ABSTRACT
BACKGROUND: Studies have showed that transforming growth factor-β1 (TGF-β1) could yield to the collagen synthesis and adhesion formation of tendon cells at the process of healing. OBJECTIVE: To investigate the preventive effect of TGF-β1 neutralizing antibody on the collagen production and adhesion formation of flexor tendon. DESIGN, TIME AND SETTING: Randomized grouping observational experiments were performed in the Experimental Animal Center of Tongji Medical College between September 2005 and June 2006. MATERIALS: New Zealand white rabbits aged 2-5 months, weighing 3.5-4.5 kg. TGF was offered by Santa Cruz Biotechnology, USA. METHODS: Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Cells were divided into two groups at random. In the experiment group, each cell culture was supplemented with 1 μg/L of TGF-β at increasing dose (0.1, 0.5, 1.0 mg/L) of TGF-β1 neutralizing antibody. No reagents were given in the control group. Collagen Ⅰ production was measured by enzyme-linked immunoabsorbent assay. Eighty-four adult New Zealand white rabbit forepaws underwent sharp transection of middle toe flexor digitorum profundus, followed by immediate repair. Thirty-six adult New Zealand white rabbit were divided into three groups randomly (n=12), injecting with the saline, 1.0 mg/L TGF-β1 neutralizing antibody and 2.0 mg/L TGF-β1 neutralizing antibody into tendon sheath respectively. Tendons were harvested at 4 and 8 weeks to conduct adhesion detection, biomechanical testing, histological evaluation and scanning electron microscopy observation. The remaining 48 New Zealand white rabbits were divided into two groups randomly (n=24), undergoing the saline and 1,0 mg/L TGF-β1 neutralizing antibody injection in tendon sheath respectively. Tendons were harvested at an increasing time interval (1, 2, 4, 8 weeks) and analyzed by in situ hybridization to determine the mRNA expression of TGF-β1 and collagen Ⅰ. MAIN OUTCOME MEASURES: Collagen production and adhesion of rabbit tendon cells. RESULTS: ELISA exhibited that TGF-β1 increased collagen Ⅰ production and the addition of neutralizing antibody significantly reduced TGF-β-induced collagen Ⅰ production in all cell cultures. The effect between antibody and collagen Ⅰ was dose dependent. At 4 and 8 weeks after operation, the gliding excursion ratio of the tendon was shortened and the simulated active flexion ratio were less in saline group compared with 1.0 and 2.0 mg/L TGF-β1 groups (P < 0.05). The tendon anastomosis breaking strength was shown no significant differences among 3 groups (P > 0.05). Scanning electron microscopy and histological observation showed that collagen fibers arranged irregularly in saline group, but arranged regularly in 1.0 and 2.0 mg/L TGF-β1 groups at 4 and 8 weeks after operation. The in situ hybridization examination revealed that TGF-β1 and collagen Ⅰ mRNA expression in 1.0 mg/L TGF-β1 group was lower than that in saline group at each time (P < 0.05). CONCLUSION: TGF-β1 neutralizing antibody can inhibit the function of the TGF-β1 effectively following the flexor tendon injury and repair, and can prevent adhesion formation.
ABSTRACT
BACKGROUND: Tendon cells possess collagen-secreting function, which plays an important role in the wound healing and adhesion. Little is known about the biological effects of lactate on tendon cells. OBJECTIVE: This study was designed to study the proliferation and collagen production of tendon sheath fibroblasts,epitenon tenocytes, and endotenon tenocytes and investigate the effect of lactate on cell proliferation, collage production and secretion of transforming growth factor- β1(TGF- β 1),basic fibroblast growth factor (bFGF), and interleukin-8 (IL-8) by each of these 3 cell types in rabbit flexor tendon.DESIGN, TIME AND SETTING: This study, a randomized controlled animal experiment, was performed at the Animal Laboratory Center, Affiliated Hospital of Qingdao University Medical College between September 2005 and July 2006.MATERIALS: Six adult New Zealand clean rabbits of either gender were included in the present study.METHODS: Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendon and cultured. Each cell type was identified by observing morphological structure through methylene blue staining.Three types of cells were cultured with media supplemented with 25 mol/L lactate to obtain cell number. The collagen production and secretion of TGF- β 1, b-FGF and FL-8 were compared after adding culture media supplemented with 25, 50,100, and 200 mmol/L lactate, respectively. At the same time, the above-mentioned indices measured after adding medium were compared with those measured without adding lactate.MAIN OUTCOME MEASURES: Type Ⅰ, Ⅱ, and Ⅲ collagen production was detected by immunohistochemistry; The effects of different concentrations of lactate on type Ⅰ collagen production as well as secretion of TGF- β 1,b-FGF, and IL-8by an enzyme linked immunosorbent assay (ELISA).RESULTS: 25 mmol/L lactate reduced 3 types of cultured cells. There was no significant difference in the cell number among the 3 types of cells (P > 0.05) Lactate produced more type Ⅰ, Ⅱ ,and Ⅲ collagen tissue compared to not adding lactate (P < 0.05) When lactate concentration increased to 50 mmol/L, type Ⅰ collagen reached its peak level in the 3 types of cells. There was significant difference in type Ⅰ collagen compared with lactate concentration was 0 mmol/L(t = 4.58, 3.97,3.16, P < 0.01 ) . When lactate concentration increased to 100 mmol/L and 200 mmol/L, type Ⅰ collagen was noticeably decreased. When lactate concentration was 25 mmol/L in the 3 types of cells, the secretion of TGF- β 1 and bFGF was increased and the secretion of IL-8 was decreased. There was significant difference compared with not adding lactate (P <0.05).CONCLUSION: Lactate can increase the collagen production of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes. The increasing degree of collagen production is related to lactate concentration, in particular at 50 mmol/L. Such a stimulation may be related to the increase of TGF- β 1 and bFGF and the decrease of IL-8 secretion.
ABSTRACT
0.05) Lactate produced more type Ⅰ,Ⅱ,and Ⅲ collagen tissue compared to not adding lactate(P