Fatty degeneration and wnt10b expression in the supraspinatus muscle after surgical repair of torn rotator cuff tendon.

PURPOSE: In the torn rotator cuff muscles, decreased expression of wnt10b prior to elevation of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) has previously been reported. The purpose of this study is to elucidate the expression profiles of these adipogenesis-related genes after rotator cuff detachment and reattachment in a rabbit model. METHODS: We investigated gene expression profiles of PPARγ, C/EBPα, and wnt10b in different parts of rabbit supraspinatus (SSP) muscle after tendon detachment (n = 6 for each time point). In addition, we assessed expression of the same genes after SSP reattachment with different intervals from initial detachment (n = 6). Fatty degeneration of the SSP muscle was examined by Oil red-O staining. Gene expression profiles were examined by quantitative real-time polymerase chain reaction. RESULTS: After SSP detachment, Oil red-O-positive oil deposits increased after 3 weeks. In the SSP reattachment model, numerous Oil red-O-positive cells were present at 5-week reattachment, following 2- and 3-week detachment. PPARγ and C/EBPα messenger ribonucleic acid expression exhibited a significant increase at 2 and 3 weeks after SSP detachment and remained increased at 5-week reattachment after 2- and 3-week detachment. A decreased expression of wnt10b was observed from 1 week after SSP detachment. Expression of wnt10b was recovered not in the central area of the SSP muscle but in the periphery after reattachment. Adipogenic change was not observed when SSP tendon was reattached after 1-week detachment. CONCLUSIONS: These results may suggest that once the adipogenic transcription factors, PPARγ and C/EBPα, were elevated, repair surgery after rotator cuff tear could not prevent the emergence of fat in the SSP muscle.

A vitamin D analogue, eldecalcitol, enhances expression of fast myosin heavy chain subtypes in differentiated C2C12 myoblasts.

BACKGROUND: Several lines of evidence indicate that the active form of vitamin D has an anabolic effect on skeletal muscle. Eldecalcitol, an analogue of the active form of vitamin D, has the potential to increase bone density and decrease fracture risk. The objective of this study was to investigate the effect of eldecalcitol in C2C12 myogenic cells. METHODS: C2C12 cells were grown to confluency and the culture medium was replaced with low-glucose DMEM containing 2% horse serum. Eldecalcitol was added at a concentration of 1, 10 or 100 nM. Gene expression profiles of vitamin D receptor (VDR), MyoD, IGF-1, neonatal myosin heavy chain (MHC), and the fast MHC subtypes Ia, IIa, IIb and IId/x were analyzed by quantitative RT-PCR. Protein expression of MHC subtypes was evaluated by western blotting and immunostaining. RESULTS: Eldecalcitol upregulated gene expression of VDR, MyoD and IGF-1. Incubation with eldecalcitol in the absence of serum followed by the addition of serum after 1 h was associated with greater increases in the expression of these genes compared with co-incubation with eldecalcitol and serum. Gene expression of MHC subtypes IIa, IIb and IId/x was significantly increased by eldecalcitol. Protein expression of fast MHC subtypes was significantly increased by eldecalcitol at 1 and 10 nM. CONCLUSION: Similar to the active form of vitamin D, eldecalcitol had an anabolic effect on fast MHC subtypes. Taking into account its pharmacokinetic profile, eldecalcitol is expected to be beneficial for the maintenance and improvement of muscle function in elderly individuals.

Transfer of the bone morphogenetic protein 4 gene into rat periodontal ligament by in vivo electroporation.

