Introduction and Objective. Achilles tendon defect is difficult problem for orthopedic surgeon, and therefore the development of new treatments is desirable. Platelet-rich fibrin (PRF), dense fibrin scaffold composed of a fibrin matrix containing many growth factors, is recently used as regenerative medicine preparation. However, few data are available on the usefulness of PRF on Achilles tendon healing after injury. The objective of this study is to examine whether PRF promotes the healing of Achilles tendon defect in vivo and evaluated the effects of PRF on tenocytes in vitro. Materials and Methods. PRF were prepared from rats according to international guidelines on the literature. To create rat model for Achilles tendon defect, a 4-mm portion of the right Achilles tendon was completely resected, and PRF was placed into the gap in PRF group before sewing the gap with nylon sutures. To assess the histological healing of Achilles tendon defect, Bonar score was calculated using HE, Alcian-blue, and Picosirius-red staining section. Basso, Beattie, Bresnahan (BBB) score was used for the evaluation of motor functional recovery. Biomechanical properties including failure tensile load, ultimate tensile stress, breaking elongation, and elastic modulus were measured. We examined the effects of PRF on tenocytes isolated from rat Achilles tendon in vitro. The number of viable cells were measured by MTS assay, and immunostaining of ki-67 was used for detection of proliferative cells. Migration of tenocytes was evaluated by wound closure assay. Protein or gene expression level of extracellular matrix protein, such as collagen, were evaluated by immunoblotting, immunofluorescence, or PCR. Phosphorylation level of AKT, FGF receptor, or SMAD3 was determined by western blotting. Inhibitory experiments were performed using MK-2206 (AKT inhibitor), FIIN-2 (FGFR inhibitor), SB-431542 (TGF-B receptor inhibitor), or SIS3 (SMAD3 inhibitor). All p values presented are two-sided and p values < 0.05 were considered statistically significant. Results. In rat Achilles tendon defects, Bonar score was significantly improved in PRF group compared to control group. Collagen deposition at the site of Achilles tendon defect was observed earlier in PRF group. Consistent with the histological findings, BBB score was significantly improved in PRF group. PRF also significantly improved the biomechanical properties of injured Achilles tendon. Furthermore, proliferating tenocytes, labelled by ki-67 were significantly increased in PRF group. These data suggested PRF prompted the healing of Achilles tendon defect. Thus, we further examined the effects of PRF on tenocytes in vitro. PRF significantly increased the number of viable cells, the proliferative cells labelled by ki-67, and migratory ability. Furthermore, PRF significantly increased the protein expression levels of collagen-I, collagen-III, α-SMA, and tenascin-C in tenocytes. Next, we examined the signalling pathway associated with PRF-induced proliferation of tenocytes. PRF increased the phosphorylation level and induced nuclear translocation of AKT, known as key regulator of cell survival. PRF also induced the phosphorylation of FGF receptor. Inhibition of AKT or FGF-receptor completely suppressed the positive effects of PRF on tenocytes. Furthermore, we found that inhibition of FGF receptor partially suppressed the phosphorylation of AKT by PRF. Thus, PRF induced the proliferation of tenocytes via FGFR/AKT axis. We further evaluated the signalling pathway associated with PRF-induced expression of extracellular matrix. PRF increased the phosphorylation levels of SMAD3 and induced nuclear translocation of SMAD3. Furthermore, inhibition of TGF-B receptor or SMAD3 suppressed increased expression level of extracellular matrix by PRF. Thus, PRF increased expression level of extracellular matrix protein via TGF-BR/SMAD3 axis. Conclusions. PRF promotes tendon healing of the Achilles tendon defect and recovery of exercise performance and biomechanical properties. PRF increases the proliferation ability or protein expression level of extracellular matrix protein in tenocytes via FGFR/AKT or TGF-βR/SMAD3 axis, respectively

