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CHARACTERIZATION OF COLLAGEN FIBRIL DIAMETER DISTRIBUTION AND BIOMECHANICAL PROPERTIES OF HEALTHY AND INJURED RAT ANTERIOR CRUCIATE LIGAMENT

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I would like to express my gratitude to all the individuals who have made such a significant contribution to the successful completion of this research project. 27 Figure 8: Combined adjusted and unadjusted PCL fiber diameter distributions in the form of (A) histogram and (B) line graph (n=4). In animal models of cattle and sheep, ACL injuries have previously been shown to lead to a change in the distribution of collagen fibril diameter, with a shift from a bimodal distribution in the healthy ACL to a unimodal distribution after injury.

In this study, it is hypothesized that the collagen fiber diameter distribution in the ACL of rats changes from a bimodal distribution in the healthy ACL to a unimodal distribution after injury and that this change can be mimicked in scaffolds fabricated using electrospinning. Secondly, transmission electron microscopy (TEM) evaluation of the healthy and damaged ACL tissues was performed to evaluate the collagen fibril diameter distributions in the tissues. Findings show that the bimodal distribution of ACL collagen fibril diameter changed to unimodal after injury, causing a decrease in mean diameter.

INTRODUCTION)

Anterior Cruciate Ligament

  • ACL Composition and Structure
  • Function of Anterior Cruciate Ligament
  • Biomechanical properties of ACL
  • Clinical Problem and Treatment of ACL Injuries
  • Rat Anterior Cruciate Ligament

The entire kinematics of the forward and backward movement of the knee is controlled by the ACL. It prevents the tibia from slipping out of the femur and ensures knee rotation stability[5]. Because the mechanical attributes of the ACL are so closely related to the structure of collagen fibers, a change in the distribution of collagen fibers in the ACL can result in a decrease in mechanical resistance to force, which can lead to tears and fractures.

In the linear region, at the beginning of elastic deformation, the resistance pressure gradually increases. The yield area indicates the beginning of irreversible deformation, and due to the tearing of collagen fibrils at this point, stress decreases, leading to the rupture of the ligament[8]. Due to anatomical similarities between rats and humans [13], this study used rats as animal models to investigate the effect of injury on the diameter of collagen fibrils in the ACL.

Ligament Tissue Engineering

  • Nanofiber Scaffolds
  • Growth Factors
  • Electrospinning for Nanofibers
  • Mechanical Testing

Injuries due to ACL rupture are reported to occur more than 200,000 times per year in the United States [9]. Due to its hypocellular and minimally vascular nature, and the frequency of movement, ACL injuries do not regenerate, and their healing process leads to the development of a scar tissue. The scaffold forms an ideal environment for cell proliferation, while growth factors assist in the formation of new tissue [16].

Growth factors are proteins that are found in the early stages of ligament development and are thought to play a key function in the healing process. Furthermore, during electrospinning, the current is significantly elongated due to the acceleration towards the counter electrode, and the electrostatic forces overcome a surface tension when the voltage exceeds a threshold value, resulting in the release of an electrified positive current[25]. Polymer concentration is known as one of the most influential parameters to control the diameter of electrospun fibers [27].

Framework of the Study

In addition, solution viscosity and solvent evaporation rate controlled by ambient temperature and humidity can also affect the diameter, surface pore characteristics and spinnability of the fibers. The compressive, tensile, bending and shear properties of tissue can be determined using mechanical testing procedures. Uniaxial traction procedures, in which a properly harvested tissue is attached to handles and stretched in opposite directions, are the most widely used tests for investigating the mechanical properties of the ACL [30].

Tensile testing regimes can be affected by deformation as well as the degree of each of these parameters [30]. The stress-strain curve for many soft tissues undergoes a nonlinear transition as strain increases, with unfolded collagen becoming the dominant load-bearing component [ 31 ]. replicate the diameter distribution of collagen fibers and iii) compare the mechanical properties of native ACL tissue and nanofibrous scaffolds.

MATERIALS AND METHODS)

  • Materials
  • Harvesting the ACL Tissue
  • Biomechanical Tests
  • Preparing Rat ACL for TEM Characterization
  • Measurement of ACL Fibril Diameter
  • Nanofiber Scaffold Fabrication
  • Mechanical Properties of PCL Scaffolds
  • Scanning Electron Microscopy Characterization
  • Statistical Analysis

A surgical blade was used to remove all tendons and ligaments from the joint except the ACL (Figure 1A). A uniaxial material testing machine with a 1 kN load cell (MTS Criterion Model 43, MTS Systems Co., Eden Prairie, MN, USA) was used to assess the biomechanical parameters of ACL tissues. A custom jaw assembly was used to mount the Tibia-ACL-Femur joint to the unit, and the ACLs.

After reaching the state of failure, the knee joints were removed and the ruptured ACL was used to represent the damaged ACL tissue, and all five specimens were used in fibril diameter measurements to show the diameter distribution of damaged ACL tissue. First, the ACL tissue was located, it was stretched for better visibility, and it was removed from the joint to provide a sample with dimensions of approximately 2mmx2mmx2mm from the middle part of the tissue (Figure 1C, n=4). To prepare injured ACL tissue, a representative sample of size 2mmx2mmx2mm after rupture of ACL tissue was recovered from the injured ACL tissue from the region close to the rupture point (n=5).

Phosphate buffer solution (PBS) was used to wash the specimens three times for ten minutes each time. To ensure a smooth transition, a graded ethanol sequence was used to prevent any changes in tissue structure. After that, different combinations of resin and propylene oxide were used to penetrate the dried specimens.

