is very serious and can result in ligament rupture and failure [15,16]. In the case of ligament reconstruction, an osteoarthritis case associated with LARS artificial ligament after anterior cruciate ligament surgery was reported . In the histology of this report, the authors described only a few chondrocytes grew well along with the parallel fibers of the LARS ligament . High graft failures, no so-called ligamentization and severe synovitis have been reported as major disadvantages of synthetic grafts [17-20].The adhesive interactions of cells with other cells and the Extracellular Matrix (ECM) synthesis play a fundamental role in the healing process of ligament tissue engineering. At the cellular level, ligament wound healing involves cell attachment, detachment, migration, and proliferation. Cells and materials are two essential components in ligament tissue engineering, and so the interactions between them are important. Materials could interfere with cell adhesion, proliferation, and differentiation, while cell adhesion and subsequent functionality also affect properties of surrounding materials .
Pore interconnectivity throughout an implant favors the distribution of nutrients, cell migration, metabolic waste removal and the tissue ingrowth, enhancing its regenerative properties [22,23]. Contrasting to the natural materials, synthetic polymers present low immunogenicity potential and are more versatile. Polyesters has been effectively used to produce mechanically strong and biodegradable scaffolds for tendon/ligament applications [24,25]. These polymers are well characterized and have been approved by the FDA for certain human uses . However, one of the disadvantages of synthetic polymers is the lack of biological cues for promoting cell adhesion and proliferation [26,27]. The aim of this study is to evaluate the cell viability and adhesion to fibers of polyester implant (PolyTape, Neoligaments™) for ligament tissue engineering.
Mesenchymal stem cells (ATCC-PCS-500-012, isolated from human bone marrow) were seeded and cultured in an artificial prosthesis of polyester at 37 oC in a 95% air and 5% CO2 atmosphere. Before MSC seeding, the artificial prosthesis were sterilized in the flow hood with, then under sterile conditions in the mine flow hood, the polyester graft (PolyTape, Neoligaments™) was cut into 5mm fractions and placed in triplicate into wells of a 48-well plate. To initiate cell culture on the graft, 10x105 cells were seeded in each fragment (5x7mm). First, each fragment was kept immersed in DMEM culture medium (Corning) during 10 minutes. Then, the culture medium was removed and with a micropipette, the cells suspended in 50 uL of culture medium were distributing them evenly over the entire surface of the scaffold. The synthetic prosthesis fragments were sterilized into the flow hood, then the cells were covered completely with DMEM-high glucose culture medium (Corning), supplemented with 10% fetal bovine serum and 1%
antibiotic/antimycotic (Thermo Fisher Scientific). The changes of medium were carried out every two days for 2 weeks. To expose the proportion in which the cells colonized the surface of the prosthesis, the fragments were stained with calcein (caymanchem) at a concentration of 0.2 mg / mL. Cell growth was recorded with photographs at 24, 48, 72 and 120 hours, as well as later at two weeks in culture. The images were captured in a pyramid microscope Carl Zeiss Axio system image Vs 40X64 V. To obtain the percentage of fluorescence for calcein, 10 photos of each sample were taken, and Image J program (NIH) was used to obtain the mean of the fluorescence percentage for each sample.
The data of this study were stored in an Excel data base (Microsoft Office for PC) and processed with the STATISTICA version 10 software. The percentages of fluorescence were obtained for the calcein at 24, 48, 72 and 120-hours. A Kolmogorov-Smirnov test was applied to establish if the samples presented a normal behavior and as a result of this the statistical significance of the differences between groups was determined by one-way Analyses of Variance (ANOVA); p<0.05 was statistically significant.
Cell Growing at 2-Weeks
After two weeks of cell culture, the artificial prosthesis was evaluated by microscopy and was observed that cells covered the implant on average 98.57 ± 0.74% of the surface (Figure 1a).
Cell Viability Quantified by Calcein Staining
Cell survival and proliferation was measured at 24, 48, 72, and 120 hours after seeding. The percentage of cell viability was assessed following 24 hours of incubation at 37 oC and 5% CO2. Results showed a constant increase in cell density on the surface from day 1 until day 5. The porous structure was covered by a confluent layer of cells and extracellular matrix (Figure 2C). At 24 hours the 7.68 ± 1.12%, 48 hours the 19.26 ± 1.49%, 72 hours the 31.46 ± 1.43% and at the 120 hours the 60.65 ± 2.69%, on average were of percentages of green fluorescent quantified by calcein staining in the cells that were covering the superficial area of fragment plus cells (Figure 1b), we found statistically significant differences between all the times analyzed (p=0.01).
Cell Attachment to The Artificial Prosthesis
A well-defined architecture with same macropore structure of the artificial prosthesis was visualized by imaging of the green fluorescence. SEM observations allowed to determine that MSCs were able to adhere to the surface of the polyester scaffolds (Figure 2A & 2B). Cell morphology was fibroblast-like shape and those were spreading in the fibers surface. Further, it should be highlighted that there was no pore occlusion by the cells. However, by observing the pores it was shown that the cells were also capable of colonizing these areas, without occluding those.
Cell adhesion into the structure and insertion site is an important factor in artificial ligament use. Various grafts have been used so far for the treatment of ligament reconstruction. The majority of synthetic grafts used in the past for knee have exhibited poor long-term physiologic and functional out-comes, no evidence in the literature is present about the use of those prosthesis for labrum reconstruction . After trials in clinics for 20 years, most of these prostheses were no longer used because of high complication and failure rate (31% to 42%) . Generally speaking, early applications of artificial ligaments were not successful. The bleak results of follow-ups have revealed the underlying hazards: immune response, effusions, loosening, and rupture of the prostheses. However, recently novel types of artificial ligaments (Neoligaments) were also introduced in clinics, including artificial tendons and ligaments [30-34]. Neoligaments, prostheses made of polyester (Dacron), have been reported in clinical application with low rate failure . Biocompatibility and mechanical strength are two key properties when we assess a scaffold used in a tissue-engineered ligament . Tissue ingrowth is very important in artificial ligaments and is often affected by the surface topography, pore diameter, and porosity of the materials . The device should also have interconnected porosity to allow cell migration, tissue growth, and vascularization within the tunnel segments.
In this study, an artificial flat ligament prepared with polyester fibers was used to investigate the effects of porous structures on cellular adhesion and migration. We observed that cells seeded on the polyester prosthesis spread and proliferated until a confluent layer forming extracellular matrix at two weeks of in-vitro culture on average 98.57% of the implant surface. Interestingly, we observed significant improvement in cell growing through the time reaching a 60.65% implant coverage at 5 days of culture quantified by percentages of green fluorescent by calcein staining. Those findings suggest a positive biocompatibility between mesenchymal stem cells showing that the polyester fibers are not cytotoxic. Cell accommodation through the scaffold suggests that its architecture, pores and surface provide a favorable environment for cell attachment. This also demonstrates that open architecture of the scaffold facilitates the infiltration of a cell suspension into the 3D structure of synthetic prosthesis. It was shown that interconnectivity of pores allowed for uniform cell distribution throughout the ligament, resulting in high cell density and homogeneous distribution at the end of the culture period. These new options could display the biology, integration and mechanics of the original labrum or ligament while sponsoring the growth of new tissue and resisting rejection from the body.
Results showed an increase in the number of viable cells in the surface of the scaffold fibers throughout the culture period, indicating an adequate compatibility to the cells cultured in the polyester prosthesis offering a new scaffold for labrum and ligament regeneration.
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