OBJECTIVE: Regulation of alveolar bone metabolism is required in clinical dentistry. The aim of the present study was to establish a method for gene transfer into the periodontal ligament (PDL) by in vivo electroporation with a plasmid vector and to investigate the effects of BMP-4 transfer into the PDL. DESIGN: Plasmids containing mouse BMP-4 cDNA (pCAGGS-BMP4) were transfected into cultured rat PDL cells by in vitro electroporation, and BMP-4 production and secretion were detected by immunocytochemistry and western blotting. Next, pCAGGS-BMP4 was injected into the PDL of rats, and electroporation was performed in vivo, using original paired-needle electrodes. BMP-4 expression was examined by immunohistochemical staining 3, 7, 14, 21, and 28days after electroporation. Control groups were injected with pCAGGS by electroporation, injected with pCAGGS-BMP4 without electroporation, or subjected to neither injection nor electroporation. RESULTS: In vitro-transfected rat PDL cells exhibited production and secretion of the mature-form BMP-4. After in vivo electroporation of pCAGGS-BMP4, site-specific BMP-4 expression peaked on day 3, gradually decreased until day 14, and was absent by day 21. We observed no unfavorable effects such as inflammation, degeneration, or necrosis. CONCLUSIONS: Gene transfer by electroporation with plasmid DNA vectors has several advantages over other methods, including the non-viral vector, non-immunogenic effects, site-specific expression, simplicity, cost-effectiveness, and limited histological side effects. Our results indicate that the method is useful for gene therapy targeting the periodontal tissue, which regulates alveolar bone remodeling.

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.

Oxygen tension affects lubricin expression in chondrocytes.

We assessed the effects of oxygen tension on lubricin expression in bovine chondrocytes and cartilage explants and a role for hypoxia-inducible transcription factor (HIF)-1α in regulating lubricin expression was investigated using a murine chondroprogenitor cell line, ATDC5, and bovine chondrocytes isolated from superficial and middle/deep zones of femoral cartilage. ATDC5 cells and bovine chondrocytes were cultured in micromass under different oxygen tensions (21%, 5%, and 1%). ATDC5 cells and middle/deep zone chondrocytes that initially had low lubricin expression levels were also cultured with or without transforming growth factor (TGF)-ß1. Quantitative reverse transcription (RT)-PCR was used to determine lubricin and chondrogenic marker gene mRNA levels and immunohistochemistry was used to assess lubricin protein expression. Explant cartilage plugs cultured under different oxygen tensions were also subjected to immunohistological analysis for lubricin. HIF-1α gene silencing was achieved by electroporatic transfer into ATDC5 cells. A low oxygen tension reduced lubricin gene expression levels in bovine superficial chondrocytes, TGF-ß1-treated middle/deep zone chondrocytes, and TGF-ß1-treated ATDC5 cells. Lubricin expression in explant cartilage was also suppressed under hypoxia. HIF-1α gene silencing in ATDC5 cells attenuated the lubricin expression response to the oxygen tension. These results corroborate with previous studies that the oxygen tension regulates lubricin gene expression and suggest that HIF-1α plays an important role in this regulation. The normal distribution of lubricin in articular cartilage may be due to the hypoxic oxygen environment of cartilage as it is an avascular tissue. An oxygen tension gradient may be a key factor for engineering cartilage tissue with a layered morphology.

Muscle activity pattern of the shoulder external rotators differs in adduction and abduction: an analysis using positron emission tomography.

BACKGROUND: The muscle activity pattern during shoulder external rotation has not been fully clarified. This study aimed to determine the activities involved in external rotation in the adducted and abducted positions using positron emission tomography (PET). METHODS: Seven healthy volunteers underwent PET examinations after performing external rotation using an elastic band at both 0° and 90° of shoulder abduction in the frontal plane. External rotation exercise was performed before and after injection of fluorine 18 fluorodeoxyglucose, which was followed by PET examination. The protocols for external rotation exercise were identical between the 2 shoulder positions. To obtain control data, PET examination was also performed under resting conditions. The order of these 3 PET examinations was randomized, and they were performed at intervals of 1 week or greater. Each PET image was fused to the corresponding magnetic resonance image to identify each shoulder muscle. After this, the standardized uptake value was calculated in each muscle and was compared between the 2 shoulder positions. RESULTS: The infraspinatus showed the greatest muscle activity during external rotation at 0° of abduction, whereas the teres minor showed the greatest activity at 90° of abduction. The teres minor-infraspinatus ratio at 90° of abduction (mean ± SD, 1.21 ± 0.23) was significantly higher than that at 0° of abduction (0.84 ± 0.15) (P < .01). CONCLUSION: The infraspinatus and teres minor are the main shoulder external rotators. The teres minor is more important as an external rotator in abduction than in adduction.

