Chinese Journal of Plastic Surgery, Vol. 20, No. 1, Jan. 2004
Dynamic changes in collagen after acellular allogeneic dermal matrix subcutaneous transplantation
Huo Menghua, Qi Keming, Huang Jinjing, Guan Zhengyu, Zhuang Qiang, Wang Yang, Lv Xiaoyan
【Abstract】
Objective: To investigate the changes of collagen after acellular allogeneic dermal matrix ADM transplantation.
Methods: Allogeneic ADM was transplanted under the skin of SD mice, and the content of collagen in ADM and the proportion of type I and III collagen were determined.
Results: There was no significant change in the content of collagen and the proportion of type I and III. collagen after allogeneic ADM transplantation.
Conclusion: Allogeneic ADM is a good soft tissue filling material.
Key words: acellular dermal matrix; Graft; collagen
Acellular dermal matrix (acellulardermalmatrix (ADM) removes cellular components from the skin and retains the matrix components of the dermis. Collagen (collagen) is the main component of the dermal matrix, and the changes in collagen after ADM transplantation are of great significance for its function.
1. Materials and methods
1. Source of allogeneic ADM: After allogeneic SD rats are killed, they are soaked in 2% iodophor solution for disinfection, and the skin is cut and trimmed into full-thickness skin sheets, about 0.5mm thick, and decellularized, freeze-dried and hydrated respectively to make acellular dermal matrix. Then cut into 1 cm × 1 cm flakes and put them in phosphate buffer for later use.
2. Experimental animals: SD is a healthy adult rat, weighing about 250g, male and female (provided by the Laboratory Animal Center of the Chinese Academy of Agricultural Sciences). It was divided into 5 groups of 10 pieces according to the different time of collection.
3. Surgical method of subcutaneous transplantation: remove the hair of the back skin of SD mice with a depilatory agent 1 day before surgery, and clean it. On the day of surgery, the body weight was weighed, 1% sodium pentobarbital 30mg/kg body weight, and intraperitoneal injection of general anesthesia. The skin of the operation area was disinfected with 2% iodophor solution, and sterile towels were spread. An incision is made on the right side of the midline of the back, about 0.5 cm from the midline. Between the skin and the skin muscles, it separates to the right side, forming a cavity of 1 cm × 1 cm in size. Allogeneic ADM is implanted, with the basement membrane surface facing the skin and the dermal side facing the dermal muscle, and the incision is sutured after flattening within the space. Regular rearing in separate cages.
4. Quantitative determination of collagen content: 2 weeks after transplantation, 1, 2, 3, and 4 months after transplantation, the skin is incised through the original incision to reveal the ADM, and it is separated from the surrounding tissues and removed. The fresh tissue specimens were repeatedly baked into dry tissues, hydrolyzed by 6mol/LHCl, oxidized by 0.05mol/L chloramine T, and developed by 10% p-dimethylaminobenzaldehyde, and then colorimetric on a UV-1601 recording spectrophotometer with a wavelength of 560nm (a product of Shimadzu Corporation in Japan), and the absorbance value of hydroxyproline was determined, and the content of collagen was calculated according to the hydroxyproline standard curve.
5. Quantitative determination of collagen typing: use cellulose acetate film electrophoresis to separate type I and III. collagen, stain Coomassie Bright Blue R250, decolorize 75% acetic acid-50% methanol solution, remove the dye that is not bound to protein, and two clear electrophoresis bands can be seen. The stained electrophoresis bands were cut and trimmed, and the stained protein was eluted from the film with 0.12NNaOH-80% methanol solution, and the color was compared on a UV-1601 recording spectrophotometer with a wavelength of 580nm, and the absorbance A value of the sample was measured and its ratio was calculated.
6. Statistical analysis methods: Tissue specimens of different periods were expressed as mean ± standard deviations, one-way ANOVA (F-test) was performed using SPSS10.0, and ADM at different periods after transplantation was compared with ADM before transplantation (Dunnettt test).
2. Results
There was no significant difference in the content of collagen and the ratio of type I and type III collagen after transplantation (P>0.05, Table 1).
3. Discussion
The main feature of collagen molecule is that hydroxyproline (42-hydroxyproline) accounts for 12%~14% of amino acid components, which is not found in other animal tissue proteins (except elastin), so hydroxyproline can be used as a special marker, and the quantitative determination of hydroxyproline after hydrolysis is a commonly used method for collagen quantification. Coomassie Bright Blue R250 is a protein dye that binds to proteins due to the affinity of its hydrophobic groups to the hydrophobic microregions of proteins. When the protein sample is fixed on a fibrous film and Coomassie Brilliant Blue R250 is interacted with the protein, the protein is colored. The protein is eluted from the fiber film for colorimetric assay, which quantitatively measures the concentration of the protein. Collagen is an umbrella term for a group of proteins that have both common and different characteristics in structure, and there are at least five different types. The distribution in vivo is tissue-specific, and the skin contains collagen types I and III, and collagen type IV is only present in the basement membrane[1]. The total collagen content in the human body accounts for about 25%~33% of the total protein, and they are the main structural proteins of the body and an important component of various tissues. Its main function is to act as an organizational support, giving tension to the organization. In addition, collagen molecules and their fibers play an important role in biological development, growth, cell differentiation and adhesion, motility, chemical orientation, and antigen-antibody binding reactions [1].
After allogeneic ADM transplantation, host fibroblasts are transplanted and vascularized, and fibroblasts proliferate on the fiber scaffold provided by allogeneic ADM, and ADM is continuously remodeled and in dynamic equilibrium while producing collagen. Studies have shown that there is no significant change in the collagen content and the ratio of type I and III collagen after subcutaneous transplantation of allogeneic ADM, which is the necessary material basis for ADM to function. As a soft tissue filling material, ADM can retain its original tissue volume after transplantation and achieve the purpose of filling tissue defects. As a dermal substitute, it can better cover the wound after transplantation, provide tension, and promote the survival, adhesion, differentiation, and proliferation of epidermal cells [2]. It is further shown that acellular dermal matrix is a good soft tissue filler material and dermal substitute.
References
1. Li Yurui, Biochemistry and Research Methods of Extracellular Mesenchyme, Beijing: People's Medical Publishing House, 1988.2, 128.
2． Ghosh MM,Boyce S,Layton C,et al. A comparison of methodologies for the preparation of human epidermal dermal composites． Ann Plast Surg,1997,39:390-404 .
(Received:2002-08-21)