In vivo study on the repair of rat Achilles tendon injury treated with non-thermal atmospheric-pressure helium microplasma jet.

Non-thermal atmospheric-pressure plasma (NTAPP) has been widely studied for clinical applications, e.g., disinfection, wound healing, cancer therapy, hemostasis, and bone regeneration. It is being revealed that the physical and chemical actions of plasma have enabled these clinical applications. Based on our previous report regarding plasma-stimulated bone regeneration, this study focused on Achilles tendon repair by NTAPP. This is the first study to reveal that exposure to NTAPP can accelerate Achilles tendon repair using a well-established Achilles tendon injury rat model. Histological evaluation using the Stoll's and histological scores showed a significant improvement at 2 and 4 weeks, with type I collagen content being substantial at the early time point of 2 weeks post-surgery. Notably, the replacement of type III collagen with type I collagen occurred more frequently in the plasma-treated groups at the early stage of repair. Tensile strength test results showed that the maximum breaking strength in the plasma-treated group at two weeks was significantly higher than that in the untreated group. Overall, our results indicate that a single event of NTAPP treatment during the surgery can contribute to an early recovery of an injured tendon.

Bone mineral density around cementless short stems after reverse shoulder arthroplasty: changes over time and its relationship to stem positioning.

Background: There are numerous reports of bone adaptation observed on plain radiography studies after the use of cementless short stems in reverse shoulder arthroplasty. However, reports on changes in bone mineral density (BMD) around the stem using dual-energy X-ray absorptiometry (DEXA) are prominently absent. In the present study, we measured BMD around the stem using DEXA and investigated changes over time from postoperative year 1 to year 2. Additionally, the relationship between BMD changes, filling ratio, and stem alignment was examined. Methods: Forty-seven patients with short cementless stems who could be assessed via DEXA at 1-2 years postoperatively were included. After dividing the zones around the stem into 5, the BMD in each zone was measured, in addition to BMD changes and amount of change from postoperative year 1 to year 2. The relationship between filling ratio and stem alignment on postoperative plain radiography was assessed. Results: A significant decrease in BMD in zone 3 was observed between postoperative year 1 and year 2 (P = .02). Regarding filling ratio and stem alignment, a negative correlation between valgus stem alignment and BMD change observed in zone 1 (r = -0.470, P < .01). In addition, stem valgus greater than 6° correlated with a significant decrease in BMD in zone 1. (P = .01). No significant differences were found in the other zones. Furthermore, there was no correlation between filling ratio and BMD change. Conclusion: In reverse shoulder arthroplasty cementless short stems, changes that that occurred between postoperative year 1 to year 2 demonstrated a decrease in BMD in zone 3. In addition, a decrease in BMD in zone 1 was observed with a stem alignment of valgus 6° or higher, suggesting that stem alignment within valgus 6° is required to prevent a decrease in BMD.

Comparison of cost, surgical time, and clinical results between arthroscopic transosseous rotator cuff repair with lateral cortical augmentation and arthroscopic transosseous equivalent suture bridge: A propensity score-matched analysis.

BACKGROUND: To reduce the healthcare burden, the clinical results of arthroscopic rotator cuff repair and the cost of the implants used have recently been focused upon. This study compared implant cost, surgical time, short-term clinical results, and cuff repair integrity 2 years postoperatively between arthroscopic transosseous rotator cuff repair using lateral cortical augmentation (TOA) and arthroscopic transosseous-equivalent suture bridge (TOE). METHODS: This study included 220 patients with rotator cuff repairs performed by a single surgeon between December 2013 and December 2018. Overall, 70 TOA and 68 TOE cases met the inclusion criteria. The same surgeon performed the procedures at two different hospitals, and the techniques differed between the facilities. A total of 42 TOA patients were matched with 42 TOE patients. The patients were matched using a propensity score analysis by gender, age, and cuff tear size. The minimum follow-up period was 2 years. Implant cost and surgical time were compared between the two methods. The range of motion, clinical outcomes, and visual analog scale were evaluated. Magnetic resonance imaging was performed to examine cuff repair integrity 2 years postoperatively. RESULTS: The follow-up rate was 81% (112/138 patients). Implant cost was significantly lower with TOA ($1,396 vs. $2,165; p < 0.001) than with TOE. The average surgical time in the TOA method was significantly shorter than that in the TOE method (82 vs. 109 min; p = 0.001). At a minimum 2-year follow-up, the mean active elevation, abduction, and clinical outcomes improved with both methods, although no improvements in external and internal rotations were observed with either method. There were no significant differences in the postoperative variables and retear rate (TOA, 12%; TOE, 19%; p = 0.548) between the two methods. CONCLUSIONS: TOA and TOE achieved comparable clinical results; however, TOA was more cost-effective and had a shorter surgical time than TOE. LEVEL OF EVIDENCE: Level Ⅲ, retrospective matched control study.

