Development of arthrogenic joint contracture as a result of pathological changes in remobilized rat knees.

This study aimed to elucidate how rats recover from immobilization-induced knee joint contracture. Rats' right knees were immobilized by an external fixator at a flexion of 140° for 3 weeks. After removal of the fixator, the joints were allowed to move freely (remobilization) for 0, 1, 3, 7, or 14 days (n = 5 each). To distinguish myogenic and arthrogenic contractures, the passive extension range of motion was measured before and after myotomy of the knee flexors. Knee joints were histologically analyzed and the expression of genes encoding inflammatory or fibrosis-related mediators, interleukin-1ß (1L-1ß), fibrosis-related transforming growth factor-ß1 (TGF-ß1), and collagen type I (COL1A1) and III (COL3A1), were examined in the knee joint posterior capsules using real-time PCR. Both myogenic and arthrogenic contractures were established within 3 weeks of immobilization. During remobilization, the myogenic contracture decreased over time. In contrast, the arthrogenic contracture developed further during the remobilization period. On day 1 of remobilization, inflammatory changes characterized by edema, inflammatory cell infiltration, and upregulation of IL-1ß gene started in the knee joint posterior capsule. In addition, collagen deposition accompanied by fibroblast proliferation, with upregulation of TGF-ß1, COL1A1, and COL3A1 genes, appeared in the joint capsule between days 7 and 14. These results suggest the progression of arthrogenic contracture following remobilization, which is characterized by fibrosis development, is possibly triggered by inflammation in the joint capsule. It is therefore necessary to focus on developing new treatment strategies for immobilization-induced joint contracture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1414-1423, 2017.

Regulation of the microvascular circulation in the leg muscles, pancreas and small intestine in rats.

To study the microvascular circulation, we examined the proportion of open and functioning capillaries in the leg muscles, pancreas and small intestine of anesthetized rats. Fluorescein isothiocyanate (FITC)-labeled Lycopersicon esculentum lectin was injected into the heart and allowed to circulate for 3 min to label open and functioning capillaries. Specimens were removed, frozen, sectioned and double-immunostained. Using one section, open and functioning capillaries were detected by immunostaining for this lectin bound to endothelial cells, while all capillaries were visualized by immunostaining for platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31). These capillaries were semi-automatically detected and counted by fluorescence microscopy. The percentages of open and functioning capillaries were as follows: the soleus muscle, 93.0 ± 5.5%; superficial zone of the gastrocnemius muscle, 90.8 ± 6.2%; deep zone of the gastrocnemius muscle, 95.6 ± 4.0%; the plantaris muscle, 94.1 ± 2.7%; the pancreas, 86.3 ± 11.7%; and the small intestine, 91.1 ± 4.9% (n = 8, each). There was no significant difference among these data by the Kruskal-Wallis test. This study clearly demonstrated that the proportions of open and functioning capillaries are high and similar among the leg muscles, pancreas and small intestine in spite of their structural and functional differences. This finding agrees with previous studies and supports the notion that the microvascular circulation is mainly controlled by changing of the blood flow in each capillary rather than changing the proportion of open and functioning capillaries.

Analysis of Gene Expression in Experimental Pressure Ulcers in the Rat with Special Reference to Inflammatory Cytokines.

