Effects of culture on PAMPS/PDMAAm double-network gel on chondrogenic differentiation of mouse C3H10T1/2 cells: in vitro experimental study

BACKGROUND: Recently, several animal studies have found that spontaneous hyaline cartilage regeneration can be induced in vivo within a large osteochondral defect by implanting a synthetic double-network (DN) hydrogel, which is composed of poly-(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) and poly-(N,N’-dimethyl acrylamide) (PDMAAm), at the bottom of the defect. However, the effect of hydrogel on hyaline cartilage regeneration remains unexplained. The purpose of this study was to investigate the chondrogenic differentiation of C3H10T1/2 cells on PAMPS/PDMAAm DN gel. METHODS: C3H10T1/2 cells of 1.0 × 10(5) were cultured on PAMPS/PDMAAm DN gel in polystyrene tissue culture dishes or directly on polystyrene tissue culture dishes. We compared cultured cells on PAMPS/PDMAAm DN gel with those on polystyrene dishes by morphology using phase-contrast microscopy, mRNA expression of aggrecan, type I collagen, type II collagen, Sox 9 and osteocalcin using real-time RT-PCR, and local expression of type II collagen using immunocytochemistry. RESULTS: C3H10T1/2 cells cultured on the PAMPS/PDMAAm DN gels formed focal adhesions, aggregated rapidly and developed into large nodules within 7 days, while the cells cultured on the polystyrene surface did not. The mRNA levels of aggrecan, type I collagen, type II collagen, Sox 9 and osteocalcin were significantly greater in cells cultured on the PAMPS/PDMAAm DN gel than in those cultured on polystyrene dishes. In addition, C3H10T1/2 cells cultured on PAMPS/PDMAAm DN gel expressed more type II collagen at the protein level when compared with cells cultured on polystyrene dishes. CONCLUSIONS: The present study showed that PAMPS/PDMAAm DN gel enhanced chondrogenesis of C3H10T1/2 cells, which are functionally similar to mesenchymal stem cells. This suggests that mesenchymal stem cells from the bone marrow contribute to spontaneous hyaline cartilage regeneration in vivo in large osteochondral defects after implantation of PAMPS/PDMAAm DN gels. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2474-15-320) contains supplementary material, which is available to authorized users

In Vivo Imaging of Particle-Induced Inflammation and Osteolysis in the Calvariae of NFκB/Luciferase Transgenic Mice

Wear debris causes biological response which can result in periprosthetic osteolysis after total joint replacement surgery. Nuclear factor-kappa B (NFκB), a representative transcription factor involved in inflammation, is believed to play an important role in this event by regulating the production of proinflammatory mediators and osteoclastogenesis. In this study, we sought to determine whether activation of NFκB in response to stimulation by particles could be visualized by in vivo imaging. We loaded polyethylene (PE) particles onto the calvaria of NFκB/luciferase transgenic mouse, and detected luminescence generated by activation of NFκB. On day 7 after loading, the level of luminescence was maximal. Levels of luminescence were significantly correlated with the levels of luciferase activity, proinflammatory mediator mRNAs, and bone resorption parameters. This system, which enabled us to evaluate particle-induced inflammation and osteolysis without sacrificing mice, constitutes a useful tool for evaluating the efficacy of prophylaxis or treatments for particle-induced osteolysis

Change in sensory integration and regularity of postural sway with the suspensory strategy during static standing balance

Background and aimThe suspensory strategy, a method for controlling postural balance in the vertical direction of the center of mass (COM), is considered by the elderly as a means of balance control. The vertical COM control might alter the sensory integration and regularity of postural sway, which in turn impacts balance. However, to date, this was not confirmed. Thus, this study aimed at investigating the influence of the suspensory strategy achieved through knee flexion on the static standing balance.MethodsNineteen participants were monitored at knee flexion angles of 0°, 15°, and 65°. Time-frequency analysis and sample entropy were employed to analyze the COM data. Time-frequency analysis was utilized to assess the energy content across various frequency bands and corresponding percentage of energy within each frequency band. The outcomes of time-frequency are hypothesized to reflect the balance-related sensory input and sensory weights. Sample entropy was applied to evaluate the regularity of the COM displacement patterns.ResultsKnee flexion led to a decreased COM height. The highest energy content was observed at 65° knee flexion, in contrast with the lowest energy observed at 0° in both the anterior–posterior (AP) and medial-lateral (ML) directions. Additionally, the ultra-low-frequency band was more pronounced at 65° than that at 0° or 15° in the ML direction. Furthermore, the COM amplitudes were notably higher at 65° than those at 0° and 15° in the AP and ML directions, respectively. The sample entropy values were lower at 65° and 15° than those at 0° in the ML direction, with the lowest value observed at 65° in the vertical direction.ConclusionThe suspensory strategy could enhance the sensory input and cause sensory reweighting, culminating in a more regular balance control. Such suspensory strategy-induced postural control modifications may potentially provide balance benefits for people with declining balance-related sensory, central processing, and musculoskeletal system functions

Is the increase in type III collagen of the patellar tendon graft after ligament reconstruction really caused by "ligamentization" of the graft?

