Abstract
This study evaluated the effectiveness of silk fibroin materials for wound repair confined to the buccal mucosa in a rat model by assessing several key clinical parameters and the associated local and systemic immune response. Ninety male SD rats were subjected to microscopic oral surgery to establish a full thickness wound on the buccal mucosa. Rats were randomly divided into three groups based on the treatments received: group A, covered with polyporous silk fibroin scaffold; group B, repaired with crosslinking silk fibroin film; and group C, control. Visual observation of the wounds suggests that wound shrinkage 5 days after the operation was significantly lower in both silk fibroin repaired groups (A and B) than that in the controls. The distribution of inflammatory neutrophils in group A was significantly lower than those in the control group throughout the entire study. The percentage of fibroblasts and capillary endothelia (CD34 + ), and the subgroups of peripheral lymphocytes (CD3 + , CD4 + , CD8 + ) were similar amongst the groups. The results revealed that placement of silk fibroin in an oral buccal defect can reduce the degree of wound shrinkage and enhance the growth of mucosal epithelial cells without any local or systemic immunological incompatibility.
Over the past three decades the options for reconstructive surgery of mandible defects have increased to enable the re-establishment of mandible continuity and optimal soft-tissue reconstruction . Numerous advances in microsurgical techniques, plating technology and instrumentation, and an understanding of donor site angiosomes have made consistent and reliable mandibular reconstruction possible . Vascularized osseous free tissue transfer is the state-of-the-art treatment for mandibular reconstruction which ultimately results in a patient with oral competence and limited swallowing deficits, and the ability to communicate effectively and interact with their community in a meaningful manner .
Employing a free flap means there are some unavoidable drawbacks in mandibular reconstruction including: poor external skin colour and texture restoration that compromises the aesthetic results; and inadequate reconstruction of functional deficits that include loss of sensation and motor function. Investigations are continuing into new techniques and materials, such as distraction osteogenesis and tissue engineering, which should improve the ability to reconstruct the mandible. Many natural and synthetic polymers have been considered for use as biomaterial scaffolds in reconstruction. The utility of most polymers is limited by the challenging combination of biocompatibility, biodegradability, controllable porosity, stability for an extended time-period during neotissue growth, and processibility into porous matrices .
Of the natural and synthetic polymers, silk fibroin has been evaluated in tissue engineering and recommended as one of the potential biomedical matrices to be employed as guided tissue regeneration and for dressing burn wounds . Silk fibres consist of two proteins: fibroin and sericin. Fibroin is extruded from the silkworm gland in the form of filaments embedded in a sericin rubbery coating . It is a group of polymeric proteins produced for the construction of cocoons by several species in the order Lepidoptera. The secondary structure of fibroin proteins, imparted during the spinning of cocoons, is responsible for the mechanical properties of silk fibres and has led to these proteins being considered as biomaterials for tissue engineering and other biomedical applications. Although silk has been used commercially for textiles and industrial purposes, it is only recently that the biological potential of solubilized silk fibroin been extensively explored as a natural biomaterial scaffold/matrix for cell culture and tissue engineering . The importance of a scaffold/matrix has been well established due to its key role in transducing environmental cues to cells seeded within it, acting in essence as a translator between the local environment and the developing tissue (neotissue), hence aiding the development of biologically viable functional tissue .
Recently, a number of independent studies have investigated the biological features of silk fibroin and determined that it has good haemocompatibility , biocompatibility , oxygen and water permeability , minimal inflammatory reaction, and supports the attachment and proliferation of human and animal cell lines . In general, the attachment and spreading of cells on a solid surface is a prerequisite for their proliferation, performance and aggregation . It has been demonstrated that silk fibroin membranes, nets and scaffolds could support the attachment and proliferation of endothelial, smooth muscle, epithelial and osteoblast cells . For instance, the alignment and elongation of human coronary artery smooth muscle cells occurred within 5 days after seeding on electrospun silk fibroin scaffolds. Short cord like structures formed from human aortic endothelial cells on the scaffolds by day 4, and a network of capillary tubes with lumen was observed by day 7 . Biomaterial design and the existence of other types of cells were shown to have a high impact on cellular responses with regard to cellular morphology, proliferation and formation of intercellular contacts. A higher level of adhesion molecule integrin-β1 in endothelial cells was observed on nanofibrous fibroin nets compared to the microfibrous samples . In co-cultures of human dermal microvascular endothelial cells (HDMEC) and primary human osteoblast cells or the human osteoblast-like cell line MG-63 on three dimensional (3D) bone biomaterials, endothelial cells formed microcapillary-like structures containing a lumen and gave strong endothelial cell-specific platelet–endothelial cell adhesion molecule-1 (PECAM-1) expression at cell interfaces. The life span of HDMEC was also significantly enhanced by co-culture; with HDMEC being present for up to at least 42 days, compared to the monoculture in which cells began to die rapidly after 1 week .
