Inhibition of TGF-beta-induced collagen production in rabbit flexor tendons

PURPOSE: Postoperative adhesions frequently compromise the success of flexor tendon repair. Manipulation of growth factors responsible for scar formation may be a method of decreasing adhesion formation. Transforming growth factor beta (TGF-beta) is a key cytokine in the pathogenesis of tissue fibrosis. The purpose of this study was to examine the effectiveness of TGF-beta neutralizing antibody in blocking TGF-beta-induced collagen I production in rabbit flexor tendons in vitro. METHODS: Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-beta along with increasing doses of TGF-beta neutralizing antibody (0.1-2.0 microg/mL). Collagen I production was measured by enzyme-linked immunoabsorbent assay and TGF-beta bioactivity was measured by the luciferase assay. Results were compared with TGF-beta alone and unsupplemented controls. RESULTS: The addition of neutralizing antibody significantly reduced TGF-beta-induced collagen I production in a dose-dependent manner in all 3 cell cultures. TGF-beta bioactivity was also reduced by its neutralizing antibody. CONCLUSIONS: This study shows that TGF-beta inhibition through its neutralizing antibody was effective in cultured flexor tendon cells. The results encourage further experiments that use such agents to modulate flexor tendon wound healing in in vivo models in the hope of eventually blocking the effect of TGF-beta on flexor tendons clinically.     

Flexor tendon wound healing in vitro: the effect of lactate on tendon cell proliferation and collagen production

Flexor tendon repair in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. Lactate is an early mediator of wound healing known to play an important role in stimulation of collagen production after cellular injury. Little attention has been paid to the role of lactate in flexor tendon wound healing. In this study tendon and tendon sheath were excised from rabbit forepaws. We examined proliferation of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes; collagen production by each of these 3 cell types; and effects of lactate on cell proliferation and collagen production. Three cell lines, tendon sheath, epitenon, and endotenon, were isolated and cultured. Tendon sheath fibroblasts showed the greatest proliferation. All 3 cell lines produced collagen I, II, and III. Lactate significantly increased collagen production by all 3 cell lines. We show that cells of the tendon sheath, epitenon, and endotenon produce collagen in vitro. Modulation of lactate levels may provide a means to modulate collagen production.     

Mannose-6-phosphate, an inhibitor of transforming growth factor-beta, improves range of motion after flexor tendon repair

BACKGROUND: Adhesion formation between the flexor tendon and its surrounding fibro-osseous sheath results in a decreased postoperative range of motion in the hand. Transforming growth factor-beta (TGF-beta) is a key cytokine in the pathogenesis of tissue fibrosis. In this study, the effects of two natural inhibitors of TGF-beta, decorin and mannose-6-phosphate, were investigated in vitro and in vivo. METHODS: In the in vitro investigation, primary cell cultures from rabbit flexor tendon sheath, epitenon, and endotenon were established and each was supplemented with TGF-beta along with increasing doses of decorin or mannose-6-phosphate. Collagen-I production was measured with enzyme-linked immunosorbent assay (ELISA). For the in vivo study, rabbit zone-II flexor tendons were transected and then immediately repaired. Single intraoperative graded doses of decorin, mannose-6-phosphate, or phosphate-buffered saline solution (control) were added to the repair sites, and the forepaws were tested for the range of motion and repair strength at eight weeks postoperatively. RESULTS: Decorin and mannose-6-phosphate both reduced TGF-beta upregulated collagen production. Intraoperative application of low-dose mannose-6-phosphate significantly improved the range of motion of the operatively treated digits. The effect on breaking strength of the tendon repair was inconclusive. CONCLUSIONS: Mannose-6-phosphate is effective in reducing TGF-beta upregulated collagen production in an in vitro model. This finding correlated with our in vivo finding that a single intraoperative dose of mannose-6-phosphate improved the postoperative range of motion.     

Tendon healing in vivo and in vitro: neutralizing antibody to TGF-β improves range of motion after flexor tendon repair

Adhesion formation between the flexor tendon and its surrounding fibro-osseous sheath results in a decreased postoperative range of motion (ROM) in the hand. Transforming growth factor-beta (TGF-β) is a key cytokine in the pathogenesis of tissue fibrosis. In this study, the effects of TGF-β1 neutralizing antibody were investigated in vitro and in vivo. In the in vitro investigation, primary cell cultures from rabbit flexor tendon sheath, epitenon, and endotenon were established and each was supplemented with TGF-β along with increasing doses of TGF-β1 neutralizing antibody. Collagen I production was measured with enzyme-linked immunosorbent assay. In the in vivo study, rabbit zone-II flexor tendons were transected and then immediately repaired. Transforming growth factor-β1 neutralizing antibody or phosphate-buffered saline solution (control) was added to the repair sites, and the forepaws were tested for ROM and repair strength at 8 weeks postoperatively. Transforming growth factor-β1 neutralizing antibody reduced TGF-β upregulated collagen production. Intraoperative application of TGF-β1 neutralizing antibody significantly improved the ROM of the operatively treated digits. The effect on breaking strength of the tendon repair was inconclusive.     

