Koshi N. Kishimoto

Effect of partial hydrolysis of octacalcium phosphate on its osteoconductive characteristics.

The present study was designed to investigate whether the stoichiometry of octacalcium phosphate OCP affects its osteoconductive and immune response characteristics in rat bone marrow. Those characteristics of synthetic, well-grown OCP but with a non-stoichiometric composition were compared with those of a slightly hydrolyzed OCP (low crystalline OCP: LC-OCP), the fully hydrolyzed apatitic product of OCP or biodegradable beta-tricalcium phosphate (beta-TCP) ceramic, by their implantation in rat tibia for 56 days. The physicochemical aspect of implants and biological responses were analyzed by X-ray diffraction, histomorphometry, immunohistochemistry and expression of mRNA around the implants. The remarkable findings were that: (1) the highest bone formation rate was obtained for beta-TCP whereas the lowest for LC-OCP at Day 14; (2) the rates were reversed and reached the highest for LC-OCP until Day 56; (3) the early expression of ostoeoclast markers TRAP and cathepsin-K was suppressed with LC-OCP; (4) the expression of inflammatory markers IL-beta1 and TNF-alpha was suppressed with LC-OCP. The results confirmed that the partially hydrolyzed OCP with Ca/P molar ratio 1.37 (LC-OCP) enhances bone formation most, suppressing early osteoclast activity and reducing inflammation.

Ectopic Bone Formation by Electroporatic Transfer of Bone Morphogenetic Protein-4 Gene

Orthopedic surgeons have long awaited the clinical application of bone morphogenetic proteins (BMPs) for bone regeneration. However, such possible applications involving proteins or genes transferred with virus vectors have encountered many problems, including high cost, immunological reactions, viral infection, etc. We adopted a new gene transfer system of in vivo electroporation with a plasmid expression vector. A solution of plasmid DNA containing mouse BMP-4 (pMiw-BMP4) was injected into the gastrocnemius of BALB/cA mice, and electric pulses were applied through paired-needle electrodes inserted percutaneously. As a control plasmid, LacZ-containing plasmid (pMiwZ) was transferred by electroporation. A control group in which pMiw-BMP4 was injected and not electroporated was also introduced. In these groups, the gastrocnemius was harvested at 7, 14, 21, and 28 days after electroporation (n = 6 in each). As nonplasmid controls, electroporation with saline injection (n = 6), electroporation without injection (n = 6), and saline injection only (n = 3) were prepared. In these groups, the mice were killed 7 days after experimentation. Ectopic calcification or ossification was examined by histology as well as soft X-ray. In all electroporated groups (pMiwZ, pMiw-BMP4, saline injection, and without injection), dystrophic calcification of muscle bundles and infiltration of mesenchymal cells were observed histologically. Ectopic bone formation was observed only in the pMiw-BMP4 electroporation group. At 7 days after pMiw-BMP4 electroporation, extracellular eosinophilic matrix in a collection of mesenchymal cells was observed. Between 14 and 28 days after electroporation, ectopic bone was observed in 44% of mice, and bone marrow-like cells observed in 22%. The newly formed bone was woven. Injection of pMiw-BMP4 or saline induced neither calcification nor ossification. Our findings indicate that BMP-4 transferred by electroporation can induce in vivo and in situ ectopic bone formation in skeletal muscle.

Molecular mechanism of fatty degeneration in rotator cuff muscle with tendon rupture

