The literature that addresses the degradation of SF materials in vivo lacks uniform and effective standards and experimental evaluation methods. A common method is the morphological description, which includes HE staining and scanning electron microscopy[13, 17]. The strength of the material was evaluated based on its mechanical properties[18]. The biocompatibility and in vivo absorption of the material were detected through cell biology[14, 19]. Functional indicators could also be tested for the materials that were implanted in special parts of the body[20]. Although these methods have a certain effect on understanding the reaction process and the mechanism of degradation in vivo, it is often impossible to accurately quantify the material degradation dynamically and in real-time. Most of the tests that are described in the literature are invasive tests; however, they are not applicable to living organisms. Although imaging inspection methods such as x-ray inspection and computed tomography inspection are non-invasive[21], their application range is limited and they are only sensitive to denser materials. They are not suitable for the study of SF materials with a lower density. Therefore, finding a dynamic and non-invasive degradation evaluation method is also an urgent problem that needs to be solved in the clinical applications of SF materials.
In a previous study[14], we developed a semi-quantitative approach to assess the in vivo degradation rate and biocompatibility of the 3D RSFs that have different pore sizes. The semi-quantitative method was used to evaluate the biodegradation by measuring the thickness of the residual scaffolds, fibrous capsules, and infiltrated tissues by performing a histological analysis. Since the scaffold’s volume change is irregular after implantation, the comparison method that is based on the thickness of the scaffold cannot accurately reflect the degradation of the scaffold. In fact, the best way to estimate the rate of the scaffold biodegradation is to measure the residual volume of the scaffold in three dimensions. However, this method is difficult to implement in vivo. Wang et al.[18] compared the biodegradation of several scaffolds (5 mm in diameter and 2.5 mm in thickness) by measuring the cross-sectional area of the residual scaffolds in two dimensions. This is a relatively accurate method. However, since this method was technologically limited at that time, only small scaffolds could be measured. In this study, we made improvements based on the previous studies. HE-stained sections were made by selecting the largest cross-section of the scaffold at each time point after the implantation. As long as the size of the scaffold does not exceed the size of the slice of the HE stain, the image on the slice can be completely recorded by the scanning system. A semi-quantitative comparison was performed on the cross-sectional area of the scaffolds to evaluate the degradability of the RSFs. Histological methods can intuitively obtain the changes in size of the scaffolds; thus, evaluating the scaffold degradation is more reliable. However, this method cannot be applied to study scaffold degradation in living organisms. In order to facilitate the research after the scaffold is implanted in living organisms, we urgently need a non-invasive, real-time, repetitive method that is applicable.
Ultrasound is a non-invasive imaging method that is more sensitive to the detection of soft tissues. It has been used in research that involves degrading biological materials; however, it is rarely used when researching SF scaffolds. Li et al.[22] investigated the neovascularization and biodegradation of an SF gel in vivo using multiple mode ultrasound by quantifying the echo intensity, volume, and contrast enhancement of the SF gel implants. It showed that the silk gel implants appear as hypoechoic nodules in the early stages of the degradation, and there are clear boundaries under the skin. With the passage of time, the echogenicity of the silk hydrogel implants gradually increased, and it was demonstrated through medium grayscale images. In the late stage of the degradation, the echogenicity of the silk hydrogel implant was equal to that of the adjacent border, and it could not be clearly identified until the 20th day. In this study, the degradation of the RSF scaffold was investigated in vivo by applying B-mode ultrasound. With the passage of time, the echoes of the SF scaffold gradually decreased. In the early stages of the degradation, the echo of the sample was higher than that of the muscle. In the middle of the degradation, the echo was equal to the that of the muscle. In the later stage, the echo of the sample was lower than that of the muscle. The echo of the SF scaffold during ultrasound in this study was different from the findings by Li et al[22]. This is possible because SF can be processed into different constructs (e.g., porous scaffolds, films, hydrogels, and nano/microspheres). The echo intensity of the different forms of the SF during ultrasound is different. The reason for the changes in the echo in this study might be related to the degree of tissue cells that invade the scaffold and the degradation of the scaffold. During the early stage of implantation, the scaffolds were filled with pores, only a small number of tissue cells invaded the scaffolds, and the scaffolds were hardly degraded. As a result, the echo of the scaffolds was higher than that of the muscle in the early stage. As the degradation progresses, tissue cells invade the scaffolds, and the pores in the scaffolds gradually degrade; therefore, the echo of the scaffolds is reduced. In the later stage, the scaffolds were filled with cells, which form a relatively uniform whole; hence, it appears to be hypoechoic when applying ultrasound. In these experiments, local hypoechoic echoes were observed in the scaffolds. We believe that this may be due to the penetration of the host tissue into the scaffolds and the degradation and absorption of the local scaffolds. Therefore, it can be inferred that the degradation of the SF scaffold and tissue proliferation occur simultaneously. The performance of the tissue deep into the scaffold could also be observed in the HE-stained sections, which further confirmed this idea.
In this study, we implanted two types of 3D RSFs (SF-A group and SF-B group) that were subcutaneously placed into the back of SD rats. For a semi-quantitative comparison, we used B-mode ultrasound and HE staining to measure the cross-sectional areas of two groups of 3D RSFs and performed a statistical analysis. The statistical results show that there were significant differences in the degradation of the two groups of 3D RSFs. The degradation rate of the SF-B group was significantly higher than that of the SF-A group. Regression analysis showed that the results of the B-mode ultrasound and HE staining were correlated in both groups. This indicates that B-mode ultrasound is reliable for the degradation of the SF scaffolds in vivo. In this study, we determined that the volumes of the two types of 3D RSFs increased after one week of implantation by applying two detection methods. This is possible because a fibrous capsule appeared around the scaffold after one week of implantation, and the scaffold was not significantly degraded at that time. Therefore, the volume of the scaffold is larger in comparison with when it was implanted. In addition, the infiltration of the inflammatory cells and the penetration of small blood vessels also slightly increased the volume of the scaffold. It can be observed from the figure that the cross-sectional areas of the ultrasound detection are slightly larger than those observed during HE detection (Fig. 4). A possible reason for this deviation is that ultrasound can observe the material in situ when it is placed in the body. After the SF scaffolds are implanted in the body, a fibrous membrane gradually forms on the surface of the SF scaffold. In other words, a material-tissue complex is formed, which shows a similar echo to the scaffold; thus, it can be detected as a scaffold with ultrasound. HE staining was conducted after the materials were removed from the body, and could clearly distinguish the material and the fiber envelope; therefore, the detected value in HE staining was less than that of ultrasound. In addition, dehydration during HE staining of the implanted materials may also lead to a reduction in the volume. In the experiment, it was determined that the material-tissue complex of SF-A was thicker than the material-tissue complex of SF-B. This may be related to the fact that SF-A is denser and does not easily degrade. We believe that the material-tissue complex is a common whole in the body's repair process. In addition, the detection of the material-tissue complex with ultrasound can better reflect the true situation of the repair process. In addition, ultrasound can also detect inflammation and exudation around the material in the early implantation stage, which is not possible with other invasive examinations.
In this study, it was determined that the application of ultrasound to evaluate the degradation of SF scaffolds has some limitations. In the early and middle stages of the degradation process, the SF scaffolds can be clearly detected by applying B-mode ultrasound in vivo. The echo changes accompanying the material degradation and host tissue infiltration can be observed. However, when the material was gradually degraded and absorbed in the later stage of the degradation, the echo was reduced. In other words, when there was not much material left, it was difficult to distinguish the material from the surrounding tissues, and applying ultrasound during the later stages of implantation led to the loss of its advantages.