Introduction. The interstitial fluid of bone fluid flow is supplied by flowing blood. Blood flow is determined by three kinds of muscles: cardiac, smooth, and skeletal. Cardiac muscle establishes baseline blood pressure. Smooth muscle controls vessel diameter and skeletal muscle creates intermittent intravascular pressure pulses. For the tibia the relevant skeletal muscle is the gastrocnemius which functions as a muscle pump. This study tested the hypotheses: 1) skeletal muscle-caused pressure pulses increase cortical blood flow, 2) extravasation of vascular fluid and, consequently, interstitial bone fluid flow are enhanced by resultant increased microvascular pressure and 3) bone healing is enhanced by increased bone fluid flow. Methods. Eighteen skeletally mature female New Zealand white rabbits were implanted with bone chamber windows (BCIs) as described previously. The windows were exposed at three weeks and observed weekly until Week 10 using intravital microscopy. During observation, the subject was suspended in prone position in a mesh fabric torso sling jacket so as to eliminate gravity-based reaction forces. Electrodes of a transcutaneous electrical nerve stimulator (TENS) were gel-glued at each rabbits gastroc-soleus position; but activated only in the 11 experimentals. A 4Hz 2.8 ± 1.3V impulse was delivered for 60 minutes. Still and video images were obtained at 0, 2, and 60 minutes following injection of 1μm fluorescent microspheres. Each such injection was followed by injection of 70 kD FITC- or RITC-dextran to define vascularity and capillary filtration. Additional still images were obtained at 5, 30, and 55 minutes. Muscle contraction forces during TENS were obtained acutely following the Week 10 observation with a Futek force transducer cell through an attached nylon suture. Bone mineral density was obtained at Week 3 and Week 10 with a Stratec pQCT and associated software. Data were analyzed statistically using a Wilcoxon signed rank test. Results. All three hypotheses were supported statistically by the data. The average force produced by TENS stimulated gastrocnemius contraction was 18.98 ± 9.42 N/kg muscle. This produced a microstrain of 192μe in bone around the BCI. Bloodflow results are shown in the figure. On average, flow decreased in controls by 12.6% and increased in experimentals by about 2%. Capillary filtration in experimentals was about 34.6% higher than controls after 60 minutes of TENS. Bone formation rate was 62.5% higher with TENS. Conclusion. In order to understand the role of fluid flows in bone physiology, we need to know the how and where of movement. These results suggest the part played by skeletal muscle in bone fluid movement cannot be ignored. As with many evolutionary adaptations, the muscle pump's hydrodynamic contribution to bone may be redundant and merely serve as a backup to percolation from poroelastic deformation. On the other hand, it may be crucial in disuse osteoporosis instigating conditions such as microgravity. The measured increases in capillary filtration and blood flow suggest that intravascular pressure which drives the former and resultant percolation has been increased by the muscle pump. It follows that fluid shear on cortical bone cells also increased. The challenge now is to obtain local flow measurements that would tell us how much

Objectives

This study aimed to examine the effects of SRT1720, a potent SIRT1 activator, on osteoarthritis (OA) progression using an experimental OA model.

Objectives

Ubiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown.

Objectives

This study aimed to evaluate the histological and mechanical features of tendon healing in a rabbit model with second-harmonic-generation (SHG) imaging and tensile testing.

We evaluated two reconstruction techniques for a simulated posterolateral corner injury on ten pairs of cadaver knees. Specimens were mounted at 30° and 90° of knee flexion to record external rotation and varus movement. Instability was created by transversely sectioning the lateral collateral ligament at its midpoint and the popliteus tendon was released at the lateral femoral condyle. The left knee was randomly assigned for reconstruction using either a combined or fibula-based treatment with the right knee receiving the other. After sectioning, laxity increased in all the specimens. Each technique restored external rotatory and varus stability at both flexion angles to levels similar to the intact condition. For the fibula-based reconstruction method, varus laxity at 30° of knee flexion did not differ from the intact state, but was significantly less than after the combined method.

Both the fibula-based and combined posterolateral reconstruction techniques are equally effective in restoring stability following the simulated injury.

Bone marrow mesenchymal stromal cells were aspirated from immature male green fluorescent protein transgenic rats and cultured in a monolayer. Four weeks after the creation of the osteochondral defect, the rats were divided into three groups of 18: the control group, treated with an intra-articular injection of phosphate-buffered saline only; the drilling group, treated with an intra-articular injection of phosphate-buffered saline with a bone marrow-stimulating procedure; and the bone marrow mesenchymal stromal cells group, treated with an intra-articular injection of bone marrow mesenchymal stromal cells plus a bone marrow-stimulating procedure. The rats were then killed at 4, 8 and 12 weeks after treatment and examined.

The histological scores were significantly better in the bone marrow mesenchymal stromal cells group than in the control and drilling groups at all time points (p < 0.05). The fluorescence of the green fluorescent protein-positive cells could be observed in specimens four weeks after treatment.

Using an osteotomy of the olecranon as a model of a transverse fracture in 22 cadaver elbows we determined the ability of three different types of suture and stainless steel wire to maintain reduction when using a tension-band technique to stabilise the bone. Physiological cyclical loading simulating passive elbow movement (15 N) and using the arms to push up from a chair (450 N) were applied using an Instron materials testing machine whilst monitoring the osteotomy site with a video extensometer. Each osteotomy was repaired by one of four materials, namely, Stainless Steel Wire (7), No 2 Ethibond (3), No 5 Ethibond (5), or No 2 FiberWire (7).

There were no failures (movement of > 2 mm) with stainless steel wire or FiberWire and no significant difference in the movements measured across the site of the osteotomy (p = 0.99). The No. 2 Ethibond failed at 450 N and two of the five of No. 5 Ethibond sutures had a separation of > 2 mm at 450 N.

FiberWire as the tension band in this model held the reduction as effectively as stainless steel wire and may reduce the incidence of discomfort from the hardware. On the basis of our findings we suggest that a clinical trial should be undertaken

This study was undertaken to elucidate the mechanism of biological repair at the tendon-bone junction in a rat model. The stump of the toe flexor tendon was sutured to a drilled hole in the tibia (tendon suture group, n = 23) to investigate healing of the tendon-bone junction both radiologically and histologically. Radiological and histological findings were compared with those observed in a sham control group where the bone alone was drilled (n = 19). The biomechanical strength of the repaired junction was confirmed by pull-out testing six weeks after surgery in four rats in the tendon suture group. Callus formation was observed at the site of repair in the tendon suture group, whereas in the sham group callus formation was minimal. During the pull-out test, the repaired tendon-bone junction did not fail because the musculotendinous junction always disrupted first.