Pressure ulcers have been investigated in a few animal models, but the molecular mechanisms of pressure ulcers are not well understood. We hypothesized that pressure results in up-regulation of inflammatory cytokines and those cytokines contribute to the formation of pressure ulcers. We measured genome-wide changes in transcript levels after compression, and focused especially on inflammatory cytokines. The abdominal wall of rats was compressed at 100 mmHg for 4 hours by two magnets. Specimens were obtained 12 hours, 1, or 3 days after compression, and analyzed by light microscopy, microarray, Real-Time PCR, and ELISA. The skin and subcutaneous tissue in the compressed area were markedly thickened. The microarray showed that numerous genes were up-regulated after the compression. Up-regulated genes were involved in apoptosis, inflammation, oxidative stress, proteolysis, hypoxia, and so on. Real-Time PCR showed the up-regulation of granulocyte-macrophage colony stimulating factor (GM-CSF), interferon γ (IFN-γ), interleukin 1ß (IL-1ß), interleukin 1 receptor antagonist gene (IL1Ra), interleukin 6 (IL-6), interleukin 10 (IL-10), matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinase 1 (TIMP-1), and tumor necrosis factor α (TNF-α) at 12 hours, IFN-γ, IL-6, IL-10, MMP-3, and TIMP-1 at 1 day, and IFN-γ, IL-6, and MMP-3 at 3 days. Some genes from subcutaneous tissue were up-regulated temporarily, and others were kept at high levels of expression. ELISA data showed that the concentrations of IL-1ß and IL-6 proteins were most notably increased following compression. Prolonged up-regulation of IL-1ß, and IL-6 might enhance local inflammation, and continuous local inflammation may contribute to the pressure ulcer formation. In addition, GM-CSF, IFN-γ, MMP-3, and TIMP-1 were not reported previously in the wound healing process, and those genes may have a role in development of the pressure ulcers. Expression data from Real-Time PCR were generally in good agreement with those of the microarray. Our microarray data were useful for identifying genes involved in pressure ulcer formation. However, the expression levels of the genes didn't necessarily correspond with protein production. As such, the functions of these cytokines need to be further investigated.

Effects of the magnitude of pressure on the severity of injury and capillary closure in rat experimental pressure ulcers.

Experimental pressure ulcers were successfully produced in the rat abdominal wall at 100 mmHg in our previous study. We hypothesized that injury is less severe when pressures are lower than 100 mmHg and explored a critical pressure in the production of pressure ulcers. At 70 and 60 mmHg, repeated compressions for 4 h daily for 5 consecutive days resulted in partial skin necrosis and eschar formation in the majority of rats, whereas skin injuries were absent or very mild in most of the rats at 50 mmHg. The extent of ischemia was also examined by visualization of capillary blood flow using intravascular infusion of Lycopersicon esculentum lectin. Rat abdominal walls were compressed in the range from 0 (control) to 100 mmHg. The percentages of open capillaries were 62.8 ± 10.1% at 0 mmHg and 34.7 ± 18.5% at 10 mmHg. The ratio of open capillaries was further decreased with increasing pressure, but not pressure dependently. In conclusion, the severity of injury at 50 mmHg was drastically milder than that at 60 mmHg or higher, whereas the extent of ischemia (capillary closure) was not significantly different. The pressure is vitally important; however, other factor(s) besides ischemia is likely to promote the development of pressure ulcers.

Intermittent whole-body vibration attenuates a reduction in the number of the capillaries in unloaded rat skeletal muscle.

BACKGROUND: Whole-body vibration has been suggested for the prevention of muscle mass loss and muscle wasting as an attractive measure for disuse atrophy. This study examined the effects of daily intermittent whole-body vibration and weight bearing during hindlimb suspension on capillary number and muscle atrophy in rat skeletal muscles. METHODS: Sixty male Wistar rats were randomly divided into four groups: control (CONT), hindlimb suspension (HS), HS + weight bearing (WB), and HS + whole-body vibration (VIB) (n = 15 each). Hindlimb suspension was applied for 2 weeks in HS, HS + WB, and HS + VIB groups. During suspension, rats in HS + VIB group were placed daily on a vibrating whole-body vibration platform for 20 min. In HS + WB group, suspension was interrupted for 20 min/day, allowing weight bearing. Untreated rats were used as controls. RESULTS: Soleus muscle wet weights and muscle fiber cross-sectional areas (CSA) significantly decreased in HS, HS + WB, and HS + VIB groups compared with CONT group. Both muscle weights and CSA were significantly greater in HS + WB and HS + VIB groups compared with HS group. Capillary numbers (represented by capillary-to-muscle fiber ratio) were significantly smaller in all hindlimb suspension-treated groups compared with CONT group. However, a reduction in capillary number by unloading hindlimbs was partially prevented by whole-body vibration. These findings were supported by examining mRNA for angiogenic-related factors. Expression levels of a pro-angiogenic factor, vascular endothelial growth factor-A mRNA, were significantly lower in all hindlimb suspension-treated groups compared with CONT group. There were no differences among hindlimb suspension-treated groups. Expression levels of an anti-angiogenic factor, CD36 (receptor for thrombospondin-1) mRNA, were significantly higher in all hindlimb suspension-treated groups compared with CONT group. Among the hindlimb suspension-treated groups, expression of CD36 mRNA in HS + VIB group tended to be suppressed (less than half the HS group). CONCLUSIONS: Our results suggest that weight bearing with or without vibration is effective for disuse-derived disturbance by preventing muscle atrophy, and whole-body vibration exercise has an additional benefit of maintaining microcirculation of skeletal muscle.