Fatty degeneration often occurs in rotator cuff muscle with tendon rupture. However, the molecular mechanism underlying this change has not been fully clarified yet. We investigated the gene expression of Wnt10b and adipogenic marker gene, PPARγ and C/EBPα in C2C12 myogenic cell line under inhibition of Wnt10b by adipogenic induction medium, isobutylmethylxanthine, dexamethasone, and insulin (MDI). The role of Wnt‐signal was confirmed by adding Lithium chloride (LiCl), which mimics Wnt signaling to the cultured cell with MDI. We also assessed the expression profiles of same genes in the rat rotator cuff tear model in vivo. MDI induced Oil red‐O staining positive adipocytes and upregulated PPARγ and C/EBPα expression. LiCl inhibited adipogenic induction of MDI. Rotator cuff muscle with tendon rupture showed positive staining for Oil red‐O. Real‐time polymerase chain reaction analyses revealed decreased expression of Wnt10b followed by increased PPARγ and C/EBPα gene expression in the supraspinatus muscle. Fatty degeneration and its molecular events were remarkably seen in the distal one‐third of the detached supraspinatus muscle versus control. Wnt signaling may regulate adipogenic differentiation both in the myoblasts in vitro and the muscle in vivo. Our results indicate that the reduction of Wnt10b in muscle with a rotator cuff tear is a key signal in fatty degeneration of the muscle.

Effect of addition of hyaluronic acids on the osteoconductivity and biodegradability of synthetic octacalcium phosphate.

The present study was designed to investigate whether three sodium hyaluronic acid (HyA) medical products, Artz(®), Suvenyl(®) and a chemically modified derivative of sodium HyA Synvisc(®), can be used as suitable vehicles for an osteoconductive octacalcium phosphate (OCP). OCP granules (300-500 μm diameter) were mixed with these sodium HyAs with molecular weights of 90 × 10(4) (Artz(®)), 190 × 10(4) (Suvenyl(®)) and 600 × 10(4) (Synvisc(®)) (referred to as HyA90, HyA190 and HyA600, respectively). OCP-HyA composites were injected using a syringe into a polytetrafluoroethylene ring, placed on the subperiosteal region of mouse calvaria for 3 and 6 weeks, and then bone formation was assessed by histomorphometry. The capacity of the HyAs for osteoclast formation from RAW264 cells with RANKL was examined by TRAP staining in vitro. Bone formation was enhanced by the OCP composites with HyA90 and HyA600, compared to OCP alone, through enhanced osteoclastic resorption of OCP. HyA90 and HyA600 facilitated in vitro osteoclast formation. The results suggest that the osteoconductive property of OCP was accelerated by the HyAs-associated osteoclastic resorption of OCP, and therefore that HyA/OCP composites are attractive bone substitutes which are injectable and bioactive materials.

Low-energy extracorporeal shock wave therapy promotes vascular endothelial growth factor expression and improves locomotor recovery after spinal cord injury

OBJECT Extracorporeal shock wave therapy (ESWT) is widely used for the clinical treatment of various human diseases. Recent studies have demonstrated that low-energy ESWT upregulates the expression of vascular endothelial growth factor (VEGF) and promotes angiogenesis and functional recovery in myocardial infarction and peripheral artery disease. Many previous reports suggested that VEGF produces a neuroprotective effect to reduce secondary neural tissue damage after spinal cord injury (SCI). The purpose of the present study was to investigate whether low-energy ESWT promotes VEGF expression and neuroprotection and improves locomotor recovery after SCI. METHODS Sixty adult female Sprague-Dawley rats were randomly divided into 4 groups: sham group (laminectomy only), sham-SW group (low-energy ESWT applied after laminectomy), SCI group (SCI only), and SCI-SW group (low-energy ESWT applied after SCI). Thoracic spinal cord contusion injury was inflicted using an impactor. Low-energy ESWT was applied to the injured spinal cord 3 times a week for 3 weeks. Locomotor function was evaluated using the Basso, Beattie, and Bresnahan (BBB) Scale (open field locomotor score) at different time points over 42 days after SCI. Hematoxylin and eosin staining was performed to assess neural tissue damage in the spinal cord. Neuronal loss was investigated by immunostaining for NeuN. The mRNA expressions of VEGF and its receptor, Flt-1, in the spinal cord were assessed using real-time polymerase chain reaction. Immunostaining for VEGF was performed to evaluate VEGF protein expression in the spinal cord. RESULTS In both the sham and sham-SW groups, no animals showed locomotor impairment on BBB scoring. Histological analysis of H & E and NeuN stainings in the sham-SW group confirmed that no neural tissue damage was induced by the low-energy ESWT. Importantly, animals in the SCI-SW group demonstrated significantly better locomotor improvement than those in the SCI group at 7, 35, and 42 days after injury (p < 0.05). The number of NeuN-positive cells in the SCI-SW group was significantly higher than that in the SCI group at 42 days after injury (p < 0.05). In addition, mRNA expressions of VEGF and Flt-1 were significantly increased in the SCI-SW group compared with the SCI group at 7 days after injury (p < 0.05). The expression of VEGF protein in the SCI-SW group was significantly higher than that in the SCI group at 7 days (p < 0.01). CONCLUSIONS The present study showed that low-energy ESWT significantly increased expressions of VEGF and Flt-1 in the spinal cord without any detrimental effect. Furthermore, it significantly reduced neuronal loss in damaged neural tissue and improved locomotor function after SCI. These results suggested that low-energy ESWT enhances the neuroprotective effect of VEGF in reducing secondary injury and leads to better locomotor recovery following SCI. This study provides the first evidence that low-energy ESWT can be a safe and promising therapeutic strategy for SCI.

