**4.1 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.

### **4.2 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). Fibroblast grows adherently on the surface of the material. The surrounding tissue hyperplasticly changes, inflammatory cell infiltrate is visible, most of which are lymphocytes and macrophages. No signs of tissue necrosis are found.(figure 3,4)

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 slow-

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

**Laboratory animals:** 8 healty female Sprague-Dawley rat, body weight 220g~250g, get from

**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

**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

**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.

**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.

**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). Fibroblast grows adherently on the surface of the material. The surrounding tissue hyperplasticly changes, inflammatory cell infiltrate is visible, most of which are lymphocytes and macrophages. No signs of tissue necrosis are found.(figure 3,4)

controllable degradation, mechanical robustness, and inherent biocompatibility[18-21].

biocompatibility and degradation of porous fibroin scaffolds in vivo [4,22].

**4. Our in vivo research on the histocompatibility of silk fibroin** 

drying. The average pore size of the scaffold was between 10 and 20um.

department of laboratory animal medicine, Fudan University

materials into 0.9% NaCl water for 24 hours.

And no obvious granulation was found.(table 1)

**3. Biocompatibilty research of silk fibroin** 

**4.1 Materials and methods** 

**4.2 Result and discussion** 

Fig. 1. The wound healed up well while taking samples

Fig. 2. Thin connective tissue wrap could be found on the scaffold, and the inflammatory reaction surrounding the tissue was slight.


Table 1. general observation

**The fourth week**: There is few parts with eosinophilic staining inside the material disappears 4 weeks after operation, which shows crumbling phenomenon, while it's general structure remains(figure 5). Fibroblasts grow along the surface of the scaffold or into the pores. There are lymphocytes and macrophages infiltrate. Vessel hyperplasia is visible,

A Preliminary In Vivo Study on the Histocompatibility of Silk Fibroin 419

Fig. 5. There is few parts with eosinophilic staining inside the material disappears 4 weeks after operation, which shows disaggregation phenomenon, while it's general structure

Fig. 6. Fibroblasts grow along the surface of the scaffold or into the pores. There are lymphocytes and macrophages infiltrate. Vessel hyperplasia is visible, while new capillary

vessel inside the material is obvious. No signs of tissue necrosis are found. (×400)

remains. (×200)

while new capillary vessel inside the material is obvious. No signs of tissue necrosis are (figure 6).

**The sixth week**: There are a few parts with eosinophilic staining inside the material disappears 6 weeks after operation, which shows crumbling phenomenon, while it's general structure remains.Inflammatory cell infiltrate is visible 6 weeks after operation. Histocytes gradually grow deep into the scaffold. No signs of tissue necrosis are.(figure 7,8)

**The eighth week**: Histocytes grow more deeply into the scaffold. Inflammatory cell infiltrate is visible, most of which are macrophages. Vessel hyperplasia reduces inside the tissue, while hyperblastosis still obvious. No signs of tissue necrosis are.(figure 9,10)

Fig. 3. Fibroblast grows adherently on the surface of the material two weeks after operation. The surrounding tissue hyperplasticly changes, inflammatory cell infiltrate is visible, most of which are lymphocytes and macrophages. No signs of tissue necrosis are found. (×200)

Fig. 4. Fibroin scaffold is eosinophilic staining. The surface is not regular while there are big or small pores inside. Fibroblasts grow adherently on the surface of the material. Inflammatory cell infiltrate is visible, most of which are lymphocytes and macrophages.(×400)

.

while new capillary vessel inside the material is obvious. No signs of tissue necrosis are

**The sixth week**: There are a few parts with eosinophilic staining inside the material disappears 6 weeks after operation, which shows crumbling phenomenon, while it's general structure remains.Inflammatory cell infiltrate is visible 6 weeks after operation. Histocytes

**The eighth week**: Histocytes grow more deeply into the scaffold. Inflammatory cell infiltrate is visible, most of which are macrophages. Vessel hyperplasia reduces inside the

Fig. 3. Fibroblast grows adherently on the surface of the material two weeks after operation. The surrounding tissue hyperplasticly changes, inflammatory cell infiltrate is visible, most of which are lymphocytes and macrophages. No signs of tissue necrosis are found. (×200)

Fig. 4. Fibroin scaffold is eosinophilic staining. The surface is not regular while there are big

or small pores inside. Fibroblasts grow adherently on the surface of the material. Inflammatory cell infiltrate is visible, most of which are lymphocytes and

gradually grow deep into the scaffold. No signs of tissue necrosis are.(figure 7,8)

tissue, while hyperblastosis still obvious. No signs of tissue necrosis are.(figure 9,10)

(figure 6).