Impact of constrained humeral liner on impingement-free range of motion and impingement type in reverse shoulder arthroplasty using a computer simulation.

BACKGROUND: Dislocation is a major complication of reverse total shoulder arthroplasty (RSA). The humeral liner may be changed to a constrained type when stability does not improve by increasing glenosphere size or lateralization with implants, and patients, particularly women with obesity, have risks of periprosthetic instability that may be secondary to hinge adduction on the thorax, but there are few reports on its impact on the range of motion (ROM). This study aimed to determine the influence of humeral liner constraint on impingement-free ROM and impingement type using an RSA computer simulation model. METHODS: A virtual simulation model was created using 3D measurement software for conducting a simulation study. This study included 25 patients with rotator cuff tears and rotator cuff tear arthropathy. Impingement-free ROM and impingement patterns were measured during flexion, extension, abduction, adduction, external rotation, and internal rotation. Twenty-five cases with a total of 4 patterns of 2 multiplied by 2, making a total of 100 simulations: glenosphere (38 mm normal type vs. lateralized type) and humeral liner constraint (normal type vs. constrained type). There were 4 combinations: normal glenosphere and normal humeral liner, normal glenosphere and constrained humeral liner, lateralized glenosphere and normal humeral liner, and lateralized glenosphere and constrained humeral liner. RESULTS: Significant differences were found in all impingement-free ROM in 1-way analysis of variance (abduction: P = .01, adduction: P < .01, flexion: P = .01, extension: P = .02, external rotation: P < .01, and internal rotation: P < .01). Tukey's post hoc tests showed that the impingement-free ROM was reduced during abduction, external rotation, and internal rotation with the combination of the normal glenosphere and constrained humeral liner compared with the other combinations, and improved by glenoid lateralization compared with the combination of the lateralized glenosphere and constrained humeral liner. In the impingement pattern, the Pearson χ2 test showed significantly greater proportion of impingement of the humeral liner into the superior part of the glenoid neck in abduction occurring in the combination of the normal glenosphere and constrained humeral liner group compared with the other groups (P < .01). Bonferroni post hoc tests revealed that the combination of the normal glenosphere and constrained humeral liner was significantly different from that of the lateralized glenosphere and constrained humeral liner (P < .01). Using constrained liners resulted in early impingement on the superior part of the glenoid neck in the normal glenosphere, whereas glenoid lateralization increased impingement-free ROM. CONCLUSION: This RSA computer simulation model demonstrated that constrained humeral liners led to decreased impingement-free ROM. However, using the lateralized glenosphere improved abduction ROM.

Reliability and validity of a new deltoid muscle area measurement method after reverse shoulder arthroplasty.