Effects of chitosan on cell proliferation and collagen production of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes

Flexor tendon repair in zone II is often complicatedby adhesions to the surrounding fibro-osseoustendon sheath. Adhesion between the tendon andtendon sheath will impair the gliding function oftendons and result in marked impairment of handfunction. Experimental strategies to decrease adhesionformation, with the long-term goal of improving clinicaloutcome after tendon injury and repair, have been thesubject of numerous studies over the past 20 years.Biochemical agents such as antihistamines, s…     

Flexor tendon healing in vitro: effects of TGF-beta on tendon cell collagen production

Flexor tendon healing is complicated by adhesions to the surrounding sheath. Transforming growth factor beta (TGF-beta) is a cytokine with numerous activities related to wound healing. We examined the effects of TGF-beta-1, -2 and -3 on tendon cell proliferation and collagen production. Three separate cell lines--sheath fibroblasts, epitenon and endotenon tenocytes--were isolated from rabbit flexor tendons and cultured separately. Cell culture media was supplemented with 1 or 5 ng/mL of TGF-beta-1, -2, or -3. Cell number and collagen I and III production were measured and compared with unsupplemented control cultures. The addition of TGF-beta to cell culture media resulted in a decrease in cell number in all 3 lines that did not reach statistical significance. There was a significant increase (p <.05) in collagen I and III production with the addition of all 3 TGF-beta isoforms. Modulation of TGF-beta production may provide a mechanism to modulate adhesion formation clinically.     

Tendon healing in vivo: effect of mannose-6-phosphate on flexor tendon adhesion formation

The purpose of this study was to investigate the preventive effect of mannose-6-phosphate on flexor tendon adhesion formation. From a total of 84 adult New Zealand White rabbits, 36 were randomly divided into 2 groups, the normal saline group and the mannose-6-phosphate group, after anastomosis of the flexor tendons. Tendons were harvested at 4 weeks, and biomechanics testing was conducted. The other 48 rabbits were randomly divided into 2 groups, the normal saline group and the mannose-6-phosphate group, after anastomosis of the flexor tendons, and tendons were harvested at 7, 14, 28, and 56 days and analyzed by in situ hybridization to determine the mRNA expression of transforming growth factor (TGF)-β1 and collagen I. The results of biomechanics testing indicated that mannose-6-phosphate can effectively prevent flexor tendon adhesion formation after anastomonsis. The in situ hybridization examination revealed that TGF-β1 and collagen I mRNA expression in the mannose-6-phosphate group was lower than that in the normal saline group at each time point. Mannose-6-phosphate can effectively inhibit the function of TGF-β1 and prevent adhesion formation after flexor tendon injury.     

A comparison of tenocytes and mesenchymal stem cells for use in flexor tendon tissue engineering

PURPOSE: Tissue-engineered tendon grafts will meet an important clinical need. To engineer tendons, we used acellularized allogeneic tendon as scaffold material. To determine the ideal cell type to seed the scaffolds, we studied in vitro characteristics of epitenon tenocytes, tendon sheath fibroblasts, bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived mesenchymal stem cells (ASCs). Subsequently, we implanted reseeded acellularized tendons in vivo as flexor tendon grafts. METHODS: Tenocytes, sheath fibroblasts, BMSCs, and ASCs were obtained from adult rabbits. For all cell lines, collagen 1, 2, and 3 immunocytochemistry was performed, and proliferation was assessed by hemacytometry and senescence by beta-galactosidase staining. Flexor tendons were acellularized after harvest. Tendons were assessed by histology after in vitro reseeding with each of the cell types after 1, 4, and 8 weeks. Finally, reseeded tendons and controls were implanted in a flexor profundus tendon defect. After 6 weeks, the reseeded tendons were harvested and assessed by histology. Statistical analysis for cell proliferation was performed using analysis of variance and t-tests with Bonferroni correction. RESULTS: All cell types had similar collagen expression. Cell proliferation was higher in ASCs in late passage compared with early passage and in ASCs compared with epitenon tenocytes at late passage. The other cell types were similar in growth characteristics. No senescence was detected. In vitro assessment of reseeded constructs showed the presence of cells on the construct surface. In vivo assessment after implantation showed viable cells seen within the tendon architecture in all cell types. CONCLUSIONS: This study suggests that the four cell types may be successfully used to engineer tendons. Adipoderived mesenchymal stem cells proliferate faster in cell culture, but the cell types were similar in other respects. All could be used to successfully repopulate acellularized tendon in vivo as flexor tendon grafts.     

