I declare that this manuscript entitled "In-vivo Testing of Bimodal Scaffolds in a Rabbit Model" is the result of my own work, except for citations and citations that have been properly cited. 27 Figure 9: MicroCT images of rabbit knee joint after ACL replacement with PET&PCL bimodal graft (A) Bone tunnel in tibia (B) Bone tunnel in femur. The research hypothesis is that a graft created by aligning polyethylene terephthalate and polycaprolactone (PET&PCL) nanofibers with a bimodal diameter distribution will resemble the diameter distribution of collagen fibers (CFDD) in healthy rabbit ACL.
The histocompatibility of the ligament and bone growth would be increased with the biodegradable part of PCL, while the non-biodegradable PET part would maintain the integrity to prevent deterioration of the mechanical strength of the graft. They then assessed the alignment of the nanofibers and their fiber diameter and compared them to a native rabbit ACL. As a third step, the mechanical quality of the synthesized grafts was evaluated and compared with native rabbit ACL.
The novelty of the study is that the nanofiber grafts of PET and PCL resembling CFDD of ACL tissue harvested from healthy rabbits were presented for the first time.
INTRODUCTION)
- Anterior cruciate ligament tears and current treatment methods
- Biomaterial selection and method of graft fabrication
- Selection of animal model
- Problem statement, study hypothesis and aims
In this thesis, a method for biomimetic graft fabrication is proposed and the structural and mechanical properties of the graft were compared with those of native ACL tissue harvested from rabbits. Biomaterials are one of the essential components of the regenerative engineering approach and must have appropriate chemical and physical properties. One of the reported studies [11] claimed that PCL is a suitable material for use in regenerative medicine due to its biodegradable, biocompatible and bioresorbable nature.
In terms of structural similarity of the PET&PCL graft to the native ACL, the graft was designed to exhibit a bimodal fiber diameter distribution (a diameter distribution with two peaks). Tissue engineering provides a regenerative approach to ACL injuries through the use of biomaterials, which can be processed to integrate some of the structural properties of the native ACL tissue into the scaffold/graft. We also hypothesize that the mechanical properties of the PET&PCL graft will be comparable to those of the native ACL tissue, and that its mechanical performance will be maintained or improved after its reconstruction in rabbits.
To test PET&PCL graft performance in a rabbit ACL reconstruction model.
MATERIALS AND METHODS)
- Materials
- Fabrication of nanofiber based membranes with an electrospinning device
- Scanning Electron Microscope characterization of membranes
- Determine diameter distribution of PET&PCL fibers
- Fabrication of grafts and tensile tests
- Testing the graft in a rabbit animal model
- Micro-CT characterization
- Statistical Analysis
The bimodal distribution of PET&PCL fibers was produced using PET solutions prepared in trifluoroacetic acid (TFA) and dichloromethane (DCM) while PCL solutions prepared in acetic acid (AA), formic acid (FA) and pyridine as shown in Table 1. Voltages were adjusted to 16 kV and 9 kV for PET and PCL solutions, respectively. In addition, each graft was divided into three sections, which increased the sample size and the accuracy of the measurements.
In each section and cohort, the diameter of fibrils crossing the reference lines was measured. The diameter distribution, mean diameter (calculated as arithmetic mean) and range (difference between the largest and smallest diameter) were determined for each cohort separately. The electrospun aligned membranes were cut into pieces 3 cm wide, which is the dimension in the fiber direction.
Using uniaxial material testing equipment (MTS Criterion Model 43, MTS Systems Co., Eden Prairie, Minnesota, United States), the mechanical characteristics of the rolled PET&PCL and rolled PCL grafts were evaluated (Figure 3B). The dimensions of the grafts, including length, width and thickness, were measured using a caliper (Faithful Quality Tool, UK, Faicaldig digital caliper 150 mm). At a test speed of 5 mm/min, constant extension was applied to both PET&PCL and PCL grafts until incorporation.
ACL reconstruction of the right knee using bimodal rolled graft was performed on a four-month-old male rabbit weighing about 4 kg. After shaving the right knee, betadine was used to clean the surface and the knee was aseptically draped. The native ACL was excised at its femoral and tibial origins following a medial para-patellar incision and lateral patellar dislocation. At the tibia and lateral femoral condyle at. anatomical position of the original ACL 2.5 mm drill hole was created.