Microvascular circulation at cool, normal and warm temperatures in rat leg muscles examined by histochemistry using Lycopersicon esculentum lectin.

Local cooling and/or warming of the body are widely used for therapy. For safer and more effective therapy, microvascular hemodynamics needs to be clarified. To examine blood circulation in rat leg muscles at 20, 30, 37 and 40°C, fluorescein isothiocyanate (FITC)-labeled Lycopersicon esculentum lectin was injected into the cardiac ventricle. Endothelial cells of open and functioning blood vessels were labeled by this lectin for 3 min and detected by immunostaining for lectin. The percentage of open and functioning capillaries of leg muscles by the avidin-biotin method was 89.8±3.3% at 37°C, while capillaries were unclear or unstained at 20 and 30°C, probably due to a decrease of blood flow. The results using the tyramide-dinitrophenol method were 58.6±15.0% at 20°C, 68.5±12.3% at 30°C, 83.8±5.7% at 37°C and 83.3±7.8% at 40°C. The value at 20°C was significantly different from those at 37 and 40°C. The results by the tyramide-biotin method were 85.5±5.3% at 20°C, 87.3±9.7% at 30°C, 94.7±3.6% at 37°C and 92.5±2.1% at 40°C. Based on these results, it was concluded that the blood flow of each capillary considerably decreased at 20 and 30°C and probably increased at 40°C, whereas the proportion of open and functioning capillaries was essentially unchanged.

Regulation of connective tissue remodeling in the early phase of denervation in a rat skeletal muscle.

Denervation alters the metabolism of the extracellular matrix (ECM) in skeletal muscle; however, the underlying mechanisms of ECM remodeling are not fully understood. The aim of this study was to elucidate the dynamic features of the ECM regulatory process in the early phase of denervated skeletal muscle in male Wistar rats. We investigated the expression of collagens (total, type I, and type III), transforming growth factor beta 1 (TGF-ß1), and matrix metalloproteases (MMPs) together with their endogenous inhibitors (TIMPs), at the mRNA and/or protein level in the soleus muscles of control animals and at days 3, 7, and 14 post-denervation. Expression of mRNA encoding collagens was decreased at days 3 and 7, and had recovered by day 14, in parallel with total collagen protein content. Content of TGF-ß1 protein was elevated sequentially, up to a maximum of 158% at day 14 post-denervation (P < 0.05), as was TIMP-2 mRNA expression (272% at day 14), whereas MMP-1, MMP-2, and TIMP-1 mRNA expression was not affected at any stage. The initial reduction of collagen mRNA may be responsible for hypoactivity caused by the disappearance of contractile function. Recovery of collagen mRNA/protein at day 14 may be due mainly to the suppressive effects of TGF-ß1 on collagen degradation via TIMP-2 upregulation.

Histological findings of uveal capillaries in rabbit eyes after multiple intravitreal injections of bevacizumab.