Most applications have been considered with respect to reconstructing musculoskeletal and or vascular tissues, but a small number of recent studies have begun exploring the potential use of fibroin for engineering more functionally complex tissues such as those found within the oral and maxillofacial field. To the best of the authors’ knowledge, no study has been conducted to evaluate the inflammatory reaction and biocompatibility of silk fibroin on oral mucosa defects. The goal of this study was to evaluate the effectiveness of silk fibroin materials for wound repair confined to the buccal mucosa in a rat model by assessing several key clinical parameters and the associated local and systemical immune response.
Materials and methods
Ninety 3-month-old male SD rats, weighing 180–200 g (animal service centre of Suchow University) were randomly separated into three groups. The buccal mucosal defects were treated with polyporous silk fibroin scaffold in group A ( n = 30); crosslinking silk fibroin film in group B ( n = 30) and Vaseline gauze only in group C ( n = 30). The experiments were approved by the Board for Animal Experiments of the Soochow University.
Surgical procedure
The animals were anaesthetized with an intraperitoneal injection of 3.6% chloral hydrate (1 ml/100 g). The anaesthetized animals were placed in a supine position and the full thickness defects of 10 mm in diameter were demarcated and created by surgical incision using a standard surgical blade on the buccal mucosa under microscopic guidance. Figure 1 shows different views of wound creation. Sterilized polyporous silk fibroin scaffolds and crosslinking silk fibroin films were trimmed into round membranes with diameters of 10 mm and loaded onto single layered Vaseline gauze squares. The defects were covered with the gauze and secured by suturing to the adjacent mucosa with 7-0 silk suture. Two additional Vaseline gauze squares were placed on top and sutured with 1-0 silk suture.
At 5 days postoperatively, 10 rats were selected randomly from each group and anaesthetized with an intraperitoneal injection of 3.6% chloral hydrate (1 ml/100 g). The colour, exudation and healing of the wounds were observed and recorded. The diameter of the wounds was measured at three different locations using Vernier callipers. The selected rats were marked and macrographic observation and diameter measurements were made on the marked rats on days 10 and 15.
On days 10, 20 and 30 after surgery, 10 rats were selected from each group. 0.5 ml tail blood was obtained for peripheral blood T lymphocyte analysis under anaesthesia. These rats were killed and 5 mm × 5 mm newly grown mucosal tissues were harvested and fixed in 10% formalin, prior to haematoxylin–eosin (H–E) staining and subsequent histological and immunohistochemical analysis.
Histological study
Tissues were taken from the surgical area and fixed in 10% formalin for 48 h. H–E staining was done according to a standard protocol. Five microscopic views from each rat were randomly selected and the number of inflammatory cells and fibroblasts were counted by two independent pathologists using ×400 magnification.
Determination of newly formed blood vessels in the wounds was verified by CD34 staining . Briefly, the tissues were taken from the surgical area and fixed in 10% formalin for 48 h. Samples were cut into 3 mm × 3 mm × 3 mm cubes and embedded in paraffin and then sectioned to tissue slides with a thickness of about 3 μm. Immunohistochemical studies were performed using primary rabbit antibody against rat. Briefly, the tissue slides were deparaffinized and rehydrated, then submerged in hydrogen peroxide for peroxidase quenching. Before using the primary antibody, the slides were incubated with goat serum for 10 min to block non-specific binding. After 1 h incubation with the primary antibody, the secondary goat against rat antibody was added and incubated at room temperature for 10 min. Fifty microlitres streptovidin biotin complexes were incubated for 10 min at room temperature. Staining was performed by diaminobenze dinetetrahydrochloride (DAB) substrate and slides were counterstained with haematoxylin and mounted.
Endothelial cells of newly formed blood vessels were stained with anti-CD34 monoclonal antibody (Boster Biological Technology, Ltd, Wuhan, China). The positive cells appeared as clear brown in cytoplasm. Density of microvascular endothelia was expressed as the average cell numbers of 5 views at ×400 magnification.
Cytokeratin (CK) expression was determined to assess the density of newly formed oral epithelial cells in the wounds using immunohistochemistry described previously . Briefly, animals were killed and tissue samples were taken from the surgical area and fixed in 10% formalin for 48 h. Samples were cut into 3 mm × 3 mm × 3 mm cubes and embedded in paraffin and then sectioned to tissue slides with a thickness of about 3 μm. Immunohistochemical studies were performed using mouse against rat monoclonal CK antibody (Boster Biological Technology, Ltd, Wuhan, China). Briefly, the tissue slides were deparaffinized and rehydrated, then submerged in hydrogen peroxide for peroxidase quenching. Mouse against rat monoclonal antibody was added and kept overnight at 4 °C. Horseradish peroxidase conjugated goat anti-mouse polymer was added and incubated at 37 °C for 30 min. Staining was performed by DAB substrate and slides were counterstained with haematoxylin and mounted. The positive newly formed oral epithelial cells appeared as clear brown in cytoplasm. Magnification of ×100 was employed to assess the overall morphological features whilst ×400 was used for observations of the multi-layered epithelial cells with thick rete pegs .
