New-Generation Hybrid Tissue Material That Heals Meniscus Tears and Acts as a Cushion Produced Using Nanotechnology
The meniscus is a fibrocartilaginous organ that redistributes stress and increases the stability of the knee joint. Meniscus damage is common and remains a challenging problem for orthopedic surgeons. Tissue engineering is currently the most promising method for meniscus repair. Electrospinning is a technique for manufacturing fibers at small scale.
With different materials and parameters, electrospun materials can have different mechanical properties, porosity, and orientation that can mimic the architectural and mechanical properties of natural meniscus [1]. Therefore, electrospun materials can be used in meniscus regeneration and treatment.
In this study, a nanotechnology-based composite material was obtained from polycaprolactone (PCL) incorporated with comfrey root, ginger (Zingiber officinale), and self-heal herb using a combination of electrospinning and freeze-drying techniques.
Two membranes produced by electrospinning were stacked within a prepared chitosan (CTS) solution and freeze-dried. The resulting nanocomposites underwent characterization studies including morphological (FEGSEM, field emission gun scanning electron microscope), biological (cell culture), and mechanical (tensile) analyses. When the study results were evaluated, the produced composite material was found suitable for use in treating meniscus tear disease.
Meniscus tear is the name given to a type of cartilage injury that occurs most frequently in the knee, causing pain and disability. In the human knee, there are two C-shaped pieces of cartilage that act as a cushion between the tibia and femur. These are called the meniscus.
They protect the constantly moving bones from wear and tear. However, meniscus tearing can occur through knee bending. In some cases, a piece of worn cartilage breaks off and becomes trapped between the joint surfaces, causing the knee to lock and movement to be restricted. It can develop from many causes such as sports, trauma, and discoid meniscus in all age groups. Injury during sports activity is the most common occurrence and is typically associated with anterior cruciate ligament rupture [2].
Meniscus surgery is the most frequently performed surgical procedure by orthopedists, particularly those specializing in sports surgery. The meniscus is an important part of the knee joint and is vital for continued knee function. With recognition of the meniscus's supporting effect on joint stability, shock absorption effect, its effect on balanced load transmission by improving the fit of the femur and tibia joint surfaces, its effects on proprioception, and its positive effects on cartilage nutrition, interest in protecting and repairing meniscus tissue has also increased [3].
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Ginger (Zingiber officinale) is a spice and plant that those experiencing tissue damage should definitely consume. Ginger has shown successful results in relieving pain caused by meniscus tear. When you massage the knee area by applying warm ginger water, you can enable faster tissue repair. To maintain general muscle and bone health, you should regularly drink ginger tea. Ginger prevents bone and cartilage damage [7]. The image of ginger (Zingiber officinale) is shown in Figure 1.2.
Self-heal herb, known by many different names, is also known among the public as nerve herb. This beneficial plant, known for its ability to heal wounds and tissues, also quickly heals tissue wounds that occur along with meniscus damage. There are many cell-renewing creams containing self-heal herb. Research results include information that self-heal herb heals the meniscus [8]. The image of self-heal herb is shown in Figure 1.3.
Polycaprolactone (PCL) is a biodegradable polymer with potential applications for bone and cartilage repair. PCL is a biologically degradable polyester with a melting point of approximately 60°C and a glass transition temperature of approximately −60°C. The most common use of PCL is in the production of specialty polyurethanes. PCL demonstrates good resistance to water, oil, solvent, and chlorine (Cl) compared to the produced polyurethane (PU) [9].
PCL has certain advantages over other polymers such as polylactic acid (PLA). PCL is more stable under ambient conditions, is significantly less expensive, and can be easily obtained in large quantities. Today, many researchers have focused on the use of PCL biocomposites and copolymers with both natural and synthetic polymers. PCL polymer is preferred in composite material production in literature for meniscus tear applications due to its elastic properties [10]. The chemical structure of PCL is shown in Figure 1.4.
Nanobiotechnology is the use of biological materials, biomimetic or biologically inspired, inorganic, organic molecules in nanotechnology devices for the purpose of controlling and imaging biological processes. Nanobiotechnology is a scientific discipline applied to alter or improve the physical and chemical properties of materials and devices.