PURPOSE: To evaluate the potential toxicity of multiple intravitreal injections of bevacizumab on the uveal capillaries of rabbit eyes. MATERIALS AND METHODS: Nine eyes of nine rabbits that received single intravitreal injections of bevacizumab (IVB) constituted the single IVB group, while nine eyes of nine rabbits that received three injections of IVB, with an interval of 28 days between injections, constituted the repeat IVB group. Seven eyes of seven rabbits constituted the control group. The rabbits in the single and repeat IVB groups were sacrificed 7 and 28 d after the single and third IVB injection, respectively. Uveal specimens were compared between groups after immunohistochemical staining. Ultrastructural findings were evaluated by electron microscopy. Control group rabbits were sacrificed 7 d after saline injection. Clinical examination and fundus fluorescein angiography were performed at baseline, 7 d after the first injection, and after the last injection. RESULTS: Differences in the CD31-positive areas of the iris, ciliary body and choroid 7 d after IVB were not statistically significant among the single IVB, repeat IVB and control groups (p = 0.0749, p = 0.7237 and p = 0.7346, respectively; analysis of variance). Endothelial cell fenestrations (ECFs) in the choriocapillaris and ciliary body observed by electron microscopy on day 7 in the single and repeat IVB groups were decreased by 50% (p < 0.0001) and 33% (p < 0.0001), respectively, in both IVB groups compared with those in the control group. However, ECFs observed on day 28 in both groups were comparable to those observed in the control group. CONCLUSIONS: Single IVB and repeated IVB did not have any effect on normal vessel endothelium density as per immunohistochemical findings. Ultrastructural findings revealed that IVB transiently decreased the ECFs in the choriocapillaris and ciliary body.

Articular cartilage repair using an intra-articular magnet and synovium-derived cells.

The purpose of this study was to investigate the chondrogenic potential of magnetically labeled synovium-derived cells (M-SDCs) and examine whether M-SDCs could repair the articular cartilage using an intra-articular magnet after delivery to the lesion. Synovium-derived cells (SDCs) were cultured from the synovium of a rat knee, and were magnetically labeled with ferumoxides. M-SDCs were examined with a transmission electron microscope. A pellet culture system was used to evaluate the chondrogenic potential of M-SDCs in a magnetic field. In a rat model, allogeneic M-SDCs were injected into the knee after we made an osteochondral defect on the patellar groove and implanted an intra-articular magnet at the bottom of the defect. We histologically examined the defects at 48 h, 4 weeks, 8 weeks, and 12 weeks after treatment. Electron microscopy showed the transfection of ferumoxides into SDCs. The pellet cultures revealed the chondrogenic potential of M-SDCs in a magnetic field. M-SDCs accumulated in the osteochondral defect at 48 h after treatment, and we confirmed the regeneration of the articular cartilage at 4 weeks, 8 weeks, and 12 weeks after treatment using an intra-articular magnet. We demonstrated that articular cartilage defects could be repaired using an intra-articular magnet and M-SDCs. We believe that this system will be useful to repair human articular cartilage defects.

Reduced soleus muscle injury at long muscle length during contraction in the rat.

Muscle injury was studied to test the hypotheses that maintaining the soleus muscle at a long muscle length during contraction prevents muscle injuries and that the prevention of initial muscle injuries reduces subsequent muscle damage. The rat sciatic nerve was stimulated for 30 min with plantar or dorsal flexion of the foot, and the time course of contraction-induced injuries was examined. The soleus muscle injuries were first classified into one of five types, and the percentages of aberrant sarcomere areas observed in the soleus muscle were then separately quantified by electron microscopy at 0, 1, 6, 12, and 24 h (n = 3) post-stimulation. At a short muscle length (plantar flexion) during contraction, the soleus muscle showed sarcomere hypercontraction (9.8 ± 2.5%, mean ± standard error) and Z-band disarrangement (31.0 ± 4.5%) at 0 h, sarcomere hypercontraction (6.7 ± 1.9%), Z-band disarrangement (28.0 ± 4.9%), and sarcomere hyperstretching (1.3 ± 1.3%) at 1 h, the absence of sarcomere hypercontraction, but Z-band disarrangement (6.7 ± 1.9%) and sarcomere hyperstretching (5.0 ± 1.8%) at 6 h, and myofilament disorganization at 12 and 24 h (5.2 ± 1.5 and 2.5 ± 1.0%, respectively). In contrast, the soleus muscles at a long muscle length (dorsal flexion) during contraction using a self-made brace showed alterations in 1.2-2.4% of sarcomeres at 0 h and afterwards. Desmin disappeared, and α-actinin immunostaining was weaker in areas of sarcomere hypercontraction, whereas dystrophin was always detected along the sarcoplasmic membrane, suggesting that the integrity of the sarcolemma was intact. These results indicate that initial and subsequent muscle injuries were significantly reduced at long muscle length during contraction, probably through the prevention of sarcomere hypercontraction, and that initial muscle injuries rapidly progress to other injuries or normal structure.