In order to understand the factors that influenced callus formation at the site of repair, four further groups were evaluated. The nature of the sutured tendon itself was investigated by analysing healing of a tendon stump after necrosis had been induced with liquid nitrogen in 16 cases. A proximal suture group (n = 16) and a partial tenotomy group (n = 16) were prepared to investigate the effects of biomechanical loading on the site of repair. Finally, a group where the periosteum had been excised at the site of repair (n = 16) was examined to study the role of the periosteum. These four groups showed less callus formation radiologically and histologically than did the tendon suture group.

In conclusion, the sutured tendon-bone junction healed and achieved mechanical strength at six weeks after suturing, showing good local callus formation. The viability of the tendon stump, mechanical loading and intact periosteum were all found to be important factors for better callus formation at a repaired tendon-bone junction.

We evaluated the possible induction of a systemic immune response to increase anti-tumour activity by the re-implantation of destructive tumour tissue treated by liquid nitrogen in a murine osteosarcoma (LM8) model. The tumours were randomised to treatment by excision alone or by cryotreatment after excision. Tissue from the tumour was frozen in liquid nitrogen, thawed in distilled water and then re-implanted in the same animal. In addition, some mice received an immunological response modifier of OK-432 after treatment. We measured the levels of interferon-gamma and interleukin-12 cytokines and the cytotoxicity activity of splenocytes against murine LM8 osteosarcoma cells. The number of lung and the size of abdominal metastases were also measured.

Re-implantation of tumour tissue after cryotreatment activated immune responses and inhibited metastatic tumour growth. OK-432 synergistically enhanced the anti-tumour effect. Our results suggest that the treatment of malignant bone tumours by reconstruction using autografts containing tumours which have been treated by liquid nitrogen may be of clinical value.

A rat model of lumbar root constriction with an additional sympathectomy in some animals was used to assess whether the sympathetic nerves influenced radicular pain. Behavioural tests were undertaken before and after the operation.

On the 28th post-operative day, both dorsal root ganglia and the spinal roots of L4 and L5 were removed, frozen and sectioned on a cryostat (8 μm to 10 μm). Immunostaining was then performed with antibodies to tyrosine hydroxylase (TH) according to the Avidin Biotin Complex method. In order to quantify the presence of sympathetic nerve fibres, we counted TH-immunoreactive fibres in the dorsal root ganglia using a light microscope equipped with a micrometer graticule (10 x 10 squares, 500 mm x 500 mm). We counted the squares of the graticule which contained TH-immunoreactive fibres for each of five randomly-selected sections of the dorsal root ganglia.

The root constriction group showed mechanical allodynia and thermal hyperalgesia. In this group, TH-immunoreactive fibres were abundant in the ipsilateral dorsal root ganglia at L5 and L4 compared with the opposite side. In the sympathectomy group, mechanical hypersensitivity was attenuated significantly.

We consider that the sympathetic nervous system plays an important role in the generation of radicular pain.

We have investigated in vitro the release kinetics and bioactivity of fibroblast growth factor-2 (FGF-2) released from a carrier of fibrin sealant. In order to evaluate the effects of the FGF-2 delivery mechanism on the repair of articular cartilage, full-thickness cylindrical defects, 5 mm in diameter and 4 mm in depth, which were too large to undergo spontaneous repair, were created in the femoral trochlea of rabbit knees. These defects were then filled with the sealant.

Approximately 50% of the FGF-2 was released from the sealant within 24 hours while its original bioactivity was maintained. The implantation of the fibrin sealant incorporating FGF-2 successfully induced healing of the surface with hyaline cartilage and concomitant repair of the subchondral bone at eight weeks after the creation of the defect.

Our findings suggest that this delivery method for FGF-2 may be useful for promoting regenerative repair of full-thickness defects of articular cartilage in humans.

We hypothesised that meniscal tears treated with mesenchymal stem cells (MSCs) together with a conventional suturing technique would show improved healing compared with those treated by a conventional suturing technique alone. In a controlled laboratory study 28 adult pigs (56 knees) underwent meniscal procedures after the creation of a radial incision to represent a tear. Group 1 (n = 9) had a radial meniscal tear which was left untreated. In group 2 (n = 19) the incision was repaired with sutures and fibrin glue and in group 3, the experimental group (n = 28), treatment was by MSCs, suturing and fibrin glue.

At eight weeks, macroscopic examination of group 1 showed no healing in any specimens. In group 2 no healing was found in 12 specimens and incomplete healing in seven. The experimental group 3 had 21 specimens with complete healing, five with incomplete healing and two with no healing. Between the experimental group and each of the control groups this difference was significant (p < 0.001).

The histological and macroscopic findings showed that the repair of meniscal tears in the avascular zone was significantly improved with MSCs, but that the mechanical properties of the healed menisci remained reduced.