To test the hypothesis that extrinsic cells that infiltrate the devitalized patellar tendon (PT) synthesize type III collagen even in the environmental milieu of the native PT, we conducted the present experimental study using the rat in situ frozen–thawed PTs. Tissue culture showed no cell outgrowth from the tendons immediately after the freeze–thaw treatment. Analysis by RT-PCR showed that the expression level of type III procollagen mRNA in the frozen–thawed tendon was significantly higher than that in the sham-operated tendon at 6 and 12 weeks. Immunohistological findings showed positive type III collagen staining around cells that had infiltrated the necrotized tendon at 3, 6, and 12 weeks. In addition, the elastic modulus of the in situ frozen–thawed tendon at 6 weeks was significantly less than that of the sham-operated tendon. The present study indicates that extrinsic cells that had infiltrated the devitalized PT synthesized type III collagen at least for 12 weeks even in the environmental milieu of the native PT. These findings raised the question whether the increase in type III collagen of the PT graft after ACL reconstruction is really caused by “ligamentization,” the adaptation of the PT graft to the ACL environment

In vivo effects of partial electrothermal shrinkage on mechanical properties of the anterior cruciate ligament in rabbits

Background: No studies have been conducted to clarify an in vivo remodeling of the radiofrequency-treated lesion of the anterior cruciate ligament. The purpose was to determine in vivo effects of radiofrequency shrinkage on mechanical properties of the anterior cruciate ligament. Methods: Thirty skeletally mature rabbits were used. In each group, radiofrequency energy set at non-ablative levels was applied to the posterolateral bundle of the anterior cruciate ligament with a bipolar radiofrequency generator. All animals were sacrificed at 0, 6, and 12 weeks after surgery, respectively. In each group, 7 and 3 out of the 10 specimens were used for biomechanical and histological evaluations. Findings: After shrinkage treatment, the anterior–posterior translation of the knee and the length of the posterolateral bundle were significantly reduced immediately after surgery, but that this effect disappeared at 6 weeks. The tensile strength and the tangent modulus of the treated bundle were significantly lower than that of the normal control bundle at each period. In addition, the tensile strength and the tangent modulus measured at 12 weeks were significantly lower than that at 0 week. Histological examination showed granulation-like tissues with numerous plump fibroblasts and inflammatory cells were dominantly found in the midsubstance of the posterolateral bundle at 12 weeks. Interpretations: This result suggested that the anterior cruciate ligament tissue shortened with the radiofrequency treatment is elongated gradually over time. The mechanical properties of the posterolateral bundle of the anterior cruciate ligament reduced by the radiofrequency shrinkage are not restored in vivo, but significantly deteriorated with time

The effect of intraosseous graft length on tendon-bone healing in anterior cruciate ligament reconstruction using flexor tendon.

The current study was performed to understand the relationship between graft length placed within the bone tunnel and intraosseous graft healing in anterior cruciate ligament (ACL) reconstruction. Twenty-four adult beagle dogs were divided into two groups of 12 animals each. In each animal, ACL reconstruction using a 4-mm diameter autogenous flexor tendon graft was done in the left knee. In groups I and II, the graft having a length of 15 and 5 mm, respectively, was placed within the tibial tunnel. The proximal end of the graft was placed through the over-the-top route in all animals. In each group, five animals were sacrificed immediately after surgery, and the remaining seven were sacrificed at 6 weeks postoperatively. Biomechanical and histologic evaluations were performed. In pull out testing, the ultimate failure load and the linear stiffness of the graft-tibia complex harvested at 6 weeks were significantly greater than those harvested at the time-zero period. There were no significant differences in those parameters between groups I and II at 6 weeks. In each group, the perpendicular collagen fibers connecting the tendon to the bone tunnel wall were observed only in the narrow area located close to the intra-articular tunnel outlet. In conclusion, excessively long placement of the flexor graft within the bone tunnel does not result in an additional increase of anchoring strength and stiffness of the graft in ACL reconstruction

Stabilizing effects of ankle bracing under a combination of inversion and axial compression loading

The combined effects of bracing, axial compression and inversion rotation on the ankle-subtalar complexes were evaluated. Ex vivo tests under the load-controlled condition were performed on six cadaver ankle specimens using a six degree-of-freedom fixture. Inversion rotation was measured while subjecting the ankle-subtalar complex to a 2.5 N-m inversion moment and a combination of the testing variables (brace type, no brace, 178 N axial compression load, no compression load, 0° and 20° of plantar flexion) for a total of 16 tests per specimen. Three commercially available braces (two semirigid types and one lace up type) were evaluated. An axial compression load significantly decreased ankle-subtalar motion in unbraced ankles for the tested inversion moment. The contribution of bracing to stabilization of the ankle was smaller in the axial loading condition than in the no axial loading condition. The semirigid braces had greater stabilizing effects in response to the inversion moment than the lace up brace. Stabilizing effects of bracing were significantly greater in 20° of plantar flexion than in 0° of plantar flexion. The most common mechanism for an ankle sprain injury is inversion rotation on a weight-bearing ankle. Therefore, we should not overestimate stabilizing effects of bracing from evaluations of bracing without axial compression loading

Ex vivo infiltration of fibroblasts into the tendon deteriorates the mechanical properties of tendon fascicles but not those of tendon bundles

Background: After ligament reconstruction, mechanical deterioration of the grafted tendon is observed with revascularization and cellular infiltration. However, the effect of cellular infiltration on the mechanical properties of the tendon matrix has not been fully understood. Methods: Cultured fibroblasts derived from the rabbit patellar tendon were seeded around an acellular rabbit patellar tendon that had undergone freeze–thaw treatment. At time-0, 3, and 6 weeks after seeding the cells, we evaluated cellular distribution in the tendon using a confocal laser microscope and the mechanical evaluations of the tendon fascicles and the tendon bundles. Findings: The confocal laser microscopic analysis showed fibroblast infiltration ex vivo into the acellular tendon matrix. We could not find significant effects of the cellular infiltration on the tangent modulus of the tendon bundle, although the ex vivo cellular infiltration significantly reduced the modulus of the tendon fascicle. In addition, the tangent modulus of the incubated tendon without fibroblasts significantly decreased with time, particularly in the tendon bundle levels. Interpretation: The findings of this study suggested that the effects of ex vivo cellular infiltration on the mechanical properties of the tendon bundles are relatively small, compared with its striking effect on the tendon fascicles