Growth factors have the ability to stimulate matrix synthesis and cell proliferation in rabbit flexor tendon. Maximal stimulation effects of growth factors have a wide variation. It depends upon the different anatomic sites of the tendon segment, the kinds of growth factor, the concentration of growth factors, and the time sequence. Since proliferation was an early component of intrinsic tendon healing, we investigated the short-term dose response to four different growth factors on in vitro rabbit's tendon culture. We evaluated the effects according to the various concentrations of recombinant human insulin-like growth factor 1 (IGF), recombinant human epidermal growth factor (EGF), fibroblast growth factor (FGF), and recombinant human platelet-derived growth factor-BB (PDGF). Fetal calf serum was the most potent stimulator of cell proliferation and protein synthesis in in vitro rabbit's tendon culture. Matrix synthesis and cell proliferation were stimulated dose-dependently by IGF between the doses of 50 and 150 ng/ml. The maximum mitogenic effect of EGF was observed at the concentration of 100 ng/ml (1.3 times more than the media-only control culture). The rabbit's tendon responded significantly dose-dependently to PDGF, whereas there was no significant response to FGF.
Animal ; Cell Division/drug effects ; Dose-Response Relationship, Drug ; Growth Substances/pharmacology* ; Organ Culture ; Proteins/biosynthesis ; Rabbits ; Tendons/metabolism ; Tendons/drug effects* ; Tendons/cytology

BACKGROUNDThe main goals of flexor tendon surgery are to restore digital motion by providing tendon healing and to preserve tendon gliding. Our purpose was to investigate the effects of 5-fluorouracil (5-FU) on tendon adhesions in partially divided profundus flexor tendons (flexor digitorum profundus [FDPs]) following surgical repair and in partially divided FDPs without surgical repair, and to compare the results of the repair versus the nonrepair of zone two injuries via macroscopic and biomechanical evaluations of tendon adhesions.

METHODSWe used 32 adult male European rabbits (Oryctolagus cunniculus) weighing from 2.5 to 3.5 kg. The study was performed on the deep flexor tendons of the second and third digits of the right hind paws of the rabbits; thus, a total of 64 tendons were examined in this study.

RESULTSBased on the results achieved in our experimental study, the load (N) significantly increased in subgroup 1a in which the tendons were surgically repaired and were not treated with 5-FU compared with subgroup 2a in which tendons were surgically repaired and treated with 5-FU.

CONCLUSIONSThe load (N) significantly increased in subgroup 1a in which the tendons were surgically repaired and were not treated with 5-FU compared to subgroup 2a in which the tendons were surgically repaired and treated with 5-FU. Therefore, these results revealed a decrease in adhesion formation in the subgroup that was treated with 5-FU due to increased resistance to tendon adhesions during their excursion through the tendon sheath, which in this case required greater traction force.

Animals ; Fluorouracil ; therapeutic use ; Male ; Rabbits ; Tendon Injuries ; drug therapy ; physiopathology ; Tendons ; drug effects ; pathology ; Tissue Adhesions ; drug therapy ; physiopathology ; Wound Healing ; drug effects

BACKGROUNDRecent studies showed that bone marrow-derived mesenchymal stem cells (BMSCs) had risk of ectopic bone formation. In this study, we aimed to investigate the effect of growth and differentiation factor 6 (GDF-6) on the tenogenic differentiation of BMSCs in vitro, and then combined with small intestine submucous (SIS) to promote tendon regeneration in vivo.

METHODSThe BMSCs were isolated from the green fluorescent protein (GFP) rats, and were characterized by multi-differentiation assays following our previous study protocol. BMSCs cultured with different concentrations of GDF-6, without growth factors served as control. After 2 weeks, mRNA expression and protein expression of tendon specific markers were examined by qRT-PCR and Western blotting to define an optimal concentration of GDF-6. Mann-Whitney U-test was used to compare the difference in relative mRNA expression among all groups; P ≤ 0.05 was regarded as statistically significant. The GDF-6 treated BMSCs combined with SIS were implanted in nude mice and SD rat acute patellar tendon injury model, the BMSCs combined with SIS served as control. After 12 and 4 weeks in nude mice and tendon injury model, the samples were collected for histology.

