Résumé
BACKGROUND: Based on our previous researches in mechanism studies and clinical applications of human hair keratin (HHK), a new concept "in vivol in situ tissue engineering" has been proposed. Under the guidance of this theory, a scaffold of HHK-collagan sponge (inner layer) combined with poly (2-hydroxyethyl methacrylate) (PHEMA) (outer layer as a drug delivery carrier) would be developed to investigate its feasibility to be as a dermal dressing. OBJECTIVE: To develop a scaffold composed of HHK-collagan sponge (inner layer) combined with PHEMA film containing polydatin(PD)(outer layer as a drug delivery carrier) and to evaluate the therapeutic efficacy of the HHK-collagen sponge-PHEMA/PD complex on burn wound healing. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the Department of Histology and Embryology, Southern Medical University between March and December 2005. MATERIALS: Burn was induced in 15 male Sprague-Dawiey (SD) rats, Rat models of burn were evenly randomized to 3 groups: experimental, positive control, and negative control. METHODS: ①HHK-collagen sponge was prepared through combination of a HHK meshwork (1mm × 1 mm in size for each grid) made up of three components (determined according to biochemical procedures of various degrees, i.e., light, medial, and severe) at a ratio of 4:3:3 with primary collagen sponge extracted from bovine tendons in a mould. Sponge film (used as inner layer dressing) was made by vacuum freeze-drying. ② PHEMA was prepared by polymerization. Than PD was added to prepare PHEMNPD film (used as outer layer dressing).③ Degree Ⅱ burn wound models were established in SD rats by scalding, Superficial necrotic tissue was removed from burn wounds at postnatal 3 days and leave the denatured dermis remained. The wounds were either covered with human HHK-collagen- PHEMNPD complex in the experimental group, or with glutaraldehyde-treated porcine skin in the positive control group, and sterile absorbent gauze was used in the negative control group. MAIN OUTCOME MEASURES: ① Complete epithelization was taken as the standards, and at postoperative 7, 14, and 21 days, wound healing was respectively calculated. ② At postoperative 1, 2, 4, 6, and 8 weeks, the whole wound surface and its peripheral tissue were dissected for observing granulation tissue growing under an optical microscope and detecting the collagen fiber and elastic fiber in the newly formed tissue by immunohistochemical staining. RESULTS: ① Gross observation results revealed that in the experimental group, the volume of the diffusate under the ideal moisture was less compared with the positive control group; the healing time was slightly shorter in both the experimental group and the positive control group than in the negative control group (P= 0.000); At postoperative 7, 14, and 21 days, the healing rate was higher in the experimental and positive control groups than in the negative control group (P=0.000), in addition, the experimental group exhibited higher healing rate than the positive control group at postoperative 14 days ( P ＜ 0.05). ②Optical microscope results showed that at postoperative 2 weeks, a small quantity of collagen fibers were found in the wound granulation tissue in all 3 groups, in particular in the experimental group. Immunohistochemical staining results regarding collagen protein and elastin revealed that at postoperative 2 weeks, both the fine strip-like type Ⅰ collagen fibers and a few silk-like elastic fibers were stained yellowish-brown in the dermal matrix in the experimental group, which were weakly positive in the positive control group, while there was no elastin detectable in the negative control group; at postoperative 8 weeks, burn wounds in all the 3 groups werefully recovered. Remodeling of collagen fibers was more obvious in the experimental and positive control groups than in thenegative control group, while the tendency to scar formation with derangement of epithelial cells and collagen fibers in dermis was more prominent in the negative control group than in the remaining two groups.CONCLUSION: HHK-collagen sponge-PHEMA/PD complex may be a new burn dressing via in vivo construction of tissueengineered epidermis, in which PHEMA may be a feasible drug-delivery carrier.
