In recent years, the abuse of antibiotics has posed a great challenge to modern medicine16–18. As a potentially effective medical treatment resource, antimicrobial peptides can rapidly destroy bacterial membranes and have difficultly making bacteria tolerant to the drug. However, since the discovery of antimicrobial peptides, they have not been widely used in clinical medicine due to their own shortcomings, such as unstable physicochemical properties, easy enzymatic hydrolysis and high cost, and their pleiotropic nature also limits their application as specific antibiotics19. However, the most important thing is that natural antimicrobial peptides need specific environments to be biologically active, suggesting that they have been largely influenced by host-pathogen interactions over millions of years of evolution20. Therefore, many natural antimicrobial peptides are sensitive to serum, divalent cations, pH value and protease, etc., which greatly limits their application range21–23. Whereas, synthetic antimicrobial peptides are expected to solve the above problems and become one of the hot areas of current research. KSL-W is a synthetic antibacterial peptide, which can effectively kill oral bacterial pathogens and inhibit oral biofilm formation. And the microsphere system prepared by PLGA can not only have an obviously sustained and controlled release effect on drugs, but also protect bioactive materials from the degradation of the corresponding environment and improve bioavailability, which has great potential for medical application24–26.
In this study, KSL-W@PLGA microspheres were prepared by the double emulsion method. Influence of the concentration of PLGA, the concentration of KSL-W, the concentration of PVA emulsion and the volume ratio of inner water phase to organic phase on the encapsulation efficiency of KSL-W@PLGA microspheres was studied utilizing orthogonal test. According to the orthogonal experimental results, we found that the concentration of PVA had the greatest impact on the encapsulation efficiency of the PLGA microspheres, followed by the concentration of the inner water phase KSL-W and the volume ratio of the inner water phase to organic phase, while the concentration of the organic phase was the least important factor. Therefore, the optimum technological conditions of preparation KSL-W@PLGA microspheres were determined, namely, the concentration of PLGA is 25mg/mL, the concentration of KSL-W is 10mg/mL, the concentration of PVA is 0.5%, and the volume ratio of inner water phase to organic phase is 2/1. KSL-W@PLGA microspheres prepared under above conditions had smooth surface and uniform morphology, the drug loading and encapsulation efficiency are (5.29 ± 0.05)% and (74.23 ± 0.24)%, respectively.
Numerous studies have shown that E.faecalis and P. gingivalis are the main pathogenic bacteria of chronic periapical periodontitis, in most cases of endodontic treatment failure, one of the most common pathogenic microorganisms is E.faecalis, with a positive rate of 24%-77%27. As a Gram-positive facultative anaerobe, it has a special ability to invade and colonize in the depths of the dentin tubules, withstand changes in extremes including calcium hydroxide (CH) and other drugs, and survive in starvation environment. At the same time, the formation of E.faecalis biofilm in the root canal also increases the difficulty and failure rate of root canal treatment. In patients with periapical periodontitis secondary to root canal treatment failure, 16S RNA genetic sequencing found that the number of E.faecalis was significantly increased28. P.gingivalis also plays an important role in the occurrence and development of chronic periapical periodontitis, and its detection rate in the root canal samples of patients with periapical periodontitis was up to 69.4%29. Therefore, in this experiment, E. faecalis and P. gingivalis were used as experimental strains of bacteria to study the sustained antibacterial effect of KSL-W@PLGA microspheres. The experimental results showed that there was a significant bacteriostasis effect on the 5th and 10th days, indicating that the PLGA sustained-release microspheres prepared in the experiment could achieve sustained antibacterial effects.
With the progress of current medical methods, there are many different planning treatments for the repair of bone defects. At present, the most frequently used and effective treatment plan is bone transplantation. Autogeneous bone graft does not have immunological rejection because the donor bone is derived from the patient, at the same time, the healing effect can be better through the blood vessel anastomosis. However, it still has many shortcomings, such as secondary operative trauma and limited resources, which limit its clinical application. Therefore, more and more attention is focused on allogeneic bone and artificial bone substitute materials. For example, Bio-Oss® particles are commonly used in clinic, which is an inorganic material extracted from bovine bone, mainly composed of hydroxyapatite crystals. It not only has good biocompatibility, but also can provide the cytoskeleton for cell growth30. However, it lacks osteogenesis and osteoinductive properties and is prone to collapse, also the natural absorption and degradation process in the body is very long31. Therefore, it is of great significance to develop new bone-repairing materials with good biocompatibility, suitable speed of biodegradation and antibacterial activity32− 34.
