An Experimental Study Of The Degradation Of Different Pore Sizes Silk Fibroin In Vivo And Its Relationship With The Host’s Tissue Response

Objectives:To study the morphological characters as well as the changes in molecular biology of silk fibroin scaffolds with different pore sizes in the process of degradation in vivo, to study the possible mechanism of the regulation of degradation rate of silk fibroin from the structure changes.Materials and Methods:1. Two kinds of silk fibroin scaffolds:SF-S, SF-L. Their average pore sizes were74.35±10.84μm (SF-S),139.23±44.93μm (SF-L), and their average porosities were94.2±0.1%(SF-S) and96.6±0.1%(SF-L), respectively.2. Twenty seven healthy Sprague Dawley rats were used as experimental animals. Two paravertebral incisions (2cm each) per rat were made approximately1cm lateral to the vertebral column. Subcutaneous pockets (1.5cm×3.0cm each) were created by blunt dissection. Two kinds of SF-S, SF-L were implanted into the two subcutaneous saclike lacunae to observe their degradation rate in vivo. The controls underwent the same operation, but no scaffolds implanted.3. At weeks1,4and12post-surgery, the animals were sacrificed, and the tissue-covered specimens were retrieved. The specimens were assessed by gross observation, HE staining, Masson staining and real-time reverse transcription-polymerase chain reaction (RT-PCR). Results:1. Gross observation showed that all the animals survived, and the wound healed without scar and abscess. At12weeks, a layer of pink fibrous membrane was found in growth and wrapping the SF-L group materials, while the SF-S group not.2. Histological observation showed that fibrous encapsulation occurred in all types of scaffolds, and endogenous cells which mainly include fibroblasts and leucocytes infiltrated at the interface between the scaffold and the fibrous capsule. There was a different degradation rate for two types of scaffolds (SF-S<SF-L, P<0.05). Different capsule thickness and fibroblast bands were also observed for two types of scaffolds (SF-S<SF-L, P<0.05and P<0.01,respectively).3. Cytokine relative gene expression was examined from the tissue around the scaffolds after1week,4weeks and12weeks of implantation. The expression of all the cytokines was relatively high for both groups after1week and4weeks, and a significant decrease (P<0.001) after12weeks. At every interval, different expressions of all the cytokines for two types of scaffolds were observed.Conclusions:1. The degradation rate of silk fibroin scaffolds (porosity rate>90%) with different pore sizes was different in vivo. During the observation period, the degradation rate was found faster in the large aperture silk fibroin group. However micro-porous collapsing and aperture diminishing were found in the large aperture silk fibroin group after12weeks, further study was needed.2. After implantation, a series of changes in the morphology of the host were observed and they were consistent with molecular biology results. In the earlier period of the experiment, HE staining showed that increased inflammatory cells, fibrous capsule and angiogenesis in the tissue around the material were observed; At the later stage, plenty of fibroblasts and fibrous capsule were observed. The inflammatory cells decreased, but gathered at the location where the degradation was significant. The number of micro vessels decreased but more matured.3. At gene level, the expression of profibrogenic cytokines was relatively high in both groups after1week and4weeks, and a significant decrease (P<0.001) after12weeks. At every interval, different expressions of all the cytokines in two types of scaffolds were observed.4. After12weeks, the expression of profibrogenic cytokines was up-regulated in the large aperture group compared with the small aperture one, and the fibroblasts were also increased. As the degradation of the large aperture group was greater, we concluded that the fibroblast may be involved in the degradation process of the material.5. The appearance of proliferating fibroblasts in the material surface was found neat, orderly and polar in both groups.6. There appears relevance between the degradation of the silk fibroin (139.23±44.93μm pore size) and the expression of the cytokines IL-1β, TNF-α, IL-10, TGF-β1and VEGF, especially the TNF-α and TGF-β1The significant degradation was found after the peak level of the above-mentioned factors.