Background: Accurate deltoid muscle assessment after reverse shoulder arthroplasty (RSA) is difficult using magnetic resonance imaging due to metal artifacts. We hypothesized that measuring the deltoid muscle area (DA) in the middle part of the deltoid's total length postoperatively would reduce metal artifacts and allow for an accurate assessment. This study aimed to assess the reliability and reproducibility of magnetic resonance imaging and evaluate its impact on postoperative outcomes. Methods: The DA in the middle part of the muscle's total length was measured twice by four examiners using pre and postoperative magnetic resonance imaging in 60 patients who underwent RSA (22 men, 38 women; mean age: 77.4 years). The DA at the greater tuberosity was measured preoperatively, and its correlation with the middle part of the deltoid's total length was evaluated. The Constant-Murley Score was measured at 2 years postoperatively, and its correlation with the DA in the middle part of the deltoid's total length pre- and postoperatively was assessed. Results: Intraclass correlation coefficients for intraobserver measurements of preoperative and postoperative DA in the middle part of the deltoid's total length were almost perfect, with mean values of 0.98 and 0.97, respectively. The intraclass correlation coefficients for interobserver reliability regarding the first and second DA measurements in the middle part of the deltoid's total length were 0.95 and 0.95 (preoperatively) and 0.89 and 0.90 (postoperatively). The Constant-Murley Score was assessed at 2 years postoperatively in 51 patients. Muscle strength was weakly and moderately correlated with preoperative DA (r = 0.33, P = .02) and postoperative DA (r = 0.49, P < .01), respectively. Conclusion: DA measurement in the middle part of the deltoid's total length after RSA was not affected by metal artifacts and had excellent reproducibility. This measurement method positively correlated with postoperative muscle strength, suggesting its usefulness for predicting postoperative muscle strength.

Assessment of Prevalence and Risk Factors for Central Sensitization Related to Shoulder Osteoarthritis and Rotator Cuff Tears Using the Central Sensitization Inventory: A Cross-Sectional Study in Shoulder Surgery Patients.

Shoulder disorders occasionally cause intractable pain. Central sensitization (CS) may be involved in such pain. Identifying risk factors associated with CS is crucial for effective pain control. This study aimed to determine the effects of shoulder osteoarthritis and rotator cuff tears (RCT) on CS and associated factors. This study included patients evaluated for CS using the Central Sensitization Inventory (CSI) before surgery for shoulder osteoarthritis, RCT, or cuff tear arthropathy. Patients with a CSI score of 40 or higher were defined as having CS. The relationships between glenohumeral osteoarthritis (GHOA), RCT size, and CS were statistically analyzed. Multiple regression analysis was performed to examine the factors affecting CSI scores. Subjects included 167 patients: 131 patients had RCT without GHOA, 23 had GHOA with RCT, and 13 had GHOA without RCT. The GHOA group had a significantly higher CSI score (27.5 [10.8-40.5] vs. 18.0 [10.0-27.5]) and CS prevalence (27.8% vs. 8.4%) than the RCT without GHOA group. There was no significant correlation between RCT size and CSI scores. Multiple regression analysis showed that female sex, severe pain, and long pain duration were associated with higher CSI scores. Considering the risk factors for CS might be helpful in shoulder treatment.

Parathyroid Hormone Inhibits Fatty Infiltration and Muscle Atrophy After Rotator Cuff Tear by Browning of Fibroadipogenic Progenitors in a Rodent Model.

BACKGROUND: Progressive fatty infiltration and muscle atrophy after rotator cuff tears lead to tendon repair failure and poor outcomes. Fibro-adipogenic progenitors (FAPs) are involved in fatty infiltration and muscle homeostasis of skeletal muscle. Inducing FAP differentiation into brown adipocyte-like "beige adipocytes" suppresses fatty infiltration and muscle atrophy. HYPOTHESIS: Parathyroid hormone (PTH) suppresses fatty infiltration and muscle atrophy after rotator cuff tears in a rat model by browning of FAPs. STUDY DESIGN: Controlled laboratory study. METHODS: PTH was administered subcutaneously for 4 or 8 weeks to a rotator cuff tear model in rats. After treatment, fatty infiltration of supraspinatus muscles was assessed using Oil Red O staining and muscle atrophy using wet muscle weight and muscle fiber cross-sectional area. Costaining of platelet-derived growth factor receptor α (FAP marker) and uncoupling protein 1 (browning marker) was performed to confirm FAP browning by PTH. Mouse-isolated FAPs were cultured with PTH and evaluated for browning-related gene expression and adipogenic differentiation using BODIPY staining. Myogenic differentiation of C2C12 myoblasts was evaluated using coculture of PTH-treated browning FAPs with C2C12. RESULTS: PTH inhibited fatty infiltration after rotator cuff tear at 8 weeks. Rotator cuff wet muscle loss of PTH-treated rats was inhibited at 4 and 8 weeks. Furthermore, PTH-treated rats demonstrated larger myofiber cross-sectional area than did untreated rats at 4 and 8 weeks. Costaining indicated colocalization of platelet-derived growth factor receptor α and uncoupling protein 1 and promoted PTH-induced FAP browning. PTH increased the expression of browning-related genes in FAPs and suppressed fat droplet accumulation in vitro. Coculture with PTH-treated FAPs promoted C2C12 cell differentiation into myotubes. CONCLUSION: PTH induced FAP-derived beige adipocytes by upregulating browning-related gene expression, and the browning effect of PTH on FAPs inhibited fatty infiltration and muscle atrophy in the rat rotator cuff tear model. PTH might have potential as a therapeutic drug for fatty infiltration and muscle atrophy after rotator cuff tears. CLINICAL RELEVANCE: PTH may expand treatment options for rotator cuff tears by reducing fatty infiltration and muscle atrophy after rotator cuff tears by browning of FAPs.

