A Preliminary In Vivo Study on the Histocompatibility of Silk Fibroin

Biomaterials used for tissue engineering should have the property of good histocompatibility, superb plasticity and desired degradability, so that it can be extensively applied for defect tissue repairing with excellent clinical outcome. In the past decade, silk fibroin has become one of the most favored biomaterials for its wide availability, superb performance and readiness to be shaped for different purposes in tissue engineering[1-14],. Porous scaffolds made by silk fibroin can be made into different pore size and porosity to serve for different needs of tissue repairing. The porous structure may contribute to the mass exchange in the scaffolds. However, the implanted protein scaffolds will degrade and can hardly be separated from host tissues. Therefore, little has been reported on histocompatibility experiment in vivo for silk fibroin[15-22]. In this chapter, progress in study of silk fibroin scaffold in tissue engineering application and biocompatibility research will be introduced, then our histocompatibility experiment of porous scaffolds will be reported. In our experiment, porous scaffolds were made platy and buried in subcutaneous part of the back of SD rat. Tissue reaction was observed, and the value of silk fibroin as tissue engineering scaffold material was discussed.


Introduction
Biomaterials used for tissue engineering should have the property of good histocompatibility, superb plasticity and desired degradability, so that it can be extensively applied for defect tissue repairing with excellent clinical outcome. In the past decade, silk fibroin has become one of the most favored biomaterials for its wide availability, superb performance and readiness to be shaped for different purposes in tissue engineering [1][2][3][4][5][6][7][8][9][10][11][12][13][14], . Porous scaffolds made by silk fibroin can be made into different pore size and porosity to serve for different needs of tissue repairing. The porous structure may contribute to the mass exchange in the scaffolds. However, the implanted protein scaffolds will degrade and can hardly be separated from host tissues. Therefore, little has been reported on histocompatibility experiment in vivo for silk fibroin [15][16][17][18][19][20][21][22] . In this chapter, progress in study of silk fibroin scaffold in tissue engineering application and biocompatibility research will be introduced, then our histocompatibility experiment of porous scaffolds will be reported. In our experiment, porous scaffolds were made platy and buried in subcutaneous part of the back of SD rat. Tissue reaction was observed, and the value of silk fibroin as tissue engineering scaffold material was discussed.

The application of silk fibroin in tissue engineering
It is known to all that the application of silk production as un-absorb suture has many years of history. Along with the progress of tissue engineering techniques, scientists can extract natural polymeric materials-fibroin from silk and make it into different forms to fit various needs of tissue engineering [1] . Vitro studies show silk fibroin is biodegradable. The speed and degree of degradation can be adjusted through changing physicochemical property [2][3][4] . The product of degradation is mainly free amino acid and has no toxic sideeffect on tissue. Silk fibroin can be easily made into different forms to serve for various needs such as membrane, gelatum, knitting scaffold, porous scaffold and electrospinning scaffold. Therefore, it has been gradually utilized in various medical field such as drug delivery [5,6] , nerve regeneration [7][8][9] , dermis healing [10] , artificial ligament repair [11,12] , bone or cartilage healing [13.14] , vascular tissue engineering [15,16] , otology application [17] and so on.

Biocompatibilty research of silk fibroin
Silk fibroin has been utilized as natural biomaterial in tissue engineering applications. Many researches have been made in vitro to reveal its attractive properties such as slowcontrollable degradation, mechanical robustness, and inherent biocompatibility [18][19][20][21] . For the silk fibroin scaffolds are made up of proteins, those materials which have been implanted will degrade and could hardly be separated from host tissues, that made the study of histocompatibility difficult. Therefore, few report can be found about research on biocompatibility and degradation of porous fibroin scaffolds in vivo [4,22] .