As a natural organic component in animal skin, bone tissue, tendons, and ligaments, collagen has good biocompatibility, biodegradability and low immunogenicity, and plays an important role in the adhesion, growth, migration and differentiation of cells. Collagen can guide the formation of new bone tissue and is a carriage material widely used in the regeneration of bones, blood vessels and nerves. However, due to the poor mechanical properties and the fast degradation rate of collagen, its application in tissue engineering is limited. Silk fibroin is a natural fiber, which has excellent performance in elasticity, flexibility, biocompatibility and biodegradability. In particular, the silk fibroin secreted by the silkworm has good biocompatibility and low immunogenicity, which has attracted widespread attention in the field of bone tissue engineering35–37. However, its low capacity of bone-forming limits its application in the realm of orthopaedics38. Hydroxyapatite (HA), as the main inorganic component of bone tissue, has good biocompatibility, stability and biodegradability39. It will not cause irritating effect or rejection reaction after implantation, and has good bone conductibility and the ability to induce bone formation. However, the main disadvantages of HA are easy brittleness and not easy to be degraded. Therefore, researchers try to mix different biomaterials to achieve the best therapeutic effect. Jin et al.40 evaluated the potential of the silk fibroin/hydroxyapatite porous material as a delivery vehicle for human placenta-derived mesenchymal stem cells in reconstruction of bone, and the results showed that it could markedly enhance tissue repair. Ding et al.41 used a composite scaffold composed of hydroxyapatite nanoparticles (HA) and silk fibroin (SF), while bone morphogenetic protein-2 (BMP-2) was loaded in the HA/SF scaffold to improve osteogenesis capacity, which provided preferable microenvironments for bone regeneration. Quinlan et al.42 prepared bioactive glass/collagen-glycoaminoglycan composite scaffolds containing resorbable bioactive glass particles with cobalt ions, which significantly enhanced the production and expression of VEGF in endothelial cells, meanwhile, the composite scaffolds have the ability to promote angiogenesis and proliferation of osteoblasts, and promote bone tissue regeneration.
In this study, the biocompatibility of 3D-printed bone scaffold containing KSL-W@PLGA microspheres/collagen/silk fibroin/nano-hydroxyapatite was discussed. The inoculation of MC3T3-E1 cells on the scaffolds was observed for 5-7d. HE staining and SEM were used to observe the growth of MC3T3-E1 cells. As can be seen from Fig. 4–5, MC3T3-E1 cells showed a tendency of multi-layer growth along the scaffolds aperture, and the cells adhered to the surface of the scaffold were fully stretched and pseudopodia were visible.
The good three-dimensional structure and appropriate porosity play an important role in the adhesion and growth of cells, as well as for the exchange of nutrients and metabolites. Generally speaking, the mechanical properties of scaffolds will decrease when the porosity is too high; while the porosity is too low, it will not be able to meet the needs of osteoblasts to grow into the scaffolds and affect the formation of new bone tissue. Zhang et al. analyzed the bone tissue engineering scaffolds with 50%, 60% and 70% porosity from the macroscopic characteristics, microstructure and biomechanical properties, and found that the scaffolds with 60% porosity had the best effect43. In general, the aperture sizes of scaffolds ranged from 150µm to 800µm44–46. Larger aperture size can increase the proliferation of osteoblasts and the growth of blood vessels. Meanwhile, sufficient nutrients exchange is also conducive to the formation of bone tissue, but mechanical properties are correspondingly reduced. However, when the aperture size of the scaffolds is too small, although there are certain advantages in mechanical properties, the limited adhesion and proliferation of cells and local hypoxia limit the bone repair ability. Generally speaking, the pore size suitable for bone tissue engineering is between 400µm and 600µm47–50. In this experiment, the porosity of the prepared 3D-printed scaffolds was (81.96 ± 1.83) % and the pore size was (523 ± 42) µm, which basically met the requirements of tissue engineering. HE staining and SEM also showed that the scaffold materials had good biocompatibility, which was conducive to the adhesion and proliferation of MC3T3-E1 cells.