Deep learning-based screening tool for rotator cuff tears on shoulder radiography.

BACKGROUND: Early diagnosis of rotator cuff tears is essential for appropriate and timely treatment. Although radiography is the most used technique in clinical practice, it is difficult to accurately rule out rotator cuff tears as an initial imaging diagnostic modality. Deep learning-based artificial intelligence has recently been applied in medicine, especially diagnostic imaging. This study aimed to develop a deep learning algorithm as a screening tool for rotator cuff tears based on radiography. METHODS: We used 2803 shoulder radiographs of the true anteroposterior view to develop the deep learning algorithm. Radiographs were labeled 0 and 1 as intact or low-grade partial-thickness rotator cuff tears and high-grade partial or full-thickness rotator cuff tears, respectively. The diagnosis of rotator cuff tears was determined based on arthroscopic findings. The diagnostic performance of the deep learning algorithm was assessed by calculating the area under the curve (AUC), sensitivity, negative predictive value (NPV), and negative likelihood ratio (LR-) of test datasets with a cutoff value of expected high sensitivity determination based on validation datasets. Furthermore, the diagnostic performance for each rotator cuff tear size was evaluated. RESULTS: The AUC, sensitivity, NPV, and LR- with expected high sensitivity determination were 0.82, 84/92 (91.3%), 102/110 (92.7%), and 0.16, respectively. The sensitivity, NPV, and LR- for full-thickness rotator cuff tears were 69/73 (94.5%), 102/106 (96.2%), and 0.10, respectively, while the diagnostic performance for partial-thickness rotator cuff tears was low at 15/19 (78.9%), NPV of 102/106 (96.2%) and LR- of 0.39. CONCLUSIONS: Our algorithm had a high diagnostic performance for full-thickness rotator cuff tears. The deep learning algorithm based on shoulder radiography helps screen rotator cuff tears by setting an appropriate cutoff value. LEVEL OF EVIDENCE: Level III: Diagnostic Study.

Comparison of short-term clinical outcomes and radiographic changes in Grammont reverse shoulder arthroplasty between the French and Japanese populations: A propensity score-matched analysis.