The electrospinning method is the process of converting viscous liquids into nano-sized fibers by applying a kV voltage to viscous liquids at very low flow rates. Polymer solutions or melts are used as viscous liquids in its application. The solution reaching the tip of a medical syringe in uniform flow forms a spherical droplet under the effect of surface tension, and here it thins under the effect of electric forces (Taylor cone) and is transferred to a grounded collector as nanofibers. Thus, nanofiber membrane production is achieved [11].
Many successful techniques for meniscus repair have been described. In current literature, open meniscus repair techniques are applied only in combination with open surgical procedures such as tibial plateau fractures or multiple ligament injuries. Over time, many arthroscopic surgical meniscus repair techniques have been developed and are now performed entirely arthroscopically.
These are:- Inside-out repair,
- Outside-in repair,
- All-inside repair techniques.
When recent literature studies are examined, many natural and synthetic-based materials have been used via electrospinning technique in composite production for meniscus tear applications. To date, no finalized nanotechnology-based product has emerged. In this context, there is a serious gap in meniscus tear applications [12-15].
In this study, nanotechnology-based composite material was produced through a combination of electrospinning and freeze-drying techniques using comfrey root, ginger (Zingiber officinale), self-heal herb, and PCL materials. Two membranes produced by electrospinning were stacked within a prepared chitosan (CTS) solution and freeze-dried. The produced composite material was intended for use in treating meniscus tear disease.2. Materials and Methods
2.1. Materials Used
Comfrey root, ginger (Zingiber officinale), and self-heal herb, which are plants beneficial for natural meniscus tear disease, were obtained from local herb shops and plant growers. Chitosan (CTS), medium molecular weight, and trifluoroacetic acid (TFA) were preferred for dissolving chitosan (CTS). Polycaprolactone (PCL), molecular weight (Mw): 80,000 g/mol, chloroform, and dimethylformamide (DMF) were obtained from Sigma/Aldrich. Greaseproof paper was used as the substrate surface during the electrospinning stage.2.2. Experimental Design and Techniques 2.2.1. Preparation of Electrospinning Solutions
Comfrey root, ginger (Zingiber officinale), and self-heal herb materials were first kept in a freezer at -20°C. After drying, samples were dried in a freeze-dryer until no liquid remained. After the drying process, grinding was performed in a ceramic mortar. Unground coarse particles from powder particles were separated using a 63 µm stainless steel sieve. Electrospinning solutions were prepared according to the values shown in Table 2.1.2.2.2. Composite Production via Electrospinning Method
The electrospinning parameters required for composite production are shown in Table 2.2. Table 2.2. Electrospinning parameters required for composite production The stages of composite production via electrospinning method are shown in Figure 2.1. Figure 2.1. Stages of composite production via electrospinning method2.2.3. Composite Production for Meniscus Tear Applications via Freeze-Drying Technique
All samples shown in Table 2.3 were obtained via electrospinning technique. Composite production for meniscus tear applications was carried out by freeze-drying. All samples produced via electrospinning technique were stacked as first and last layers and placed in previously prepared CTS solutions and kept in a freezer at -20°C. After drying, samples were dried in a freeze-dryer until no liquid remained. In this manner, composite production for meniscus tear applications was successfully produced. The stages of composite production for meniscus tear applications are shown in Figure 2.2.2.2.4. Characterization Studies
*Morphological Characterization
The nanofiber diameters of the produced nanofiber membranes were examined at 7 kV potential using an FEI FEGSEM QUANTA 450 instrument. The diameters of 40 nanofibers were measured and their arithmetic means were taken to determine average nanofiber diameter ranges. Within the scope of cell culture biological characterization studies, samples were placed in 96-well plates and mesenchymal stem cell seeding was performed, with cell viability values observed at 24, 48, and 72-hour intervals. Tensile strength values of all samples were determined by preparing them according to ASTM standard and conducting tensile testing at 5 mm²/min tensile speed. Samples were repeated three times each, and arithmetic means were used.3. Results and Discussion *SEM Analysis