.

macrophages.(×400)

Fig. 5. There is few parts with eosinophilic staining inside the material disappears 4 weeks after operation, which shows disaggregation phenomenon, while it's general structure remains. (×200)

Fig. 6. Fibroblasts grow along the surface of the scaffold or into the pores. There are lymphocytes and macrophages infiltrate. Vessel hyperplasia is visible, while new capillary vessel inside the material is obvious. No signs of tissue necrosis are found. (×400)

A Preliminary In Vivo Study on the Histocompatibility of Silk Fibroin 421

Fig. 9. Histocytes grow more deeply into the scaffold. Inflammatory cell infiltrate is visible, most of which are macrophages. Vessel hyperplasia reduces inside the tissue, while

Fig. 10. The border of the scaffold break down at some part 8 weeks postoperatively, while

the inside part remains as it was. (×200)

hyperblastosis still obvious. No sign of tissue necrosis is found. (×200)

Fig. 7. Inflammatory cell infiltrate is visible 6 weeks after operation. Histocytes gradually grow deep into the scaffold. No signs of tissue necrosis are found. (×200)

Fig. 8. There are a few parts with eosinophilic staining inside the material disappears 6 weeks postoperatively, which shows disaggregation phenomenon, while it's general structure remains. (×200)

Fig. 7. Inflammatory cell infiltrate is visible 6 weeks after operation. Histocytes gradually

Fig. 8. There are a few parts with eosinophilic staining inside the material disappears 6 weeks postoperatively, which shows disaggregation phenomenon, while it's general

structure remains. (×200)

grow deep into the scaffold. No signs of tissue necrosis are found. (×200)

Fig. 9. Histocytes grow more deeply into the scaffold. Inflammatory cell infiltrate is visible, most of which are macrophages. Vessel hyperplasia reduces inside the tissue, while hyperblastosis still obvious. No sign of tissue necrosis is found. (×200)

Fig. 10. The border of the scaffold break down at some part 8 weeks postoperatively, while the inside part remains as it was. (×200)

A Preliminary In Vivo Study on the Histocompatibility of Silk Fibroin 423

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

Murphy AR, Kaplan DL. Biomedical applications of chemically-modified silk fibroin. J

Rebecca L. Horan, Kathryn Antle, Adam L. Collette, etc.In vitro degradation of silk fibroin.

Cao Y, Wang B. Biodegradation of silk biomaterials. Int J Mol Sci. 2009 Mar 31;10(4):1514-24. Wang Y, Rudym DD, Walsh A,etc. In vivo degradation of three-dimensional silk fibroin

Lammel AS, Hu X, Park SH, etc. Controlling silk fibroin particle features for drug delivery.

Wang X, Yucel T, Lu Q, etc. Silk nanospheres and microspheres from silk/pva blend films

Lu yan, Chi fang-lu, Zhao Xia, etc. Experimental study on facial nerve regeneration by

Yang Y, Ding F, Wu J, Development and evaluation of silk fibroin-based nerve grafts used for peripheral nerve regeneration. Biomaterials. 2007 Dec;28(36):5526-35. Tang X, Ding F, Yang Y,etc. Evaluation on in vitro biocompatibility of silk fibroin-based

Guan G, Bai L, Zuo B, etc. Promoted dermis healing from full-thickness skin defect by porous silk fibroin scaffolds (PSFSs).Biomed Mater Eng. 2010;20(5):295-308. Bosetti M, Boccafoschi F, Calarco A,etc. Behaviour of human mesenchymal stem cells on a

Altman GH, Horan RL, Lu HH, et al . Silk matrix for tissue engineered anterior cruciate

Meinel L, Fajardo R, Hofmanna S, et al . Silk implants for the healing of critical size bone

Hofmann S,Hagenmuller H,Koch AM, et al .Control of in vitro tissue-engineered bone-like

Zhou J, Cao C, Ma X,etc. Electrospinning of silk fibroin and collagen for vascular tissue

Tamada Y. Sulfation of silk fibroin by chlorosulfonic acid and the anticoagulant activity.

Levin B, Redmond SL, Rajkhowa R, etc. Preliminary results of the application of a silk

porous silk fibroin conduit. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi.

biomaterials with primarily cultured hippocampal neurons. J Biomed Mater Res A.

polyelectrolyte-modified HEMA hydrogel for silk-based ligament tissue

structures using human mesenchymal stemcells and porous silk scaffolds.

fibroin scaffold to otology. Otolaryngol Head Neck Surg. 2010 Mar;142(3 Suppl

Biomaterials,Volume 26, Issue 17, June 2005, Pages 3385-3393

scaffolds. Biomaterials. 2008 Aug-Sep;29(24-25):3415-28.

for drug delivery.Biomaterials. 2010 Feb;31(6):1025-35.

engineering. J Biomater Sci Polym Ed. 2008;19(9):1111-23.

engineering.Int J Biol Macromol. 2010 Nov 1;47(4):514-9.

ligaments. Biomaterials, 2002, 23(20): 4131-4141.

defects. Bone, 2005, 37(5): 688-698.

Biomaterials, 2007, 28(6): 1152-1162.

Biomaterials, 2004, 25(3): 377-383.

1):S33-5.

should be made on the mechanism and degradation products.

Mater Chem. 2009 Jun 23;19(36):6443-6450.

Biomaterials. 2010 Jun;31(16):4583-91.

2006, 41(8),603-606.

2009 Oct;91(1):166-74.

**5. Conclusion** 

**6. References** 


#### Table 2. histopathology observation

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.
