Effects Of TGF-β In Tendon Wound Healing And Adhesion Formation

Part oneGene Expression of Transforming Growth Factor beta-1 in a Rabbit Zone II Flexor Tendon Wound HealingObjectives To examine the expression of transforming growth factor beta-1 mRNA in a rabbit zone II flexor tendon wound-healing model.Methods Forty-eight New Zealand White rabbit forepaws underwent complete transaction and repair of the middle digit flexor digitorum profundus tendon in zone II. Tendons were harvested at increasing time intervals (1, 7, 14, 21, 28, and 56day) and analyzed by in situ hybridization and immunohistochemistry to determine the expression patterns of transforming growth factor beta-1.Results A small number of tenocytes exhibited expression of transforming growth factor beta-1 mRNA in nonwounded control tendon specimens. The surrounding tendon sheath in these control specimens also revealed low numbers of fibroblasts expressing transforming growth factor beta-1 mRNA. In contrast, flexor tendons subjected to transaction and repair exhibited increased signal for transforming growth factor beta-1 mRNA in both resident tenocytes and fibroblasts from the tendon sheath.Conclusions The normal unwounded tenocytes and tendon sheath cells are capable of transforming growth factor beta-1 production. The cytokine is activated in the tendon wound environment. The upregulation of this cytokine in both tenocytes and tendon sheath fibroblasts are coincidence with both extrinsic and intrinsic mechanisms for tendon repair.Part twoExpression of Transforming Growth Factor beta-1 Receptors in a Rabbit Zone II Flexor Tendon Wound HealingObjectives TO analyzed the temporal and spatial distribution of TGF-[beta] receptor RI in a rabbit zone II flexor tendon wound healing model.Methods Forty-two adult New Zealand White rabbit forepaws underwent isolation of the middle digit flexor digitorum profundus tendon in zone II. The tendons underwent transection in zone II and immediate repair. The tendons were harvested at increasing time points: 1, 7, 14, 21,28, and 56 days postoperatively (n = 6 at each time point). The control flexor tendons were harvested without transection and repair (n = 6). Western blot and Immunohistochemical analysis was used to detect the expression patterns for TGF-[beta] receptors RI.Results Western blot and Immunohistochemical staining of the transected and repaired tendons demonstrated up-regulation of TGF-beta RI protein levels. TGF-beta receptor production in the experimental group (transection and repair) was concentrated in the tendon sheath, the epitenon and along the repair site. Furthermore, the TGF-beta receptor expression levels peaked at day 14 and decreased by day 56 postoperatively. In contrast, minimal receptor expression was observed in the untransected and unrepaired control tendons.Conclusions The tendon sheath and epitenon have the highest TGF-beta receptor expression after injury and repair; The peak levels of TGF-[beta] receptor expression occurred at day 14 and decreased by day 56 after wounding and repair; Understanding the up-regulation of TGF-[beta] isoforms and the up-regulation of their corresponding receptors during flexor tendon wound healing provides new targets for biomolecular modulation of postoperative scar formation.Part threeEffects of TGF-β1 on proliferation and collagen production of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytesObjective To study proliferation and collagen production of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes; and effects of TGF-β₁ on cell proliferation and collagen production by each of these 3 cell types in rabbit flexor tendon.Methods Three cell lines: tendon sheath, epitenon, and endotenon were isolated from rabbit flexor tendon and cultured. Cell culture media was supplemented with 5ng/ml of TGF-β₁. Cell number and collagen I, II and III production were measured and compared with unsupplemented control culture. Expression of type I collagen was determined by quantitative analysis of products of reverse-transcription polymerase chain reaction.Results All 3 cell lines produced collagen I, II and III. The addition of TGF-β₁ to cell media resulted in a decrease in cell number in all 3 cell1 lines that did not reach statistical significance. There was a significant increase (p<0. 05) in collagen I, II and III production and expression levels of type I collagen with the addition ofConclusions Modulation of TGF-β₁ levels may provide a means to modulate collagen production in tendon and may provide a mechanism to modulate adhesion formation clinically.Part fourEffects of lactate on TGF-β expression of flxor tendon cellsObjective To study the effects of lactate on TGF-β peptide and receptor production by flexor tendon cells.Methods Tendon sheath fibroblasts, epitennon tenocytes, and endotendonon tenocytes were isolated from rabbit flexor tendon and cultured separately. Cell cultures were supplemented with lactate and the expression of three TGF-β peptide isoforms(β1, β2, and β3) and three receptor isoforms(R1, R2, andR3) was quantified with enzyme-linked immunosorbent assay. The expression of TGF-β1 mRNA was also assessed with in situ hybridization and image analysis.Results Supplementation of the cell culture medium with lactate significantly (P<0.05) increased the expression of all TGF-β peptide and receptor isoforms in all three cell lines. Tendon sheath fibroblasts exhibited the greatest increases in β1 and β2 peptide isoform expression, Whereas epitenon tenocytes exhibited the greatest increases in receptor isoform R1 and R2 expression, endotenon tenocytes demionstrated the greatest increase in β3 peptide expression. All three tendon cell types demonstrated significant (P<0.05) increases in TGF-β1 mRNA expression when exposed to lactate.Conclusion Lactate significantly increased the expression of TGF-β peptide,TGF-β receptor and TGF-β1 mRNA. Modulation of lactate levels may provide a means of modulating the effecs of TGF-β on adhesion formation in flexor tendon wound healing.Part fiveEffects of lactate on proliferation and biological activities of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytesObjective To study proliferation and collagen production of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes; and effects of lactate on cell proliferation, collagen production and secretion of TGF-β₁, b-FGF, and IL-8 by each of these 3 cell types in rabbit flexor tendon.Methods Three cell lines: tendon sheath, epitenon, and endotenon were isolated from rabbit flexor tendon and cultured. Cell culture media was supplemented with 25mml/L of lactate. Cell number, collagen I , II and III production, and secretion of TGF-β₁, b-FGF, and IL-8 were measured and compared with unsupplemented control culture.Results All 3 cell lines produced collagen I, II and III. The addition of lactate to cell media resulted in a decrease in cell number in all 3 cell lines that did not reach statistical significance. There was a significant increase (p<0.05) in collagen I, II and III production and secretion of TGF- β₁, b-FGF,and IL-8.Conclusions Modulation of lactate levels may provide a means to modulate collagen production in tendon and may provide a mechanism to modulate adhesion formation clinically.