Effect of intravitreal bevacizumab on iris vessels in neovascular glaucoma patients.

BACKGROUND: We aimed to investigate the effects of a single 1-mg injection of intravitreal bevacizumab on iris vessels in neovascular glaucoma (NVG) patients. METHODS: Twenty-two surgically resected irises from glaucoma patients were obtained during trabeculectomy. Eight were from patients with NVG who received a 1-mg injection of intravitreal bevacizumab (IVB) before glaucoma surgery, eight were from patients with primary open-angle glaucoma (POAG), and six were from patients with NVG who were not administered IVB. The collected iris specimens were compared after immunohistochemical staining with anti-CD34 monoclonal antibodies and anti-VEGF monoclonal antibody, and the percentage of CD34-positive and VEGF-positive regions in the total area of the specimens from the three groups was compared. RESULTS: The difference in the CD34-positive area between all groups was statistically significant (p = 0.0061, Kruskal-Wallis test). There was no significant difference in the CD34-positive area between the NVG with IVB group and the POAG group (p = 0.3017, Mann-Whitney U test with Bonferroni correction). The POAG group had significantly fewer CD34-positive regions than the NVG without IVB group (p = 0.0019, Mann-Whitney U test with Bonferroni correction). Many vessels remained in the iris stroma, and there was no significant difference in the CD34-positive area between the NVG with IVB and NVG without IVB groups (p = 0.0357, Mann-Whitney U test with Bonferroni correction). The ratio of the length of CD34 expression on the iris surface in the NVG without IVB group was significantly longer than that in the NVG with IVB group (p = 0.0002, Mann-Whitney U test). The difference in VEGF expression between all groups was statistically significant (p = 0.04, Kruskal-Wallis test). There was no significant difference between the NVG with IVB group and the NVG without IVB group (p = 0.7963 Mann-Whitney U test with Bonferroni correction). The frequency of hyphema and fibrin formation in the anterior chamber 1 day after surgery between the two NVG groups was not statistically significant. CONCLUSION: A single intravitreal dose of IVB at 1 mg/0.04 ml to eyes with rubeotic glaucoma reduced the neovascularization in the human iris surface, but could not eliminate completely neovascularization in iris stroma. This finding implies that the prevention of hyphema and fibrin formation based on the slit-lamp examination can not be predicted, even if neovascularization in iris surface seems to be eliminated by a single dose of IVB.

Regeneration of peripheral nerve after transplantation of CD133+ cells derived from human peripheral blood.

OBJECT: Despite intensive efforts in the field of peripheral nerve injury and regeneration, it remains difficult to achieve full functional recovery in humans following extended peripheral nerve lesions. In this study, the authors examined the use of blood-derived CD133(+) cells in promoting the repair of peripheral nerve defects. METHODS: The authors transplanted phosphate-buffered saline (control), mononuclear cells, or CD133(+) cells embedded in atelocollagen gel into a silicone tube that was used to bridge a 15-mm defect in the sciatic nerve of athymic rats (12 animals in each group). At 8 weeks postsurgery, molecular, histological, and functional evaluations were performed in regenerated tissues. RESULTS: The authors found that sciatic nerves in which a defect had been made were structurally and functionally regenerated within 8 weeks after CD133(+) cell transplantation. From macroscopic evaluation, massive nervelike tissues were confirmed only in rats with CD133(+) cell transplantation compared with the other groups. Morphological regeneration in the samples after CD133(+) cell transplantation, as assessed using toluidine blue staining, was enhanced significantly in terms of the number of myelinated fibers, axon diameter, myelin thickness, and percentage of neural tissue. Compound muscle action potentials were observed only in CD133(+) cell-treated rats. Furthermore, it was demonstrated that the transplanted CD133(+) cells differentiated into Schwann cells by 8 weeks after transplantation. CONCLUSIONS: The results show that CD133(+) cells have potential for enhancement of histological and functional recovery from peripheral nerve injury. This attractive cell source could be purified easily from peripheral blood and could be a feasible autologous candidate for peripheral nerve injuries in the clinical setting.