RESULTSAfter the BMSCs were treated with different concentration of GDF-6 for 2 weeks, the fold changes of the specific markers (Tenomodulin and Scleraxis) mRNA expression were significantly higher in GDF-6 (20 ng/ml) group (P ≤ 0.05), which was also confirmed by Western blotting result. The BMSCs became parallel in orientation after GDF-6 (20 ng/ml) treatment, but the BMSCs in control group were randomly oriented. The GDF-6 (20 ng/ml) treated BMSCs were combined with SIS, and were implanted in nude mice for 12 weeks, the histology showed neo-tendon formation. In the SD rat patellar tendon window injury model, the histology also indicated the GDF-6 (20 ng/ml) treated BMSCs combined with SIS could promote tendon regeneration.

CONCLUSIONSGDF-6 has tenogenic effect on the tenogenic differentiation of BMSCs, and GDF-6 (20 ng/ml) has better tenogenic effect compared to other concentrations. The GDF-6 (20 ng/ml) treated BMSCs combined with SIS can form neo-tendons and promote tendon regeneration.

Animals ; Cell Differentiation ; drug effects ; Growth Differentiation Factor 6 ; pharmacology ; Male ; Membrane Proteins ; genetics ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Mice ; Mice, Nude ; Rats ; Rats, Sprague-Dawley ; Regeneration ; drug effects ; Tendons ; drug effects ; physiology

OBJECTIVETo investigate the effect of Panax Notoginseng Saponins (PNS) on tendon healing in bone tunnel.

METHODSThe experiment was performed in the animal laboratory, Wangjing hospital from April to August, 2010. All the experiment procedures were accorded with the animal ethical requirements. Twenty New Zealand rabbits were randomly divided into blank control group and PNS group with 10 animals in each group. Anterior cruciate ligament reconstruction with toe extensor tendon was done in knee joints of rabbits by suspend fixation model. PNS was injected in bone tunnel of rabbits in PNS group, and nothing was given to blank group. Specimens were collected at 4 and 8 weeks after surgery. Sections were stained with HE stain to observe the changes of interface tissue between bone tunnel and tendon graft. Interface types were classified according to Yamakado method.

RESULTSAt 4 weeks after surgery, the interface was filled with connective tissue in both group, while at 8 weeks, there was not obvious gap between bone and tendon graft in both groups under macrography. Under microscope, there were more fibroblast in the PNS group. There was larger new bone formation area in PNS group. The classification on the Yamakado type was significantly different between two group (P < 0.05).

CONCLUSIONPNS can enhance tendon-bone healing in bone tunnel, and promote the connection between tendon and bone. The study lacks quantitative analysis of those criteria, and the mechanics of the promotion of tendon-bone healing has not been totally clear.

Animals ; Bone and Bones ; drug effects ; pathology ; physiopathology ; Cell Count ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Fibroblasts ; drug effects ; pathology ; Male ; Neovascularization, Physiologic ; drug effects ; Panax notoginseng ; chemistry ; Rabbits ; Saponins ; pharmacology ; Tendons ; drug effects ; pathology ; physiopathology ; transplantation ; Transplantation, Autologous ; Wound Healing ; drug effects

OBJECTIVETo optimize the culture media by adding the growth factors required to maintain tenocytes survival and promote their differentiation without fetal bovine serum (FBS) supplementation, in order for the approach to be used for any future tendon tissue engineering.

METHODSThe human tenocytes were cultured in α-MEM media by adding FBS at various concentrations and supplementing both insulin-like growth factor 1 (IGF-1) and transforming growth factor β-3 (TGFβ-3). A number of growth factors were selected that could support tenocytes expansion at reduced differentiated state with the minimum FBS. By employing fractional factorial design, different treatment groups went through AlamarBlue(TM) tests to evaluate the cell number growth whilst collagen quantification by real time RT-PCR technique and tenocyte differentiation were also studied.

RESULTSThe tenocytes cultured for 14 days with 0% FBS, 50 ng/ml IGF-1 and 10 ng/ml TGFβ-3 maintained survival over 14 days, the Cell count were 6228.68 ± 43.87. They were higher than the other experimental groups, but less than 10% FBS control group (13 576.74 ± 286.75, t = 41.29, P < 0.05). The tenocytes cultured in the treated group also showed enhanced collagen synthesis ((0.322 ± 0.003) ng, t = 4.13 - 5.93, P < 0.05).