BACKGROUND: Although Grammont-style reverse total shoulder arthroplasty (RSA) showed excellent clinical results in Europe, its utility for Asian populations remains unclear. This study aimed to compare the French and Japanese populations in terms of range of motion (ROM), functional outcomes, and scapular notching rates in patients who underwent standard Grammont-style RSA. We hypothesized that RSA for the Japanese population may not provide as good ROM and functional results at the final follow-up as that for the French population. METHODS: A total of 25 Japanese patients undergoing RSA were propensity score matched to 25 French patients undergoing RSA. The patients were matched for four different covariates using a propensity score analysis. The minimum follow-up period was 2 years. We investigated differences between the populations with respect to body size and shoulder joint ROM and Constant score (CS) measured preoperatively and at the final follow-up. Scapular notching was examined using radiographs at the final follow-up. RESULTS: The average height and weight of the French and Japanese patients were 164 cm and 70 kg and 152 cm and 56 kg, respectively. Anterior elevation (AE), external rotation (ER) at the side, internal rotation (IR), and CS total changed from 101° to 145°, 17° to 15°, 4.5 points to 5.5 points, and 36 points to 72 points, respectively, in the French population and from 63° to 119°, 8.5° to 13°, 4.6 points to 4 points, and 28 points to 58 points, respectively, in the Japanese population. AE improved in both the groups; ER and IR remained unchanged before and after surgery. The frequency of scapular notching (>grade 1) was higher in the Japanese population (56%) than in the French population (20%) (p = 0.019). CONCLUSIONS: Grammont-style RSA improved AE and CS in both the populations, but AE and CS were significantly higher in the French population than in the Japanese population at the final follow-up. Scapular notching frequently occurs in the Japanese population.

Comparison of Occurrence of Bone Tunnel Laceration, Clinical Results, and Cuff Repair Integrity of Transosseous Arthroscopic Rotator Cuff Repair With and Without Lateral Cortical Augmentation.

PURPOSE: To compare the occurrence of bone tunnel laceration, the short-term clinical results, and cuff repair integrity of transosseous arthroscopic rotator cuff repair (ARCR) using a tunneling device, with and without lateral cortical augmentation. METHODS: A retrospective review of patients who underwent transosseous ARCR from May 2012 to December 2017 was conducted. The inclusion criterion was repairable medium- to massive-sized full-thickness rotator cuff tear. This study included 2 consecutive series of patients undergoing transosseous ARCR with and without lateral cortical augmentation, called the ITO method and AT method, respectively. The incidence of bone tunnel laceration was evaluated intraoperatively. Patients were assessed through a range of motion and Constant scores preoperatively and at final follow-up. Further, magnetic resonance imaging was performed at 24 months postoperatively to examine the repaired rotator cuff integrity. RESULTS: A total of 121 subjects were included: 33 in the AT group and 88 in the ITO group. The intraoperative bone tunnel laceration occurrence rate was 67% and 4% for the AT and ITO methods, respectively; the difference was significant (P = .001). Anatomic failure rate (Sugaya Ⅲ, Ⅳ, and Ⅴ) rate for medium- to large-sized tears was significantly lower for the ITO than for the AT method (29% vs 65%, P = .004), but not for massive tears (61% vs 69%, P = .515). The mean forward elevation, abduction, external rotation, internal rotation, and Constant score were significantly improved at final follow-up from preoperative values. There were no significant differences between the 2 methods. CONCLUSIONS: Transosseous ARCR using a tunneling device with and without lateral cortical augmentation is a reliable method of improving clinical results at a minimum follow-up of 2 years. The intraoperative occurrence rate of bone tunnel laceration occurrence rate and the anatomic failure rate of medium- to large-sized cuff tear were lower with lateral cortical augmentation than without it. LEVEL OF EVIDENCE: Level Ⅳ, therapeutic cases series.

The accelerated effect of recombinant human bone morphogenetic protein 2 delivered by ß-tricalcium phosphate on tendon-to-bone repair process in rabbit models.

BACKGROUND: Bone morphogenetic protein 2 (BMP-2) plays an important role in the tendon-to-bone repair process. However, there is no previous literature on acceleration of the tendon-to-bone repair process by BMP-2 delivered by ß-tricalcium phosphate (ß-TCP). The aim of this study was to investigate the accelerated effect of recombinant human BMP-2 (rhBMP-2) delivered by ß-TCP on the tendon-to-bone repair process. METHODS: The infraspinatus tendon of elderly female Japanese white rabbits was detached from its insertion site on the humerus. A bone tunnel (4.2 mm) was created at the original insertion site of the tendon, which was repaired using the McLaughlin procedure after filling in ß-TCP (porosity 75%) without BMP-2 (control group) or with 10 µg rhBMP-2 (BMP group). The rabbits were sacrificed at the second, fourth, and eighth weeks after surgery for histologic analysis and biomechanical testing. We also evaluated the maturity of the tendon-to-bone junction using the tendon-to-bone maturity score. RESULTS: Histologic analysis revealed no significant difference between the groups at 2 and 8 weeks but a more abundant organized fibrocartilage at the tendon-to-bone junction in the BMP group at 4 weeks. The tendon-to-bone maturity score improved sequentially. The interface of the BMP group at 4 weeks had significantly improved biomechanical properties than that of the control group. CONCLUSION: The tendon-to-bone repair process was facilitated by the use of rhBMP-2 delivered by ß-TCP at 4 weeks.