1α,25-dihydroxyvitamin D3 enhances fast-myosin heavy chain expression in differentiated C2C12 myoblasts

We investigated the effect of VD3 (1α,25‐dihydroxyvitamin D3) on the proliferating, differentiating and differentiated phases of C2C12 myoblasts, a mouse skeletal muscle cell line. VD3 treatment in 10% FBS (fetal bovine serum) inhibited the proliferation and viability of the cells in a dose‐dependent manner. It also dose‐dependently increased the percentage of cells in the G0/G1 phase as shown by flow cytometry. In the differentiating phase, VD3 treatment inhibited the formation of myotubes and the expression of total myosin heavy chain at both the mRNA and protein levels. In the differentiated phase, treatment had no significant effect on the amount of total myosin heavy chain, as Western blot analysis with MF20 antibody [DSHB (Developmental Studies Hybridoma Bank)] showed. However, significantly greater expression of fast myosin heavy chain in 1 nM VD3 was found by Western blot analysis with MY‐32 (Sigma). Thus VD3 inhibited the proliferation of myoblasts during proliferating and differentiating phases, whereas it increased the expression of the fast myosin heavy chain isoform in the differentiated phase. The data indicate that an adequate concentration of VD3 might have an anabolic effect on differentiated skeletal muscle.

Function of the shoulder muscles during arm elevation: an assessment using positron emission tomography

Although 2‐deoxy‐2‐[18F]fluoro‐D‐glucose (FDG) positron emission tomography (PET) has been used for the assessment of skeletal muscle activities, its application to the shoulder muscles is only sparse. The purpose of this study was to investigate the activities of the shoulder muscles during arm elevation using PET. Six healthy volunteers performed an arm elevation exercise before and after FDG injection. The exercise consisted of 200 repetitions of arm elevation in the scapular plane with a 0.25‐kg weight fixed to the wrist on both arms. PET examination was performed 50 min after FDG injection. For control data, PET scan was repeated for each subject on a separate day without any exercise. The volume of interest was established for each shoulder muscle. The subscapularis was divided into three portions (superior, middle, and inferior). The standardized uptake value (SUV) was calculated in each muscle to quantify its activity. The SUVs increased significantly after exercise in the deltoid, supraspinatus, and the superior portion of subscapularis. Among three divided portions of the subscapularis, the SUV of the superior one‐third was significantly greater than the rest of the muscle after exercise. Our current study clearly indicated that there were two functionally different portions in the subscapularis muscle and the superior one‐third played an important role during arm elevation in the scapular plane.