CONCLUSIONThese findings have shown for the first time that human tenocytes could be maintained survival for a long period of time in the culture media without FBS, having this approach a suitable one for tendon tissue engineering.

Cell Differentiation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Culture Media ; pharmacology ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Tendons ; cytology ; drug effects ; metabolism ; Transforming Growth Factor beta3 ; pharmacology

OBJECTIVETo study the effect of platelet-derived growth factor-BB (PDGF-BB) in different concentrations on proliferation of tendon cells cultured in vitro.

METHODSRat tendon cells were cultured and identified in vitro. The rat tendon cells were cultured in PDGF-BB nutrient solution in different concentrations. They were then divided into 1, 5, 10, 20, 50, 100, 150, 200, 250 ng/mL PDGF-BB groups (cultured with 0.1 mL 0.5% PBS with addition of 1, 5, 10, 20, 50, 100, 150, 200, 250 ng/mL PDGF-BB respectively). Tendon cells in control group were cultured with 0.1 mL 0.5% FBS. Proliferation of tendon cells was detected by MTT test. The absorbance values of tendon cells in control group and 20 ng/mL PDGF-BB group before culture and after cultured for 12, 24, 36, 48, 60, 72 hs were determined.

RESULTSThe isolated cells were identified to be rat tendon cells as they were Type I collagen staining positive and TypeIII collagen staining negative. Compared with that of control group, the absorbance values of other groups were all increased, except for that of 250 ng/mL PDGF-BB group (P < 0.05 or P < 0.01). Besides, the absorbance value rose gradually with the increase of the concentration of PDGF-BB on, and then diminished gradually with the increase of the concentration of PDGF-BB from 20 ng/mL on. Tendon cells in 20 ng/ml PDGF-BB group began to increase in number when cultured for 12 hs, and it reached the highest level (0.53 +/- 0.04) at 48 h, which were obviously higher than those of control group at 24 - 72 h (P < 0.01). The absorbance value of tendon cells in 20 ng/mL PDGF-BB group was significantly higher than that of control group at 24, 36, 48, 60, 72 h after culture (P < 0.01).

CONCLUSIONSPDGF-BB can promote the proliferation of tendon cells in a definite range of concentration and time.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; Dose-Response Relationship, Drug ; Platelet-Derived Growth Factor ; administration & dosage ; pharmacology ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Sprague-Dawley ; Tendons ; cytology ; drug effects

Adolescent ; Adult ; Combined Modality Therapy ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Radiography ; Tendon Injuries ; diagnostic imaging ; drug therapy ; surgery ; therapy ; Tendons ; diagnostic imaging ; drug effects ; surgery ; Young Adult

In this study we examined the in vitro characteristics of tenocyte adhesion to biologically-modified surface of polymer. Polylactic-co-glycolic acid (PLGA) 85/15 films were prepared by a solvent-casting technique. Each film was adhered onto the bottom of a chamber. The film was precoated with poly-D-lysine (PDL), and then coated with serum-free F12 medium containing various concentrations of fibronectin (FN), type I collagen (CN I), and insulin-like growth factor1 (IGF-1). The monoclonal antibodies (to FN and to CN I) with various dilutions were used to inhibit attachment of tenocytes to surface precoated with FN or CN I. Human embryonic tendon cells (HETCs) and transformed human embryonic tendon cells (THETCs) were used as the seeding cells. The system used for the measurement of adhesion force was the micropipette aspiration experiment system. The micropipette was manipulated to aspirate a small portion of the tenocyte body by using a small aspiration pressure. Then the pipette was pulled away from the adhesion area by micromanipulation. The minimum force required to detach the tenocyte from the substrate was defined as the adhesion force. The results showed that modification of FN or CN I by precoating significantly enhanced attachment of tenocytes to surface of polymer (P < 0.05). As antibodies to FN or CN I were added to a polymer film precoated with FN or CN I, the adhesion force decreased significantly (P < 0.05). We concluded that the specific adhesion forces of tenocytes to extracellular matrix adhesion proteins (FN and CN I) had coordinated action and showed good dependence on their precoating concentrations, and were inhibited by the antibodies to these adhesion proteins. Films precoated with IGF-1 strongly accelerated the adhesion of tenocytes to polymer. These results indicate that the specific adhesion of tenocytes to polymer can be promoted by coating extracellular matrix adhesive proteins and insulin-like growth factor1. It is of great importance to construct tissue-engineered tendon.
Biocompatible Materials ; chemistry ; Cell Adhesion ; drug effects ; physiology ; Cells, Cultured ; Extracellular Matrix Proteins ; pharmacology ; Growth Substances ; pharmacology ; Humans ; Lactic Acid ; chemistry ; Polyglycolic Acid ; chemistry ; Polylysine ; pharmacology ; Polymers ; chemistry ; Tendons ; cytology ; embryology ; physiology ; Tissue Engineering