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.

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.

Vitamin D receptor gene silencing effects on differentiation of myogenic cell lines.

INTRODUCTION: The active form of vitamin D (1,25-dihydroxy-vitamin D3) is known to increase fast-type myosin heavy chain expression in differentiated myogenic cell lines. The mechanisms for this effect are not fully understood. The aim of this study was to determine the role of signals transmitted through the vitamin D receptor (VDR) during differentiation of myoblasts. METHODS: Electroporation was used to introduce VDR siRNA molecules into C2C12 and G8 murine myoblast cell lines. Gene and protein expression profiles of VDR-gene silenced cells were analyzed in vitro. RESULTS: Suppressing VDR expression by RNA interference resulted in inhibition of myogenic differentiation of C2C12 and G8 cell lines at both mRNA and protein levels. CONCLUSIONS: Our results suggest that myoblasts require signals transmitted through VDR for differentiation into myocytes and emphasize the importance of VDR expression in skeletal muscles for maintaining muscle volume in the elderly.

Lithium chloride enhances cathepsin H expression and BMP-4 degradation in C3H10T1/2 cells.

The effect of canonical Wnt/ß-catenin signaling on chondrogenic differentiation induced by transfection of BMP4 expressing plasmid was analyzed. Lithium chloride (LiCl) which mimics canonical Wnt/ß-catenin signaling was added to cells transfected with BMP4 expressing plasmid. Although BMP4 mRNA expression was not affected by LiCl, LiCl decreased BMP4 protein accumulation. Gene expression analysis exhibited upregulation of cathepsin H by LiCl treatment. Gene silencing of cathepsin H enhanced BMP4 protein accumulation from BMP4 expressing cells. These results suggested that cathepsin H is regulated by Wnt/ß-catenin signaling and plays an important role in the regulation of BMP4 biological activity.

Effects of alfacalcidol on back extensor strength gained through back extensor exercise in postmenopausal women with osteoporosis.

OBJECTIVE: The aim of this study was to investigate the additive effect of the active form of vitamin D3 on the gain in back extensor strength through a back extensor exercise. DESIGN: A total of 107 postmenopausal women with osteoporosis were randomly divided into two groups: the D3 group and the control group. Both groups were treated with calcium and alendronate and undertook the back extensor exercise. Alfacalcidol was prescribed only to the D3 group. RESULTS: There was no significant difference in the demographic data between the two groups. Ninety-four participants who completed a 4-mo intervention were subjected to per-protocol analysis. There was no significant difference in the improvement in back extensor strength between two the groups (P = 0.349). All subjects were further categorized into two subgroups by age. In the older subgroup (≥68 yrs), no significant difference was found in the improvement in back extensor strength (P = 0.316). In the younger subgroup (<68 yrs), the back extensor strength in the D3 group was significantly more improved than in the control group (P = 0.034). CONCLUSIONS: The results of this study suggest that the administration of the active form of vitamin D3 enhances the beneficial effects of the back extensor exercise in patients younger than those in their late 60s.

Nicotine reduced MMP-9 expression in the primary porcine tenocytes exposed to cyclic stretch.