Flexor tendon healing and restoration of the gliding surface. An ultrastructural study in dogs

Healing canine flexor tendons were treated with either total immobilization and were studied by light, scanning, and transmission electron microscopy at ten, twenty-one , and forty-two days. The immobilized tendons healed by ingrowth of connective tissue from the digital sheath and cellular proliferation of the endotenon. The ingrowth of reparative tissue from the digital sheath overwhelmed the epitenon response. At the ultrastructural level, collagen resorption was prominent whereas protein synthesis was limited. This was observed at all study-intervals. In contrast, the mobilized tendons healed by proliferation and migration of cells from the epitenon. Ingrowth of reparative tissue from the tendon sheath was notably lacking in this group. The epitenon cells exhibited greater cellular activity and collagen production at each interval compared with cells of the immobilized repairs.     

Hyaluronan in flexor tendon repair

This study assesses the effect of a preparation of hyaluronan (hyaluronic acid) applied topically at the time of flexor tendon repair in a well-established model. The hypothesis is that hyaluronic acid applied topically at the time of flexor tendon repair will decrease adhesions, and will improve clinically the gliding function of the repaired flexor tendon. After transection and repair of the second and fifth flexor tendons of the left forepaw of four mongrel dogs, the second flexor tendon was treated with hyaluronic acid of molecular weight 3.6 x 10(6) daltons applied topically between the synovial sheath and the repair site. The left forepaws were completely immobilized for 5 weeks to optimize the formation of adhesion ingrowth. After death, the repaired tendons and sheaths were removed en bloc, fixed, and dissected. Gross inspection and histologic evaluation of all tendons showed that the quality and quantity of adhesions from the wound repair to the synovial sheath appeared to have been consistently affected by hyaluronan. Hyaluronic acid had a beneficial effect on both the repair site and synovial sheath by decreasing the peripheral inflammatory response and promoting a contact healing process via epitenon and endotenon cell involvement in the repair process.     

HGF拮抗TGF-β1诱导腱鞘成纤维细胞α-SMA及细胞外基质合成

目的 研究HGF能否阻抑TGF-β1诱导的腱鞘成纤维细胞α-SMA及细胞外基质过度合成.方法 选取成年新西兰大白兔7只,体重3.75～4.00 kg,无菌切取前肢中趾趾深屈肌腱,进行腱鞘成纤维细胞的分离和培养,待细胞生长成单层后,以胰蛋白酶消化传代.取第3代成纤维细胞用于实验,当细胞达到70%融合时,培养液中加入TGF-β1(5 ng/ml)及HGF(10～40 ng/ml).培养72 h后,利用Westernblot检测α-SMA表达;ELISA测定细胞Ⅰ型胶原及纤维结合素的表达.结果 TGF-β1能显著诱导α-SMA表达,半定量分析提示,TGF-β1作用后的成纤维细胞α-SMA表达量是对照组的1.8倍.随HGF的同时加入,α-SMA的表达则明显受抑制(P＜0.05),且随HGF浓度的升高其阻抑作用呈逐渐增强趋势.TGF-β1同样能诱导Ⅰ型胶原及纤维结合素的表达(P＜0.01),而HGF则可以有效地阻抑其表达,其效应呈剂量依赖性(P＜0.05).结论 HGF可以有效阻抑TGF-β1诱导的腱鞘成纤维细胞α-SMA、Ⅰ型胶原及纤维结合素的表达,这为利用HGF预防和治疗屈指肌腱损伤后粘连及瘢痕在细胞和分子水平提供了依据.     