Résumé
BACKGROUND: The grafted human hair keratin(HHK) artificial tendon,being degradable and absorbable, can induce formation of neotendon. In the present study, the main biomechanical indexes of HHK artificial tendon and its components were measured so as to determine a proper ratio for the artificial tendon composition according to different needs of the body sites and make it possess tensile stress required for functional activities.OBJECTIVE: To determine the main biomechanical indexes of HHK artificial tendon and its components.DESIGN: An observational study based on HHK artificial tendon.SETTING: Department of Biochemistry and Molecular Biology, Medical Biomechanical Key Laboratory of Chinese PLA, and Department of Histology and Embryology, a military medical university; Foshan Kangxing Medical Technology Co., Ltd.MATERIALS: The experiment was completed at the Medical Biomechanical Key Laboratory of the First Military Medical University of Chinese PLA in October 2001. The material of HHK artificial tendons was uncontaminated black hair collected from healthy young women in remote mountainous areas free from endemic diseases. There were three components of HHK artificial tendons with different in vivo absorption velocities Z, B and F. The in vivo absorption was the slowest for Z, faster for B, and the fastest for F. Different kinds of HHK artificial tendon were the mixture of Z, B and F at a proper ratio. Each group had 3 kinds, namely, Z3.0-20, B3.0-20, and F3.0-20. HHK artificial tendon consisted of 6 kinds: ZBF1. 0-20,ZBF1.5-20, ZBF3.0-20, ZBF6. 0-20, ZBF9. 0-20, and ZBF12.0-20.METHODS: Both ends of an HHK artificial tendon or each of its components were fixed on MTS 858 Moni Bionix checker to determine such biomechanical indexes as fracturing pull and tensile stress.MAIN OUTCOME MEASURES: Fracturing pull and tensile stress of HHK artificial tendon of different kinds and its components.RESULTS: Fracturing pull in Z3.0-20, B3.0-20 and F3.0-20 groups was (211.8 ± 12. 1 ), ( 178.8 ± 8.2), and ( 151.3 ± 6. 7) N, respectively; tensile stress was(71.3 ±3.9), (59.9 ±2. 7), and(50. 3 ±2.3) Mpa, respectively. Fracturing pull and tensile stress were decreased gradually in the same cross-section area of the 3 kinds of materials; Fracturing pull in ZBF1.0-20,ZBF1.5-20, ZBF3.0-20, ZBF6.0-20, ZBF9.0-20, and ZBF12.0-20 groups was(75.0 ± 3.0), (107.8±4.2), (194.1±5.3), (375.9±12.7),(508.2 ±21.3), and(670.8 ±25.4) N, respectively, while tensile stress was(75.0 ±3.0), (72.0 ±2.8), (65.1 ± 1.8), (62.9 ±2.2), (56. 3 ±2.4),and(55.8 ± 1.5) Mpa, respectively.CONCLUSION: Fracturing pull of HHK artificial tendon is increased with the increase of cross-section area, whereas tensile stress is slightly decreased with the increase of cross section area. With the increased extent of human hair treatment, its absorption in vivo is accelerated, and fracturing pull and tensile stress of HHK artificial tendon are decreased.
Résumé
BACKGROUND: Numerous experiments and clinical practice show that human hair keratin artificial tendon induces the organism to form autogenous tendon. The process of autogenous tendon formation mainly involves the synthesis, secretion and package of collagen subtype I.OBJECTIVE: To explore the role of collagen subtype I mRNA expression in autologous tendon formation after human hair keratin artificial tendon implantation.DESIGN: A completely randomized controlled experiment based on the experimental animals.SETTING: Department of Histology and Embryology and Department of Biochemistry and Molecular Biology of Southern Medical University.MATERIALS: The experiment was conducted in the Experimental Animal Center of the First Military Medical University of Chinese PLA from May 2003 to September 2004. Totally 33 New Zealand rabbits of either gender,weighing 2.0 to 2. 5 kg, were provided by the center. The animals were randomly divided into experiment 3, 6, 9, 12, 16 and 20 weeks groups, negative control 9 and 20 weeks groups and normal control group. Among them,experiment 3 and 6 weeks group and normal control group had 3 rabbits in each and the other groups had 4 rabbits. Human hair keratin artificial tendons were normal human hair treated by a series of biochemical methods and were supplied by the Department of Biochemistry & Molecular Biology of the university. The human hair keratin artificial tendons were divided into three groups with different degradation rates, namely, fast(F), medium(B) and slow(Z). The tendons were made up of the fast, medium and slow degradation groups mixed at the ratio of 4: 3: 3.off by 1.0- 2.0 cm, human hair keratin artificial tendon was grafted by end-to-end anastomosis with both ends of the broken tendon before sewing control group, no artificial tendon was implanted although the animals ungroup was normal rabbits' tendon. Sampling was carried out at 3, 6, 9, 12, 16and 20 weeks after human hair keratin artificial tendon implantation in experiment groups, and at 9 and 20 weeks after operation in negative control group, respectively. The expression of collagen subtype I mRNA was detected at weeks 3, 6, 9, 12, 16 and 20 after grafting using reverse transcription polymerase chain reaction technique.MAIN OUTCOME MEASURES: The ratio of collagen subtype I mRNA to Glyceraldehyde-3-phosphate dehydrogenase(GADPH) mRNA in normal tendon and autogenous tendon induced by human hair keratin artificial tendon at all time points was calculated, and significance test between all these paired groups were performed.collagen subtype I mRNA/GADPH mRNA expression was 0.96 ±0.02 in expression of collagen subtype I mRNA/GADPH mRNA in autogenous tendon induced by human hair keratin artificial tendon in experiment group appeared at week 3, increased rapidly at week 3 to 6, peaked at week 6, and remained stable at week 9 to 20. The expression at week 6 was significantly higher in experiment group than in normal control group( F = 6. 254, P ＜ 0.05); the expression at other weeks was also significantly higher in experiment group than in normal control group( F= 1. 258 - 1. 987, P ＞ 0.05).CONCLUSION: The activation, proliferation and secretion of collagen protein as well as the synthesis of collagen subtype I by tenocytes may be responsible for autologous tendon formation after human hair keratin artificial tendon implantation.