Nicotine is one of the major chemical components of the cigarette smoke, which has been known as a risk factor for tendon ruptures including rotator cuff tears. This study investigated the effect of nicotine on tenocytes under cyclic-stretched condition. Particularly, we focused on the morphologic changes of tenocytes and their expression of MMPs. Primary porcine tenocytes were obtained from the infraspinatus tendon. The cells were cultured on elastic chambers under static or cyclic-stretched condition for 24 h in the existence of nicotine (0, 1, 10, and 100 µM). Cell shape, gene expression of collagen type I and III, MMPs (-1, -2, -3, -9, and -13) and TIMPs (-1, -2, and -3) and enzyme activity of MMP-9 were analyzed using immunohistochemistry, RT-PCR, and zymography. Tenocytes exposed to nicotine represented significantly decreased gene expressions in MMP-9 (p < 0.001) and TIMP-3 (p < 0.05) under the cyclic stretch. Enzymatic activity of MMP-9 was also reduced by nicotine exposure in a dose-dependent manner (p < 0.001). The down-regulation of MMP and TIMP expression by nicotine shown in our in vitro experiment might deteriorate normal metabolism of the tendon. These mechanisms might affect the mechanical properties of the extracellular matrix of the rotator cuff tendon.

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.

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.

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, nicotine caused a change in the material properties of the supraspinatus tendon. This change may predispose to a tear in the supraspinatus tendon.

Hypoxia induces adipogenic differentitation of myoblastic cell lines.

Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) beta, alpha and peroxisome proliferator activating receptor (PPAR) gamma were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

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

Although 2-deoxy-2-[(18)F]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.

Effect of low-intensity pulsed ultrasound on scaffold-free ectopic bone formation in skeletal muscle.

BACKGROUND: Low-intensity pulsed ultrasound (LIPUS) is reported to have the effects of rapid appearance and early maturation of ossification in animal models. METHOD: We examined the influence of LIPUS on bone formation in C57BL/6J mouse muscle induced by gene transfer of BMP-4 expression plasmid. Electroporation was employed to transfer plasmid DNA. First, an in vitro study was carried out to confirm that LIPUS has no effect on the forced expression of BMP-4 gene transferred by electroporation into C2C12 cells. Next, the BMP-4 plasmids were injected into mouse calf muscles, and transcutaneous electroporation was applied. LIPUS (30 mW/cm(2)) exposure was performed daily for 20 minutes on one side of hind limbs (LIPUS side). The contralateral limbs were not exposed to LIPUS (control side). Muscle samples were collected at 7, 10, 14, and 21 days after electroporation. Soft X-ray films of muscles were taken, and areas of bone formation were measured. After pepsin solubilization of the muscles, calcium and total collagen content were measured. RESULTS: Radiographical measurements showed significantly more bone formation in the LIPUS side at Day 10. The area of bone was the maximum in both sides at Day 14. The LIPUS side exhibited significant increase in the calcium content at Day 10. The total collagen content with LIPUS exposure was increased significantly over control at Day 10 and 21. CONCLUSIONS: According to these results, accelerated maturation of ectopic bone formation by LIPUS was confirmed at Day 10. Moreover, our results showed that LIPUS increases the total collagen content during osteogenesis.

Stimulation of the side population fraction of ATDC5 chondroprogenitors by hypoxia.

The influence of oxygen tension on the side population (SP) fraction sorted from ATDC5 chondroprogenitor cells was investigated. ATDC5 cells cultured in normoxia (20%) or hypoxia (1% oxygen) were compared. The SP fraction was significantly higher in the cells cultured in hypoxia. The gene expression of 3 ABC transporters, abcb1a/b (mdr1a/b) and abcg2 (bcrp1) was quantified by RT-PCR. SP cells were characterized by the higher expression of abcb1a. The expression levels of abcb1b and abcg2 were higher than abcb1a. However, there was no significant difference between SP and non-SP fractions in the expression of abcb1b and abcg2. The telomeric repeat amplification protocol assay showed that SP cells tended to show lower telomerase activity than non-SP cells. Chondrogenic properties of ATDC5 cells derived from SP or non-SP were assessed by micromass culture. There were not significant differences between SP and non-SP derived cells in Alcian blue staining and sox9, Aggrecan, Col2a1 and SZP mRNA expression. The results demonstrate that the SP fraction was stimulated by hypoxia and chondrogenic properties of SP and non-SP fraction of ATDC5 cells were similar.