Local delivery of siRNA using a biodegradable polymer application to enhance BMP-induced bone formation.

Small interfering RNA (siRNA) is useful tool for specific and efficient knockdown of disease-related genes. However, in vivo applications of siRNA are limited due to difficulty in its efficient delivery to target cells. In this study, we investigated the efficacy of a biodegradable hydrogel, poly-d,l-lactic acid-p-dioxanone-polyethylene glycol block co-polymer (PLA-DX-PEG), as a siRNA carrier. PLA-DX-PEG pellets with or without fluorescein-labeled dsRNA were implanted into mouse dosal muscle pouches. The cellular uptake of dsRNA surround the polymer was confirmed by fluorescent microscopy. The fluorescence intensity was dose-dependent of the dsRNA, and exhibited a time-dependent decrease. To investigate its biological efficiency, noggin (antagonoist to BMPs) gene-silencing with siRNA (siRNA/Noggin) was examined by the amount of suppression of BMP-2-induced noggin expression and the level of performance of BMP, indicated by ectopic bone formation. Noggin gene expression induced by BMP-2 was suppressed by addition of siRNA/Noggin to the implant, and the ectopic bone formation induced by implants with both BMP-2 and siRNA/Noggin was significantly greater than those induced by implants with BMP-2 alone. These results indicate the efficacy of local delivery of siRNAs by PLA-DX-PEG polymer, which intensified bone-inducing effects of BMP and promoted new bone formation by suppressing gene expression of Noggin.

Functional recovery period after arthroscopic rotator cuff repair: is it predictable before surgery?

BACKGROUND: Several studies have reported functional recovery of the shoulder after arthroscopic rotator cuff repair (ARCR). Preoperative estimation of the time required for functional recovery is important for determining surgical indications and for planning timing of the surgery and an appropriate postoperative physical therapy. QUESTIONS/PURPOSES: We therefore asked: (1) how long it takes to obtain functional recovery after ARCR, and (2) what preoperative factors influence functional recovery time. PATIENTS AND METHODS: We retrospectively evaluated 201 patients who had undergone ARCR. Using the Japanese Orthopaedic Association (JOA) shoulder scoring system, we defined the functional recovery period as the time required to achieve a score greater than 80% in each component. We evaluated the functional recovery periods and assessed preoperative influencing factors such as age, gender, shoulder stiffness, morphologic features of rotator cuff tears, and rotator cuff tear size. RESULTS: Sixty-three patients (31%) took less than 3 months, 81 patients (40%) took between 3 and 6 months, and 57 patients (28%) took greater than 6 months to achieve a score greater than 80% in each JOA shoulder assessment component. Younger patients without shoulder stiffness and with smaller rotator cuff tears had shorter functional recovery periods. CONCLUSIONS: One hundred forty-four patients (72%) obtained functional recovery within 6 months after ARCR. Age, shoulder stiffness, and rotator cuff tear size influenced functional recovery time.

Epinephrine accelerates osteoblastic differentiation by enhancing bone morphogenetic protein signaling through a cAMP/protein kinase A signaling pathway.