Alteration of the material properties of the normal supraspinatus tendon by nicotine treatment in a rat model

Background and purpose Several studies have shown that nicotine has a detrimental effect on the development of rotator cuff tear. However, little is known about its mechanism. We evaluated the effect of nicotine on the maximum tensile load, the maximum tensile stress, and the elastic modulus of the supraspinatus tendon in a rat model. Methods 27 rats were randomly assigned to 3 groups. Subcutaneously implanted osmotic pumps delivered two different concentrations of nicotine solution (high dose: 45 ng/mL; low dose: 22.5 ng/mL) or saline solution (controls) over a 12-week period. The level of serum cotinine, a breakdown product of nicotine, was evaluated. We performed tensile testing using the left supraspinatus tendon in each rat. The maximum load of the supraspinatus tendon was measured, and the maximum tensile stress and elastic modulus were calculated. Results Serum cotinine levels showed controlled systemic release of nicotine. The maximum tensile load and stress were similar in the three groups. The elastic modulus was, however, higher in the nicotine groups than in the control group. Interpretation In a rat model, ncotine caused a change in the material properties of the supraspinatus tendon. This change may predispose to a tear in the supraspinatus tendon.

Differences in muscle activities during shoulder elevation in patients with symptomatic and asymptomatic rotator cuff tears: analysis by positron emission tomography

BACKGROUND Differences in muscle activity patterns between patients with symptomatic and asymptomatic full-thickness rotator cuff tears have not yet been fully clarified. The purpose of this study was to investigate the muscle activity pattern by use of positron emission tomography (PET) in patients with symptomatic and asymptomatic rotator cuff tears. METHODS Ten shoulders of 9 patients with full-thickness rotator cuff tears were divided into 2 groups by a numerical pain rating scale (0-10), symptomatic (≥2) and asymptomatic (0 or 1), with 5 shoulders each. Scaption exercise of bilateral arms (200 repetitions in 10 minutes) with a weight of 0.25 kg each was performed before and after injection of fluorodeoxyglucose. After PET examination, the standardized uptake value of each muscle was calculated to quantify its activity and compared between the two groups. RESULTS The activity of the anterior and middle deltoid was significantly decreased in the symptomatic group compared with the asymptomatic group (anterior deltoid, P = .02; middle deltoid, P = .03). In contrast, the activity of the superior trapezius was significantly increased in the symptomatic group compared with the asymptomatic group (P = .02). CONCLUSION In patients with a symptomatic tear, the deltoid activity was decreased and the trapezius activity was increased. It is likely that they might have moved the painful glenohumeral joint less and instead moved the painless scapulothoracic joint more during the prescribed exercise. We conclude that patients with painful rotator cuff tears use the parascapular muscles more than those without pain do during arm elevation.

Gene Expression Analysis of Ectopic Bone Formation Induced by Electroporatic Gene Transfer of BMP4

Implantation of bone morphogenetic protein (BMP) using a carrier or by BMP gene transfer into rodent muscle can induce bone formation. Implanted BMP becomes bioactive immediately after implantation. In BMP gene transfer, there is a time-lag between the secretion of gene products and bone formation. We analyzed the gene expression of chondrogenic and osteogenic specific markers in the process of ectopic bone formation by using semi-quantitative RT-PCR. A plasmid vector containing mouse BMP4 gene (pCAGGS-BMP4) was transferred into the gastrocnemius muscles of mice using electroporation. Histological examination revealed the process of endochondral bone formation in the pCAGGS-BMP4 transferred muscles. As chondrogenic markers, aggrecan gene maximal expression was detected on day 7 and decreased by day 14, and for collagen X the gene maximal expression was on day 10. As osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP) and osteopontin (OPN) gene expressions were clearly detected from day 10 and then increased by day 14. In conclusion, the present study proved that ectopic bone formation by BMP4 gene transfer into the muscle induced endochondral ossification that corresponded well with that to that by implantation of demineralized bone matrix.