The skeletal muscle vascular supply closely correlates with the muscle fiber surface area in the rat.

The skeletal muscle capillary supply (capillarity) dynamically changes in response to muscle conditions such as growth, atrophy, and hypertrophy. The capillary number-to-fiber ratio is reported to correlate closely with the muscle fiber cross sectional area. However, little information is available regarding the capillarity of neonatal and very young skeletal muscles. In this study, the vascular endothelium was reliably stained with an anti-PECAM-1 antibody, and relationships between the capillarity and muscle fiber parameters were analyzed. For assessment of the capillarity, we used the capillary length-to-fiber ratio, due to the presence of transversely running vessels. In young and adult rats, the capillary length-to-fiber ratio was proportional to both the muscle fiber cross sectional area and muscle fiber radius. However, when these data were analyzed together with data from neonatal and very young rats, the capillary length-to-fiber ratio correlated more closely with the muscle fiber radius than the muscle fiber cross sectional area in the tibialis anterior muscle. The capillary number-to-fiber ratio demonstrated results very similar to the capillary length-to-fiber ratio. During muscle atrophy after denervation, the number of capillaries was decreased in a non-apoptotic manner as revealed by electron microscopy, maintaining the close relationship between the parameters described above. In conclusion, capillarity was closely correlated with the muscle fiber radius (which represents the perimeter) during growth and atrophy. This indicates that the capillarity is linked to the muscle fiber surface area (which is determined by perimeter and section thickness), in agreement with the essential role of the cell membrane in the transport of materials by simple diffusion or active transport.

Comparison between a weight compression and a magnet compression for experimental pressure ulcers in the rat. Histological studies and effects of anesthesia.

To develop an experimental model and evaluate the effects of the magnitude and duration of pressure, the rat abdominal wall (25x20 mm) was subjected to compression either by a weight or by two magnets. In the weight compression tests, a steel plate was inserted under anesthesia into the rat peritoneal cavity, and the abdominal wall was compressed in situ between the underlying steel plate and a weight placed on the abdominal wall. This method resulted in moderate changes in the subcutaneous connective tissue and muscle at 100 mmHg (13.3 kPa) for 4 h, while some muscle damage was observed at 50 mmHg (6.7 kPa) for 4 h and at 100 mmHg for 2 or 3 h. In the magnet pinching tests, a magnet was inserted into the peritoneal cavity, and another magnet overlaid on the skin. Then the abdominal wall was compressed by the two magnets with or without anesthesia. The compression without anesthesia produced significant edema and injuries of the abdominal wall at 50 mmHg for 4 h and at 100 mmHg for 3 or 4 h, while the injuries incurred at 100 mmHg for 2 h were mild. Susceptibility to pressure was high in the muscle, moderate in the subcutaneous connective tissue, and low in the skin. The compression with anesthesia produced significantly milder injuries than those under anesthesia. These findings indicate that the difference in the extent of injuries between the weight compression and magnet compression models are clearly attributable to the pentobarbital anesthesia induced during the compression. Results therefore show that experimental pressure ulcers should be examined in a waking condition and that magnet compression is a useful model for studying the pathogenesis of pressure ulcers.

Magnetically labeled neural progenitor cells, which are localized by magnetic force, promote axon growth in organotypic cocultures.

STUDY DESIGN: An in vitro, rat animal study was conducted. OBJECTIVE: To assess the corticospinal axon growth potential in varying concentrations of neural progenitor cells (NPCs) and in magnetically localized labeled NPCs, quantitatively using our original organotypic coculture system. SUMMARY OF BACKGROUND DATA: Transplantation of NPCs for spinal cord injury has been anticipated as a possible future treatment. It is important not only to illuminate the mechanism of NPCs for spinal cord injury, but also to develop an effective cell delivery system for clinical use. In order to develop more effective, efficient, and minimally invasive cell delivery systems, we established a new system using magnetic targeting. METHODS: Magnetically labeled NPCs were suspended with activated magnetic beads and individual NPCs, and were compared the characterization to nonlabeled NPCs in vitro. We transplanted varying concentrations of 10, 10, 10, 10, and 10 NPCs in 1 microL medium to coculture models. Then the 10 labeled NPCs were transplanted with or without magnet to the cocultures. RESULTS: Magnetically labeled NPCs had similar potential in axon growth compared with nonlabeled NPCs, so there were few toxic effects of magnetically labeling NPCs. The differential potentials were not changed whether they were localized or scattered in vitro. Corticospinal axon growth was promoted in accordance with the transplanted NPC numbers around the organotypic coculture. Localized labeled NPCs with a magnet promoted axon growth much more than scattered labeled NPCs without a magnet, so magnetically localized labeled NPCs expressed higher potential in axon growth. CONCLUSION: Magnetically labeled NPCs, which were localized by magnetic force, could promote axon growth in this organotypic coculture system.