Topical effects of a catecholamine on bone morphogenetic protein (BMP)-induced ectopic bone formation were investigated in both in vivo and in vitro experimental systems. Epinephrine enhanced bone induction by BMP-2. Thus, the mass of ossicles ectopically induced by BMP-2 (5 µg) was increased by the addition of a low dose (10, 20, 40, or 80 µg) of epinephrine into a biodegradable BMP-2 carrier, in a dose-dependent manner. To investigate the mechanism by which epinephrine enhances BMP activity, in vitro experiments were carried out using osteogenic cells. The expression level of alkaline phosphatase (ALP) in cells, a marker of osteoblastic differentiation, was consistently elevated by BMP-2 (50 ng/ml) and was further elevated by the addition of epinephrine (10(-8)M). The epinephrine-enhanced ALP elevation was specifically abolished by an antagonist to ß2-adrenergic receptors (Butoxamine) and by a protein kinase A inhibitor (H89). Furthermore, BMP-induced mRNA expression of ALP and osteocalcin (marker proteins of osteoblastic differentiation) and of Osterix (a transcription factor essential for terminal differentiation to osteoblasts) in ST2 cells was significantly enhanced by the addition of epinephrine (10(-8)M). In luciferase expression assays using the promoter sequence of the Id1 gene (an immediate early response gene to BMP), luciferase activity was elevated by BMP-2 treatment (50 ng/ml) and this activity was further enhanced by the addition of epinephrine (10(-8)M). Epinephrine-enhanced luciferase activity was abolished by mutation of the cAMP-response element (CRE) sequence in the Id1 promoter, indicating that CRE-binding transcription proteins induced by epinephrine addition may act as enhancers of Smad-mediated BMP signaling.

Parathyroid hormone enhances bone morphogenetic protein activity by increasing intracellular 3', 5'-cyclic adenosine monophosphate accumulation in osteoblastic MC3T3-E1 cells.

Intermittent subcutaneous injections of parathyroid hormone (PTH) increase bone mass in a variety of animal models and humans. The anabolic actions of PTH on osteogenic cells are mainly mediated through the protein kinase A (PKA) signaling pathway via PTH receptor 1 (PTHR1). We have already reported 3', 5'-cyclic adenosine monophosphate (cAMP)/PKA-mediated enhancement of bone morphogenetic protein (BMP) signaling. Herein, we focused on the involvement of PTH in BMP signaling pathways in the MC3T3-E1 mouse osteoblastic cell line, to elucidate a potential mechanism of the anabolic actions of PTH on bone formation. Elevation of intracellular cAMP level in MC3T3-E1 cells by addition of PTH (10(-7) M) to culture media was transient without significant effect on biological actions of BMP. Cyclic addition of PTH (10 cyclic additions of 10(-8) M PTH at 3-min intervals) maintained a high intracellular cAMP level for about 2 h and mRNA expression and enzymatic activity of alkaline phosphatase (ALP) by BMP was enhanced by this addition. Relative luciferase expression assay in MC3T3-E1 cells using the Id1 promoter, an early response gene to BMPs, enhanced elevation of transcriptional activity in response to recombinant human BMP-2 by concomitant addition of PTH and BMP. Furthermore, cyclic PTH treatment significantly further suppressed BMP-induced inhibitory Smad6 expression. H89 (PKA inhibitor) almost completely abolished PTH actions on BMP signaling. IBMX (phosphodiesterase inhibitor) enhanced PTH actions. These results suggest that PTH enhances BMP signaling when PTH-induced intracellular cAMP level is maintained for a few hours, accelerating BMP actions to promote osteoblastic function and anabolic actions of new bone formation.

RNA interference for noggin enhances the biological activity of bone morphogenetic proteins in vivo and in vitro.

Noggin is a major extracellular antagonist to bone morphogenetic proteins (BMPs) which binds to BMPs and blocks binding of them to BMP-specific receptors and negatively regulates BMP-induced osteoblastic differentiation. In this study, we investigated the effect of noggin silencing by transfection of small interfering RNA (siRNA) on BMP-induced osteoblastic differentiation in vitro and ectopic bone formation in vivo induced by recombinant human BMP-2 (rhBMP-2). Noggin mRNA expression was up-regulated in response to rhBMP-2 in C2C12 cells, a myoblastic cell line, in dose- and time-dependent fashion as determined by real-time RT-PCR assay. Silencing of noggin expression by transfection of noggin siRNA suppressed BMP-stimulated noggin expression, resulting in acceleration of BMP-induced osteoblastic differentiation. For in vivo noggin silencing, siRNA was injected locally into back muscles and transfected into local cells by electroporation, where rhBMP-2-retaining (5 microg) collagen disks had been surgically placed. The implants were harvested at 2 weeks after surgery from experimental and control group mice and analyzed by radiological and histological methods. As a result, bone mineral content of ossicles ectopically induced by rhBMP-2 was significantly increased by silencing of noggin. Our findings suggest that silencing of noggin enhances the osteoblastic differentiation of BMP-responding cells in vitro and new bone formation induced by rhBMP-2 in vivo by eliminating negative regulation of the effects of BMP. RNA interference might be useful for intensifying the effects of BMP in promoting new bone (callus) formation in repair of damaged bone.