Materials and methods
Laboratory animals: 8 healty female Sprague-Dawley rat, body weight 220g～250g, get from department of laboratory animal medicine, Fudan University Preparation method of porous silk fibroin scaffold: After degummed, the raw silk was dissolved into aqueous solution of LiBr (lithium bromide). The silk fibroin solution was adjusted to the concentration of 20% after filtration and concentration. Then n-butyl alcohol was added into the solution as volume ratio of 2: 1. Mixed solution was added into selfmade mould after low-speed agitation, and white porous scaffold was obtained after freeze drying. The average pore size of the scaffold was between 10 and 20um. Material prepration: We cut the porous silk fibroin scaffolds to 1cm×1cm squares (about 1 mm thick), infused the material into 75% ethanol solution for 0.5 hour, then immersed the materials into 0.9% NaCl water for 24 hours. Technique: After peritoneal injection anesthesia, we longitudinal incised the skin of back open, blunt dissected surrounding tissues, and buried the porous silk fibroin scaffolds subcutaeous. Then we sew up the incision and raised the animals in different captivities. Histological examination The rats were executed 2,4,6,8 weeks after operation. General observation and hematoxylin and eosin stain histological examination was performed.

Result and discussion
General observation Animals recovered well after operation. Throughout the period of implantation, all scaffolds were well tolerated by the host animals and immune responses to the implants were mild. No obvious systemic reaction, abnormal eating and sleeping behaviors were found. The implant sample in animals' back hunched mildly. The wounds and surrouding skin healed well when drawing materials at different times (figure 1). No obvious hyperemia, seepage or purulent exudates signs were found in surrounding skin and muscular tissue. The surface of the scaffolds was wrapped up by the thin, semitransparent fibrous membranes. There was no obvious change in shape and appearance after implanting(figure 2). The inflammatory reaction surrounding the tissue was slight. And no obvious granulation was found. (table 1) The second week: Fibroin scaffold is eosinophilic staining. The surface is not regular while there are big or small pores inside. Hematoxylin-eosin staining microscopic examination (table 2)    Porous scaffols made by silk fibroin can be formed into different pore size and porosity, which could meet the needs of different histocytes during repairing process. Porous structure is also helpful to the exchange of nutrient substance among different parts of scaffolds. So we choose the porous silk fibroin scaffold as the research object. Biological material will be treated as foreign matter when implanted in the body, even if its biocompatibility is perfectly good. Therefore, foreign body reaction will be inevitable surrounding the material. The strength and extent of foreign body reaction are closely related to its biocompatibility and biodegradablity. So, we evaluate the tissue reaction and material change after implantation by fibrous encapsulation, granulation formation，tissue fibrosis, type of inflammantary cells, tissue necrosis and tissue ingrowth, morphological changes and disaggregation of the scaffolds. We found that wet porous fibroin scaffold was soft in texture, and could be easily cut into different forms to fit the need of implantation while the operation was performed. Tissue reaction around the materials was slight when we removed the scaffold, and the surface of the material was wrapped up by semitransparent fibrous membranes. The porous fibroin scaffolds were eosinophilic stained by HE. Its surface was irregular. And there are lacunes of different size inside the material. Chronic inflammation reaction occurred, mainly lymphocytes and macrophages could be found around the material. And fibrous capsule could be observed. After two weeks of implantation, blood capillary could be found proliferate into the lacuna of the material, and the fibroblasts also attached to the irregular surface of the material. These phenomena became more and more obvious as time went by, which showed that the surface texture of porous fibroin was favourable for interstitial cell to grow on. Moreover, the porous structure of the scaffold made the material permeable, which could probably cause the exchange of the nutrition and metabolite through the scaffold that lead to active tissue ingrowth. This may also be a plus factor for tissue regeneration. Because the fibroin scaffolds were formed by protein and could hardly be separated from surrounding tissues, histopathology observation was performed after experiment instead of quantitative determination. During eight-weeks' observation, the scaffold disintegration started from the fourth week. But there was still no obvious change inside the scaffold structure until the eighth week, which indicated to us that we should set the observation period much longer to get more information about the degradation of fibroin porous scaffold in further research. We didn't make effective statistical analysis about the quantity and type of inflammatory cells surrounding the scaffold for the number of samples was too small. Moreover, according to the requirements of different tissue repair, the pore size and porosity of porous scaffolds should be different. And materials with different structure may cause different result in tissue reaction, tissue regeneration and material degradation. These are also challenging questions that need to be considered in further experiment.

Conclusion
To sum up, porous silk fibroin scaffold shows good histocyte attachment and has good histocompatibility. The Porous silk fibroin scaffold can degradate in vivo, but more study should be made on the mechanism and degradation products.

Acknowledgment
The chapter publication was funded by Shanghai Science and Technology Committee, 1052nm03801.