Role of osteoprotegerin in arterial calcification: development of new animal model.

OBJECTIVES: Enhanced osteoclastogenesis, increased bone resorption, and osteoporosis have been reported in osteoprotegerin-deficient (OPG (-/-)) mice. OPG (-/-) mice available in Japan usually do not show vascular calcification. We have found that arterial calcification can be quickly induced by a simple procedure in OPG (-/-) mice. METHODS AND RESULTS: Male OPG (-/-), OPG (+/-), and OPG (+/+) mice were fed a high phosphate diet from 6 to 10 weeks after birth, and then 1alpha,25-dihydroxyvitamin D3 (calcitriol) was injected for 3 days. We found that severe calcification developed in the media of the aorta in OPG (-/-) mice. Under electron microscopy, calcium deposits were observed in the cytoplasm and extracellular matrix of vascular smooth muscle cells (VSMCs). Neither apoptosis of VSMCs nor infiltration of macrophages was observed. Alkaline phosphatase (ALP) activity of aortic tissue correlated with the calcified lesion area. Mouse aorta and bone extracts revealed an identical pattern by ALP electrophoresis. CONCLUSIONS: Our results demonstrated that OPG had anticalcification activity in the aorta, probably through the downregulation of ALP activity. Because the time course of arterial calcification after the injection of calcitriol is accurate and reproducible, this mouse model will be useful for further investigation of vascular calcification.

Alteration of knee joint connective tissues during contracture formation in spastic rats after an experimentally induced spinal cord injury.

This study was made to elucidate the changes in the periarticular connective tissue that can underlie the contracture after spasticity development. Sixteen Wistar rats underwent a spinal cord injury and 16 rats were either sham- or nonoperated. The periarticular connective tissue of the knee joint was assessed with histological, histomorphometric, immunohistochemical, and biochemical analyses. Histological results showed a smaller synovial intima, a dense subintimal and posterior joint capsule without fibrosis, and a disarranged posterior capsule in the spinal cord-injured knees with the flexion contracture. The synovial intima length was shortened only at the posterior capsule. Neither the distribution nor expression of type I and III collagen was affected. Contractures after spinal cord injuries are characterized by synovial intima adhesions. A dense and disarranged capsule may lead to joint stiffness. The alteration of periarticular connective tissues exhibits properties characteristic of the contracture after spasticity development.

Changes in the rat subcutaneous connective tissue after saline and histamine injection in relation to fluid storage and excretion.

An experimental design was developed for morphometric analysis of the subcutaneous connective tissue after the subcutaneous injection of 0.1 ml of saline or a histamine solution (0.01, 0.1 or 1% histamine dihydrochloride in saline). The subcutaneous connective tissue of 4-week-old rats, originally 170.0 +/- 13.6 mum in thickness, swelled 5.2-fold at 15 min, 3.0-fold at 2 h, and 1.2-fold at 6 h after the injection of saline. The total cross sectional area of both blood and lymphatic vessels increased when compared to that at pre-injection (0.0186 +/- 0.0030 mm(2) in 6-mm-long specimen), 1.4-fold at 15 min, 2.2-fold at 2 h, and 1.1-fold at 6 h post-injection, while the total number of these vessels increased 1.1-fold at 2 h. The total cross sectional area of lymphatic vessels (0.0006 +/- 0.0002 mm(2) in 6-mm-long specimen) alone surged 7.7-fold at 15 min, 4.8-fold at 2 h, and 7.3-fold at 6 h. Collagen fibers were respectively highly, moderately, and mildly disorganized in arrangement at 15 min, 2 h, and 6 h after the saline injection. Histamine elicited an earlier, longer, and more pronounced vasodilatation, particularly at high concentrations. The transvascular permeability of Evans blue increased depending on the concentration of histamine. These findings indicate that the subcutaneous connective tissue has the ability to expand and store a considerable amount of fluid and reversibly returns to normal steady-state conditions by increasing fluid excretion into the blood and lymphatic vessels. It was also strongly suggested that the blood vessels are deeply involved in the excretion and volume regulation of the tissue fluid.