Successful nanofiber formation was observed in all samples. In addition to the polymer, the plants comfrey root, ginger (Zingiber officinale), and self-heal herb, which are beneficial for natural meniscus tear disease, and electrospinning operating parameters affected the morphology of nanofiber membranes. The nanofiber diameters of 40 fibers were calculated and arithmetic means were taken to determine the nanofiber diameter distribution range of the samples. The nanofibers of the 10% PCL sample were observed to be irregularly oriented and thick. When comfrey root, ginger (Zingiber officinale), and self-heal herb, which are beneficial for natural meniscus tear disease, were incorporated into PCL, the fiber diameters of the nanofiber membranes became thinner [16]. The finest average nanofiber in the study was observed in the 10% PCL-1.5% Comfrey Root-3.0% Ginger (Zingiber officinale)-2.5% Self-heal herb sample with a value of 50-180 nm. The nanofiber diameter distribution range values of the nanofiber membranes are shown in Table 3.1. SEM images of the skin mask nanofiber membranes are shown in Figure 3.1.*Cell Culture Analysis
Composite nanofiber membranes and disks freeze-dried for meniscus tear were placed in 96-well plates. Mesenchymal stem cells were seeded onto the samples in the plates and cell viability values were examined over 24, 48, and 72 hours. Cell viability values of the 10% PCL sample were lower compared to the other samples. However, with the addition of comfrey root, ginger (Zingiber officinale), and self-heal herb, cell viability values increased. Due to synergistic effects, when materials were combined, cell viability values reached 98% by the 72-hour period [17,18]. Cell viability values of composite nanofiber membranes and disks freeze-dried for meniscus tear over 24, 48, and 72 hours are shown in Table 3.2. Table 3.2. Cell viability values of composite nanofiber membranes and disks freeze-dried for meniscus tear over 24, 48, and 72 hours*Tensile Testing
Tensile strength values of samples were prepared according to ASTM standard and tensile testing was performed at 5 mm²/min tensile speed under room conditions. Samples measuring 1x5 cm were tested three times each, and arithmetic mean strength values were used. Although the PCL sample had the lowest strength in the study, when reviewing literature studies, it has strength values comparable to many studies. In our study, as comfrey root, ginger (Zingiber officinale), self-heal herb, and their synergistic composite materials were added to PCL, strength values increased. The additives homogeneously wrapped around the polymer, increasing its strength value. In this way, strength increased compared to PCL [19]. Tensile strength values of the nanofiber membranes are shown in Figure 3.2. Successful nanofiber formation was observed in all samples. Using a combination of electrospinning and freeze-drying techniques with comfrey root, ginger (Zingiber officinale), self-heal herb, and PCL materials, next-generation composite materials were produced. Based on the results of morphological, mechanical, and biological characterization studies of the samples, the most ideal composite was 10% PCL-1.5% Comfrey Root-3.0% Ginger (Zingiber officinale)-2.5% Self-heal herb. The source of this is the synergistic effects of the materials used. The produced composite material is intended for use in treating meniscus tear disease. Furthermore, considering the properties of the materials contained in the obtained product in literature, its use is envisioned in sectors such as healthcare, textiles, food, agriculture, filtration, and defense. Erdi Buluş Master of Science in Metallurgy and Materials Engineering Expert in Nanotechnology-Materials Technology Istanbul Arel University ArelPOTKAM (Polymer Technologies and Composite Application and Research Center) Gülseren Sakarya Buluş Specialist Nurse, Health Sciences Nanotechnology/Biotechnology, Emergency and Disaster Management Silivri District Health Directorate Assoc. Prof. Dr. Yeşim Müge Şahin Istanbul Arel University ArelPOTKAM (Polymer Technologies and Composite Application and Research Center)References [1] Chen, M., Gao, S., Wang, P., Li, Y., Guo, W., Zhang, Y., ... & Xi, T. (2018). The application of electrospinning used in meniscus tissue engineering. Journal of Biomaterials science, Polymer edition, 29(5), 461-475. [2] Guilak, F., Fermor, B., Keefe, F. J., Kraus, V. B., Olson, S. A., Pisetsky, D. S., ... & Weinberg, J. B. (2004). The role of biomechanics and inflammation in cartilage injury and repair. Clinical Orthopaedics and Related Research® , 423, 17-26. 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