Chronic periapical periodontitis is a common oral disease, mainly caused by bacteria and endotoxin spreading into the periapical. Its main manifestations are inflammatory reaction and bone destructions in the periapical region. In general, RCT can heal most of periapical periodontitis, but there are still some lesions (14%-58%) due to a large inflammatory range and severe bone destruction, which cannot be healed by RCT alone, so endodontic microsurgery is necessary. Traditional endodontic surgery can repair the bone defect by removing infected apical tissue and organizing the blood clots. However, for some offending teeth with large bone defects, the therapeutic effect is not good. As guided bone regeneration (GBR) has been widely used, it provides a scaffold for the growth of osteoblasts by implanting bone repair materials into the bone defect area, and at the same time, a collagen membrane is covered between the soft tissue and bone defect area to prevent the epithelial cells and connective tissue cells from growing into the bone defect area. At present, Bio-Oss® particles are commonly used in clinic, which can be implanted into the operative region to get a better medical prognosis. However, it still has some drawbacks, such as lack of active ingredients like osteoblasts and osteogenic factors. Therefore, this study intends to explore the healing effect of bone defects by implanting the 3D-printed KSL-W@PLGA microspheres/collagen/silk fibroin/nano-hydroxyapatite scaffold to the rabbit's mandible.
In this study, Japanese white rabbits were selected as experimental animals. Considering that rabbits are rodents and their teeth grow continuously with chewing and attrition, it is not suitable to prepare the bone defect model of periapical periodontitis at the apex of the teeth. Therefore, the bone defects were prepared at the mandible of rabbits to simulate periapical periodontitis. The experimental groups of this study were 3D-printed scaffolds, which has the functions of inducing the bone formation and providing a three-dimensional environment in which cells can grow, while the Bio-Gide® membrane covered can prevent fibroblasts from growing into the bone defect area. Since Bio-Oss® particles and Bio-Gide® membrane have been widely used clinically as the ideal bone-repair materials, which have a significant clinical effect and favorable prognosis, they were used as the positive control group in this study. According to the pictures of the gross model of rabbit mandible and the HE staining results, the 3D-printed scaffold materials prepared in this study provided a supporter for the growth of osteoblasts, which was conducive to the formation of new bone tissue and accelerated the healing process of bone defects.
X-rays, a new radiation has a strong penetrating power, discovered by Röntgen in 1895, and photographic methods are available through the outline of human body bone and metal objects internal defects. In the field of stomatology, dentists can make diagnoses in the hard tissue lesions (such as caries, cracked-teeth, and fractured-teeth), pulp and periapical lesions, and periodontal lesions though digital periapical film. For example, in patients with chronic periapical periodontitis, radiographs may show the circular or elliptical images of bone density loss, indicating absorption of alveolar bone and replacement of normal bone tissue with inflammatory tissue. Micro-CT, as a micro-computed tomography technology, has a high resolution of tissue detection and does not damage the integrity of the sample, which has unique advantages in bone imaging. And at the same time, changes in bone tissue can be quantitatively analyzed by analyzing the parameters of each index in the target area.
Among them, the commonly used parameters include tissue volume (TV), which represents the total volume of the target area, when TV is the same, bone volume (BV) and bone volume fraction (BV/TV) can also reflect the amount of bone mass. Trabecular bone thickness (Tb.Th) is used to represent the average thickness of bone trabecular, which can be used to evaluate its spatial morphology and structure. In this experiment, some parameters of positive control group, such as BV/TV, Tb.N, Tb.Th, etc., are abnormally increased, which may be attributed to the fact that Bio-Oss® particles itself are an inorganic bone matrix that removes all organic matter. To sum up, the 3D-printed scaffolds demonstrated bone healing efficacy on mandible bone defects in rabbits, showing great potential for clinical application.