培养条件下肌腱中心区域腱细胞的增殖能力

目的　研究肌腱损伤后腱中心区域组织的愈合能力。方法　选用 8只白色纯种来亨鸡 ,在无菌条件下切取双侧最长趾 区趾深屈肌腱 ,切成 4 mm长的肌腱段 ,分为两组 ,每组 12条。实验组 :切除腱外膜和外膜下腱组织的肌腱中心区域组织 ;对照组 :仅剥除腱外膜组织的肌腱段。将两组肌腱组织进行体外培养 ,于培养第 9、18及 2 7天取标本进行大体观察及组织学检查。另取 4条肌腱作正常对照组 ,直接进行大体观察和组织学检查。结果　各时间点的腱细胞数 ,对照组均明显少于实验组及培养前 ,有统计学意义 (P<0 .0 1) ;实验组比培养前均明显增多 ,有统计学意义 (P<0 .0 1) ;但培养第 18天和 2 7天时 ,实验组腱细胞数较培养第 9天少 (P<0 .0 1)。结论　屈肌腱损伤后腱中心区域组织有良好的愈合能力。     

Fibronectin in the tendon-synovial complex: Quantitation In vivo and In vitro by elisa and relative mRNA levels by polymerase chain reaction and northern blot

An enzyme-linked immunosorbent assay was used to quantitate fibronectin (Fn) levels in the outer synovia (epitenon) and internal fibrous portion (endotenon) of chicken flexor tendon and sheath. Primary cell cultures from these tissues and their secretions also were assayed for Fn levels. The polymerase chain reaction (PCR) was used to determine relative steady-state levels of Fn mRNA in primary cultures of synovial and internal fibroblasts from chicken tendon, and Northern blot analysis was performed to verify relative levels of the Fn message. The epitenon contained 3.8-fold more Fn than did the endotenon, and the sheath synovium contained 21-fold more Fn than did the internal fibrous portion of sheath. Cells cultured from the epitenon produced 9.3 and 13-fold more cell-associated and secreted Fn, respectively, than did cultured endotenon fibroblasts. Sheath synovial cells produced 17 and 3.2-fold more cell-associated and secreted Fn, respectively, than did sheath internal fibroblasts. Levels of Fn mRNA, as measured by PCR and Northern blot, were 1.6 and 1.8-fold greater, respectively, in tendon synovial cells compared with tendon internal fibroblasts. The biologic reason for increased Fn in tendon synovium is not known. We theorize that Fn may stabilize tendon synovium to shear stress and may play a role in the modulation of synovial rheology in the normal tendon. In the injured tendon, Fn may be involved in the organization of collagen deposition or may act through association with growth factors to aid healing.     

腺相关病毒载体转导基因至愈合肌腱及载体组织反应的研究

目的 探讨腺相关病毒(AAV)载体转导碱性成纤维细胞牛长因子(bFGF)基因对肌腱愈合的影响,并观察腺病毒、AAV以及脂质体一质粒三种基因治疗载体应用于肌腱所产生的组织反应.方法 取13只成年白色来亨鸡的双侧巾趾趾深屈肌腱(26根),随机分为实验组和对照组,每组13根,实验组肌腱完全切断后注射AAV2-bFGF并以改良Kessler法修复,对照组不注射AAV2-bFGF,仪以改良Kessler法修复.第4周未行免疫组织化学染色,第8周术测定趾屈曲功.将6只成年新西兰白兔的趾深屈肌腱(36根)分成二组,每组12根,分别注射10μL的腺病毒、AAV2和脂质体.质粒载体,术后第3、7、14天分别取肌腱,进行石蜡切片、HE染色.结果 AAV2-bFGF可以在术后4周显著地提高肌腱bFGF的表达,而且不增加趾屈曲阻力(粘连形成).所测屈曲功第8周末实验组为(0.052±0.031)J,对照组为(0.049±0.035)J,两组问差异无统计学意义(t=0.31266,P=0.8984).脂质体-质粒载体组肌腱组织反廊重于腺病毒载体组,AAV2载体组肌腱组织反应最轻,在腱外膜处有组织反应,而腱内膜区域几乎无组织反应.结论 用AAV2载体转bFGF基因至肌腱能有效增加愈合肌腱的bFGF.在三种所研究的载体中,腺病毒和AAV2载体引起的组织反应比脂质体.质粒载体轻.AAV2引起的组织反应最轻.AAV2可能会成为肌腱的转基因良好载体.     

Genetic expression for type I procollagen in the early stages of flexor tendon healing.