OBJECTIVETo study the effect of continuous passive motion (CPM) on basic fibroblast growth factor (b-FGF) expression during tendon-bone repair in rabbits and explore the role of stress in the postoperative repair after acute rotator cuff injury.

METHODSSixteen rabbits randomized into CPM group (n=8) and non-CPM group (n=8) were subjected to surgically induced acute rupture of the supraspinatus tendon and subsequent surgical repair, with another two rabbits serving as the control. Two weeks after the operation, the rabbits in CPM group underwent CPM training, and those in non-CPM group were normally fed only. At 2, 4, 6, and 8 weeks after the operation, 2 rabbits from each group were sacrificed and the tissue samples were obtained for detecting the changes in b-FGF expression.

RESULTSTwo weeks after the operation, b-FGF expression was detected in both groups, and the CPM group showed slightly higher and more diffusive expression. At 4 weeks, b-FGF expression was significantly higher and distributed over a greater area in CPM group and in the non-CPM group. A large number of fibroblasts positive for b-FGF expression were identified in CPM group, aligning in parallel with the tendon membrane. At 6 weeks, b-FGF in the CPM group showed no obvious changes but that in the non-CPM group became lightened. At 8 weeks, b-FGF expression was reduced in both groups, which was more obvious in the non-CPM group.

CONCLUSIONCPM can promote b-FGF expression to enhance type III collagen synthesis at the tendon-bone interface in early stage of tendon-bone repair following acute rupture of supraspinatus tendon in rabbits, thereby contributing to tendon-bone recovery after rotator cuff injury.

Animals ; Bone Remodeling ; drug effects ; physiology ; Fibroblast Growth Factor 2 ; metabolism ; Male ; Postoperative Period ; Rabbits ; Random Allocation ; Range of Motion, Articular ; Rupture ; surgery ; Tendon Injuries ; rehabilitation ; surgery ; Tendons ; metabolism ; Wound Healing ; physiology

It is crucial to improve the orientation and growth of cells on substrates in tissue engineering. In this study, we investigated the effects of micropatterned surfaces coated with type I collagen (CNI) on the orientation and growth of SD rat tenocytes. Using the technique of microcontact printing and microfluidic channels, we prepared micropatterned microgrooves with a 10 microm width and 4 microm depth on silicone membrane substrates. The microgrooves were coated with CNI at concentrations 0.25, 0.5, 0.75, 1.0, and 1.25 mg/ml, respectively. The rat tenocytes at 1 x 10(5)/ml were seeded onto the CNI-coated substrates and the control substrates (without CNI coating), and then cultured in a humidified 37 degrees C/5% CO2 incubator for 48 hours. Cell proliferation was measured by MTT method. After 1, 12, 24, 48 hrs of incubation, the tenocytes' alignment and morphology were observed by means of inverted phase microscope, scanning electron microscope and fluorescent microscope. The results showed there was obvious orientation of tenocytes in CNI-modified grooves, and most of the tenocytes spread along the grooves. The tenocyte orientation became more obvious with the increasing CNI concentration over a range from 0.25 to 1.25 mg/ml. This method could find important application in the construct of engineered tendons which need precise spatial organization of cells.
Animals ; Animals, Newborn ; Cell Culture Techniques ; methods ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coated Materials, Biocompatible ; chemistry ; pharmacology ; Collagen Type I ; pharmacology ; Guided Tissue Regeneration ; methods ; Rats ; Rats, Sprague-Dawley ; Surface Properties ; Tendons ; cytology ; Tissue Engineering ; methods