Various amounts of 812 epoxy mixing medium components, DDSA (Dodecenylsuccinic anhydride) and MNA (Methyl nadic anhydride) were used to produce the resin. Infiltration was used to fill the sample blocks with resin to make them strong enough to withstand the pressure during cutting and shearing. Over 300 typical readings were obtained for each of the two groups of ACL tissues, both in the healthy and injured states.

SEM images of the PCL framework were used similarly to calculate the diameter of the fibers in the framework. A turbo pump sputtering device (Quorum Q150T ES, UK) was used to coat the samples with a 5 nm thick gold layer at a current of 20 mA. The mechanical parameters of aligned (bimodal) and unaligned (unimodal) scaffolds were compared with the mechanical properties of normal rat ACL tissue using one-way analysis of variance (ANOVA) with Tukey HSD (Honestly Significant Difference) post-hoc test.

Table 1: Composition of the ingredients to prepare bimodal and unimodal scaffolds
Table 1: Composition of the ingredients to prepare bimodal and unimodal scaffolds

RESULTS)

Diameter of Collagen Fibrils

ACL tissue from the injured knee showed a unimodal distribution with one peak at nm, while the healthy tissue showed a bimodal (two peaks) distribution with peaks at 60 nm and 153±11.5 nm. Overall, the diameter distribution of collagen fibers in the ACL of rats changed from a bimodal to a unimodal distribution after injury, indicating a decrease in mean diameters (p>0.05).

Figure 6 shows the combined distributions of ACL fibrils from injured and healthy ACLs
Figure 6 shows the combined distributions of ACL fibrils from injured and healthy ACLs

Fiber Diameter of PCL Scaffolds

Comparison of ACL collagen fibril diameters and PCL scaffold fiber diameters are given in Figure 9. No statistically significant difference was observed between the diameters of aligned PCL fibers and healthy ACL fibrils (p>0.05). The undirected PCL scaffold fiber diameters, on the other hand, were different from the damaged ACL fibril diameters (p<0.05).

Fiber alignment of aligned and non-aligned PCL scaffolds in the form of mean angle is presented in Figure 10. As clearly seen from Figure 10, the aligned bimodal scaffolds contained fibers aligned longitudinally as depicted by a normal mean angular distribution (Figure 10.A1&C). Random unimodal scaffolds, on the other hand, depicted a flatter distribution of mean angle of fibers, indicating a clear deviation from alignment (Figure 10.B1&C).

Figure 8: Combined aligned and unaligned PCL fiber diameter distributions in the form of (A)  histogram and (B) line graph (n=4)
Figure 8: Combined aligned and unaligned PCL fiber diameter distributions in the form of (A) histogram and (B) line graph (n=4)

Biomechanical Characterization of ACL Tissue and PCL Scaffolds

DISCUSSION)

Both findings obtained by Beisbayeva et al [32] in bovine ACL tissue, Smatov et al [33] in sheep ACL tissue and our results obtained in rat ACL tissue demonstrate similar behavior in terms of diameter distribution . The electrospinning technique was used to create electrospun PCL scaffolds that represented the healthy and damaged states of mouse ACL tissue. The collagen fibril diameter distribution of healthy and injured ACLs was represented by aligned and unaligned PCL scaffolds, respectively.

ACL collagen fibrils in healthy and damaged states quantitatively and qualitatively mimicked PCL scaffolds with aligned structures. Therefore, fine-tuning of the composition of the PCL solution is required to fabricate unimodal scaffolds. There is evidence that changes in the mean diameter and distribution of collagen fibers in the ACL are important markers of tissue mechanics, and changes in fiber diameter and distribution have been shown to have a direct effect on mechanical properties [4]. ].

It should be emphasized that the mechanical parameters of the healthy ACL tissue were the only ones. Due to the lack of tools to measure the mechanical properties of injured ACL tissue, it was not possible to compare the mechanical properties of injured ACL with healthy ACL and PCL scaffolds. Each of the parameters examined in this study, including ultimate load, ultimate strain, stiffness, and modulus, showed that native ACL tissue outperformed both aligned and unaligned PCL scaffolds.

A comparison of the two groups of scaffolds showed that the aligned and unaligned PCL scaffolds were comparable in terms of all parameters tested except the ultimate tension. For example, due to a lack of appropriate equipment, it was not possible to examine the mechanical characteristics of an injured ACL. Such a measurement would, for example, allow the comparison of non-aligned PCL scaffolds and damaged ACL tissue.

The mechanical characteristics of the ACL tissue were also examined in this work using tensile loading of the anterior tibial sacrum of the femur, although biomechanical stress in the sagittal plane is the most common cause of ACL injury [38].

CONCLUSION)

Zhou and Yingfang Ao, “Ultrastructural and Morphological Characteristics of the Human Anterior Cruciate Ligament and Hamstring Tendons,” Anat. Mall et al., “Incidence and trends of anterior cruciate ligament reconstruction in the United States,” Am. Karlsson, “Treatment of anterior cruciate ligament injuries with special attention to graft type and surgical technique: a review of.

Mizuno, "Localization of growth factors in the reconstructed anterior cruciate ligament: An immunohistological study in dogs," Knee Surgery, Sports. Han et al., “3D Electrospun Nanofiber-Based Scaffolds: From Preparations and Properties to Tissue Regeneration Applications,” Stem Cells Int., vol. Woo, “Effect of growth factors on the proliferation of ligamentous fibroblasts from skeletally mature rabbits,” Connect.

Spector, “The effect of selected growth factors on human anterior cruciate ligament cell interactions with a three-dimensional collagen-GAG scaffold,” J.

Сурет

Figure 1: Native ACL harvesting and characterization
Figure 2: Setup for electrospinning scaffold fabrication in an aligned approach
Table 1: Composition of the ingredients to prepare bimodal and unimodal scaffolds
Figure 3: Unaligned random scaffold electrospinning setup
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