Distribution and change of collagen types I and III and elastin in developing leg muscle in rat.

The distribution of collagen types I and III and elastin in the developing leg muscles were studied by immunohistochemistry in rat. From 0-day to 8-weeks old, the size of the gastrocnemius and plantaris muscles increased. The muscle connective tissue developed in the order of epimysium, perimysium and finally endomysium. The epimysium contained a considerable amount of collagen types I and III and some elastin in the neonates. These components in the epimysium remained almost unchanged in their distribution during development. The perimysium had little collagen type I and III or elastin at 0 day. Collagen type I and elastin slightly increased around 2 and 1 week, respectively, and returned to the previous levels. Collagen type III, however, increased and became abundant after 1 week. In the endomysium, the amounts of collagen type I and elastin were slight during postnatal growth, while collagen type III gradually increased after 2 weeks. The intramuscular tendons consistently showed intense reactivity for collagen type I and weak staining for elastin, whereas the staining for collagen type III decreased after 1 week and was finally restricted to the surface of intramuscular tendons. This study clearly demonstrated that the distribution of collagens, but not of elastin, significantly changed during development. The increase in collagen type III in the perimysium and endomysium, and its decrease in the intramuscular tendons probably reflect functional demands imposed on these connective tissues, i.e., shear forces in the former two and tensile loading in the latter.

Effects of CD44 antibody-- or RGDS peptide--immobilized magnetic beads on cell proliferation and chondrogenesis of mesenchymal stem cells.

We evaluated the efficacy of a novel mesenchymal stem cell (MSC) delivery system using an external magnetic field for cartilage repair in vitro. MSCs were isolated from the bone marrow of Sprague Drawley rats and expanded in a monolayer. To use the MSC delivery system, two types of MSC-magnetic bead complexes were designed and compared. Expanded MSCs were combined with small-sized (diameter: 310 nm) carboxyl group-combined (0.01-0.04 micromol/mg) magnetic beads, Ferri Sphere 100C, through either anti-rat CD44 mouse monoclonal antibodies or a synthetic cell adhesion factor, arginine (R)-glycine (G)-aspartic acid (D)-serine (S) (RGDS) peptide. Both cell complexes were successfully created, and were able to proliferate in monolayer culture up to at least day 7 after separation of magnetic beads from the cell surface, although the proliferation of the complexes was slower in the early period of culture than that of non-labeled rat MSCs (after 7 days of culture: proliferation of CD44 antibody-bead complexes, approximately 50%; RGDS peptide-bead complexes, 70% versus non-labeled rat MSCs, respectively). These complexes were seeded onto culture plates with or without an external magnetic force (magnetic flux density was 0.20 Tesla at a distance of 2 mm from plate base) generated by a neodymium magnet, and supplemented with chondrogenic differentiation medium. Both complexes could be attached and gathered effectively under the influence of the external magnet, and CD44-bead complexes could effectively generate chondrogenic matrix in monolayer culture. In a three-dimensional culture system, the production of a dense chondrogenic matrix and the expression of type II collagen and aggrecan mRNA were detected in both complexes, and the chondrogenic potential of these complexes was only a little less than that of rat MSCs alone. Thus, we conclude that due to the fact that MSC-RGDS peptide-bead complexes are composed using a biodegradable material, RGDS peptide, as a mediator, the RGDS peptide-bead complex is more useful for minimally invasive clinical applications using our design of magnetic MSC delivery system than CD44 antibody-beads.