To assess T lymphocyte subsets in peripheral blood, 0.5 ml tail blood samples were collected from 10 randomly selected rats in each group. Each blood sample was divided and heparinized into four tubes (three for test, one for control) for T lymphocyte subsets analysis. Ten microlitres anti-rat CD3, CD4 and CD 8 fluorescein isothiocyanate (FITC) conjugated monoclonal antibody were added to each of the test tubes (eBioscience, Shanghai Laizee Biotech Co., Ltd, Shanghai, China) . Rat IgG1 was added to the control tube as negative controls to define background staining. An aliquot of whole blood (50 μl) was added to the test tube, mixed, and then left in the dark for 20 min at room temperature. Two hundred fifty microlitres haemolysin was added to each tube, mixed thoroughly and left for 10 min at room temperature. Two millilitres phosphate buffered saline (PBS) was used to rinse the samples twice with centrifugation at 1200 rpm for 5 min. Supernatant was discarded and 0.5 ml PBS was added before flow cytometry (FCM) analysis (Beckman-Coulter USA). FCM and scattering distribution were obtained and analysed using SYSTEMT M II software. The percentage of CD3 + , CD4 + and CD8 + was calculated.
Statistical analysis
Statistical calculations were performed with SAS8.1 software (SAS Institute Inc., Cary, NC, USA). Student’s t -test was used to determine the significance of the differences between group means. Statistical significance was defined as P < 0.05.
Results
The diameter of wounds was measured 5, 10 and 15 days after surgery at three different randomly selected locations, starting from sutures at the margins of the wound. Wound diameter was calculated as the average of the three measurements and compared amongst three time points ( Table 1 ). Some sutures were lost by day 15 after surgery. By day 5, all wound dressings were detached. In group A, the wound surface was covered with brown membrane without significant swelling and redness ( Fig. 2 ). By days 10–15, the wounds had not healed completely. In group B, silk fibroin films were still visible on the wound surface at day 5. The diameter of the wound was reduced further by days 10–15 compared to that on day 5, but the wound did not heal completely. In group C, wound contraction was significant by day 5 with marginal swelling and redness. By day 15 no significant differences amongst the three groups were observed ( Table 1 ).
| Groups | 5 days | 10 days | 15 days |
|---|---|---|---|
| Scaffold | 6.31 ± 0.15* | 3.15 ± 0.46 | 1.20 ± 0.23 |
| Film | 7.44 ± 0.14* | 4.53 ± 1.17* | 1.27 ± 0.13 |
| Control | 4.44 ± 0.32 | 2.89 ± 0.45 | 1.94 ± 0.52 |
Histology and immunohistochemistry
Newly generated buccal mucosa specimens were sectioned for H–E staining and CD34, CK immunohistochemical analysis to inspect the distribution of the inflammatory cells, fibroblasts and microvascular endothelia in three groups.
On postoperative day 10, newly generated multi-layered epithelia were found at the wound margin with thick rete pegs in group A. Inflammatory cells and fibroblasts appeared on the polyporous silk fibroin scaffolds. Newly formed capillaries were visible with a small cavity ( Fig. 3 – HE ). Compared to group A, there were fewer epithelia layers with small and sparse distributed rete pegs in group B. A swelling band between the silk fibroin and tissue surface was noticeable surrounded by dense inflammatory cells and fibroblasts, with blood vessels infiltrating into the membrane ( Fig. 3 – CK). In contrast, a significant accumulation of inflammatory cells, fibroblasts, new capillaries with large cavities in granular tissue was observed in the control group ( Fig. 3 – Cd34). The epithelium in this group had fewer layers of cells compared to the other two groups and the continuity of epithelium remained intact.
By day 20, similar observations were found in the three groups including an increased number of epithelial rete pegs, reduced cellular swelling, decreased inflammatory infiltrates and increased number of fibroblasts as well as capillary endothelia. Partial degradation of silk fibroin with short and sparse fibroin fibres was also readily detectable.
By day 30, the silk fibroin in tissue almost disappeared, inflammatory cells decreased and fibroblasts increased. The number of capillaries increased and the cavity of blood vessels enlarged. Comparison of inflammatory cells, fibroblasts and density of endothelia is demonstrated in Fig. 4 .
Peripheral T lymphocyte subsets
There were no significant differences in the percentages of CD3 + , CD4 + , and CD8 + in rat peripheral blood amongst the three groups on days 10, 20 and 30 after surgery ( Fig. 5 ).
( x ¯ ± S , n = 10 )
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