Bimodal rolled graft was passed through the hole and the ends were fixed with PGA 3/0 suture at the lateral surface of the lateral femoral condyle and the medial surface of the tibia. On the fifth day, the rabbit was euthanized to obtain the knee joint together with the femur and tibia. The image was exported and preserved in the Digital Imaging and Communications in Medicine (DICOM) format.
The mechanical properties of PET&PCL bimodal grafts, PCL bimodal grafts, and a healthy rabbit ACL were compared using a one-way analysis of variance (ANOVA) followed by the Tukey HSD (Honest Significant Difference) posthoc test.
RESULTS)
- Fiber diameter of PET&PCL grafts
- Biomechanical characteristics of PET&PCL grafts
- Evaluation of implants after ACL reconstruction in a rabbit model
- MicroCT evaluation of bone tunnels
Specifically, the first peak for ACL and PET&PCL graft observed at 95±10nm and 84.4±8.8nm, respectively. However, the results show that there is a statistically significant difference between the diameters of native ACL fibers and PET&PCL fibers in terms of mean diameter and number mean diameter. The tissue modulus of this sample was calculated from the slope of the linear component of the stress-strain curve and is 1.9±0.84 MPa.
Regarding the mechanical performance parameters of the PET&PCL grafts, it was found that the ultimate stress and strain of the PET&PCL grafts were 29.5±2.5 N/mm2 and In the same way, the final stress and strain of the PCL grafts, which represented the negative control group, were 42.7±6.9 N/mm2 and Comparison of native ACL tissue with PET&PCL and PET grafts alone showed no significant difference between ACL and PET&PCL in terms of peak stress, peak load, modulus, stiffness, yield stress, and yield stress (Figure 7 C,D).
On the other hand, PCL-only grafts had significantly lower mechanical properties than ACL tissue and. * indicates significant difference from PET&PCL graft and # indicates significant difference from PCL graft at p<0.05. The gross appearance of the PET&PCL grafts (Figure 8A) was similar to the native ACL (Figure 8B).
However, our previous PCL-only graft ( Figure 8C ) was permanently deformed and unable to stabilize the knee joint after implantation. MicroCT images of rabbit knee joints collected on Day 5 clearly show the presence of the drilled bone tunnel for ACL reconstruction.
DISCUSSION)
Processing of SEM images according to diameter size reveals a bimodal distribution of PET&PCL nanofibers with two peaks at 84.4±8.8 nm and 160 nm. According to [28], rabbit native ACL has a bimodal fiber organization with a first peak at 95±10.0 nm and a second peak at 160 nm. This indicates that our bimodal PET&PCL grafts perfectly mimic the behavior of native ACL tissue in terms of structure and that the bimodal composition would improve its mechanical ability and thereby prevent ruptures.
The bimodal distribution results in improved mechanical properties because the smaller fibers fill the space between the larger fibers, making the structure dense. The incorporation of PET, which possesses superior mechanical properties [15], further increased the tensile strength of the bimodal graft. Therefore, there are no differences between the native ACL and the two types of bimodal grafts in terms of peak stress, peak load, modulus, yield strain, and yield stress.
However, in terms of stiffness, PET&PCL showed similar results to native ACL, while PCL grafts showed significantly lower results. Both PET&PCL and PCL grafts performed worse than native ACL in terms of tension at rupture. Nevertheless, this bimodal PET&PCL graft is able to stabilize the knee joint due to its similar modulus and stiffness as native ACL tissue and due to its lower elongation than PCL grafts.
After ACL reconstruction surgery in rabbits, the bimodal rolled PET&PCL graft stabilized the knee joint, while the PCL braided graft lengthened. MicroCT analysis is a technique for assessing new bone formation [32] and tunnel enlargement for several reasons, including a mechanical factor [33] and the drilling technique. The MicroCT analysis performed 5 days after surgery to evaluate the bone tunnels created in the femur and tibia on the ACL insertion sides revealed no difference.
Possible causes include the shorter duration of the examination and the smaller diameter of the needle used to drill the bone tunnel. As some authors [34] have suggested, larger bone tunnels may be beneficial for bone formation.
CONCLUSION)
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![Figure 6 shows the averaged distributions of native ACL fibrils retrieved from [28] and](https://thumb-ap.123doks.com/thumbv2/azdokorg/10950529.0/23.894.95.413.691.1006/figure-shows-averaged-distributions-native-acl-fibrils-retrieved.webp)