To determine the precise mechanism by which contact tendon healing occurs at the cellular level, the production of pro alpha (I) collagen messenger RNA (mRNA) produced by fibroblasts of healing intrasynovial flexor tendons was determined by an in situ hybridization technique. The repair site and the proximal and distal tendon stumps of repaired tendons treated with early controlled passive mobilization were fixed and buffered in formalin, 3, 7, 10, and 17 days after repair. A complimentary DNA (cDNA) probe corresponding to alpha (I) procollagen mRNA was labeled with [32P]d-CTP. After hybridization, autoradiography, and staining of the sections, the level of procollagen mRNA was assessed by microscopic examination. Rising levels of procollagen mRNA, indicating progressively increasing levels of synthetic collagen activity, were detected in the healing tendons through 10 days. A moderate decrease in procollagen mRNA was seen at 17 days. Genetic expression for procollagen mRNA was localized specifically to the epitenon cells on the tendon surface overlying the repair site and to cells in the gap between the tendon stumps. No detectable expression was noted in endotenon fibroblasts. The finding of high levels of expression for procollagen type I mRNA in the surface layer of healing tendons demonstrates that cells intrinsic to tendon epitenon contribute the greatest quantity of native tendon collagen to the repair site during these important early intervals after tendon suture.     

Flexor tendon repair

Healing canine flexor tendons treated with either total immobilization, delayed protected mobilization, or early protected mobilization was studied by biomechanical, microangiographic, biochemical, and histologic techniques at intervals through 12 weeks. The healing characteristics of the early mobilization tendons showed higher tensile strengths and improved gliding function than the delayed mobilization and immobilization tendons. Protected passive motion brought about accelerated changes in peritendinous vessel density and configuration, as well as increased repair site total DNA content. While adhesions obliterated the space between the tendon surface and the tendon sheath of the immobilized repairs, the mobilized tendons demonstrated coverage of the repair site by cells from the epitenon by 10 days, and a smooth, gliding surface that was maintained free of adhesions through 42 days. A series of in vitro studies demonstrated the cellular processes involved in the repair: phagocytosis of cellular debris and collagenous fragments by cells from the epitenon, and collagen synthesis primarily by endotenon cells.     

Flexor tendon repair in vitro: a comparative histologic study of the rabbit, chicken, dog, and monkey

Flexor tendon healing in four different animal species was explored in a tissue culture system. Ninety percent transverse lacerations were made in 88 tendon segments obtained from rabbits, chickens, dogs, and monkeys. The tendons were removed from culture and studied by light and electron microscopy at intervals of 3, 6, 9, and 12 weeks. A characteristic sequence of repair including epitenon thickening, cellular differentiation, cell migration, and phagocytosis was seen in each of the repaired tendons. The endotenon cells of several animal tendons appeared to be synthesizing collagen. There was a consistent difference in the rate of healing between the four species. The rabbit tendons demonstrated nearly complete closure of the repair site by 12 weeks. A lesser response was seen in the chicken, followed by the dog and monkey. The differences in healing rate appeared to be due to the non-species-specific in vitro culture media. The in vitro flexor tendon culture system is particularly useful in studying the tendon repair responses of various species with the contributions of vascularity and synovial cells excluded.     

Tissue reactions of adenoviral, adeno-associated viral, and liposome-plasmid vectors in tendons and comparison with early-stage healing responses of injured flexor tendons

PURPOSE: Delivery of growth factor genes that may substantially increase the healing rate of injured digital flexor tendons is a new application of gene therapy. Adenoviral, adeno-associated viral (AAV), and liposome-plasmid vectors have been used to deliver genes to tendons, but the tendon reactions to these vectors--particularly in contrast to the healing responses in the injured tendons--were unknown. This study was designed to compare the tissue reactions of the earlier-mentioned vectors in tendons with the healing responses of injured flexor tendons. METHODS: Forty-two flexor digitorum profundus tendons of 6 New Zealand white rabbits were used. Eighteen tendons were divided into 3 groups of 6 each and injected with different vectors: adenoviral vector, AAV2-luciferase vector, or pCMV-beta vector with liposome. Another 12 tendons were cut and repaired. At 3, 7, and 14 days, the tendons were harvested and stained with hematoxylin and eosin. Normal flexor tendons were harvested as controls. RESULTS: The tissue reactions of the liposome-plasmid vector in tendons were the most prominent among the 3 vectors tested. The adenoviral vector elicited a moderate degree of tissue reaction. The AAV2 vector caused remarkable reactions in epitenon but almost no reactions in endotenon. Early-stage tissue reactions were more robust in the injured tendons. Compared with early-stage inflammatory and healing responses, the reactions elicited by these vectors were less severe. CONCLUSIONS: The 3 gene delivery systems tested elicit less severe tissue reactions in flexor tendons compared with early-stage inflammatory changes in injured tendons. Adenoviral and AAV vectors elicit less severe tissue reactions than liposome-plasmid vectors. The AAV2 vector appears to cause almost no reaction in endotenon. In terms of tissue reactions, the adenoviral and AAV2 vectors, in particular AAV2, are suitable gene delivery systems for future gene transfer to the tendon in vivo.