Osteoinductive capacity and heat stability of recombinant human bone morphogenetic protein-2 produced by Escherichia coli and dimerized by biochemical processing.

One problem associated with clinical application of CHO-derived recombinant human bone morphogenetic protein (C-BMP-2) is its high cost due to the need for use of high doses. To solve this problem, Escherichia coli-derived BMP-2 (E-BMP-2) has been examined using the technique of molecular unfolding and refolding. However, it is unclear whether the characteristics of E-BMP-2 are appropriate for clinical application. In this study, we examined the biological activity of E-BMP-2 and its heat tolerance in in vitro and in vivo systems. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the high purity of E-BMP-2. E-BMP-2-induced alkaline phosphatase expression in osteoprogenitor cells (C2C12, ST2, and primary murine calvarial osteoblast cells) was dose-dependent, and consistently elicited ectopic new ossicles of significant size in mice, also in dose-dependent fashion. In addition, E-BMP-2 induced phosphorylation of Smad1/5/8 and mRNA expression of osteoblastic differentiation markers to the same extent as C-BMP-2. On the other hand, when E-BMP-2 was exposed to increasing heat over time, its bone-inducing capacity was maintained until reaching 70 degrees C for 2 h or 90 degrees C for 15 min. Thus, E-BMP-2 will exhibit a decrease in activity with the sterilization procedures required prior to use in surgery. These findings indicate that the biological capacity and heat stability of E-BMP-2 are almost equivalent to those of currently available C-BMP-2, and suggest that E-BMP-2 might, thus, solve current problems of cost impeding routine clinical use of rhBMP-2.

Expression profiles of phosphodiesterase 4D splicing variants in osteoblastic cells.

The promotion of osteoblastic differentiation by bone morphogenetic proteins (BMPs) is accelerated by chemical compounds that increase the intracellular concentration of cyclic 3',5'-adenosine monophosphate (cAMP). cAMP is synthesized from adenosine triphosphate (ATP) by adenyl cyclase and degraded by phosphodiesterase (PDE) family enzymes. Inhibition of PDEs leads to prolonged accumulation of cAMP within cells and Camp-mediated reactions. Rolipram, a specific inhibitor of PDE4, is a compound effective in inducing osteoblastic differentiation. Four PDE4 family members are transcribed from four distinct genes (4A, 4B, 4C, and 4D). Expression of PDE4A and PDE4D has been observed in osteoblastic cells. We identified PDE4D splicing variants that expressed in ST2 or primary calvarial osteoblasts by rapid amplification of the 5'-ends of cDNA when they were cultured with BMP. PDE4D9 mRNA was identified from ST2, and PDE4D1 and -4D2 mRNAs were identified from primary calvarial osteoblasts. Expression of these three variants of PDE4D mRNA was found in ST2, MC3T3-E1, C3H10T1/2, C2C12, and primary calvarial osteoblasts by RT-PCR, but not PDE4D1 or -4D2 in ST2 or PDE4D2 in MC3T3-E1. Expression of these three variants was detectable in brain, heart, lung, liver, kidney, placenta, and femur, and was thus ubiquitous. Purified recombinant PDE4D9 protein exhibited phosphodiesterase activity, which degraded cAMP to AMP, and this activity was inhibited by rolipram. These findings suggest that PDE4D1, -2, and -9 play some roles in bone formation.