Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility.

DNA double-strand breaks (DSBs) are functionally linked to genomic instability in spermatocytes and to male infertility. The heavy metal cadmium (Cd) is known to induce DNA damage in spermatocytes by unknown mechanisms. Here, we showed that Cd ions impaired the canonical non-homologous end-joining (NHEJ) repair pathway, but not the homologous recombination (HR) repair pathway, through stimulation of Ser2056 and Thr2609 phosphorylation of DNA-PKcs at DSB sites. Hyper-phosphorylation of DNA-PKcs led to its premature dissociation from DNA ends and the Ku complex, preventing recruitment of processing enzymes and further ligation of DNA ends. Specifically, this cascade was initiated by the loss of PP5 phosphatase activity, which results from the dissociation of PP5 from its activating ions (Mn), that is antagonized by Cd ions through a competitive mechanism. In accordance, in a mouse model Cd-induced genomic instability and consequential male reproductive dysfunction were effectively reversed by a high dosage of Mn ions. Together, our findings corroborate a protein phosphorylation-mediated genomic instability pathway in spermatocytes that is triggered by exchange of heavy metal ions.

A phase III randomized, double-blind, placebo-controlled trial of the denosumab biosimilar QL1206 in postmenopausal Chinese women with osteoporosis and high fracture risk.

The current study evaluated the efficacy and safety of a denosumab biosimilar, QL1206 (60 mg), compared to placebo in postmenopausal Chinese women with osteoporosis and high fracture risk. At 31 study centers in China, a total of 455 postmenopausal women with osteoporosis and high fracture risk were randomly assigned to receive QL1206 (60 mg subcutaneously every 6 months) or placebo. From baseline to the 12-month follow-up, the participants who received QL1206 showed significantly increased bone mineral density (BMD) values (mean difference and 95% CI) in the lumbar spine: 4.780% (3.880%, 5.681%), total hip :3.930% (3.136%, 4.725%), femoral neck 2.733% (1.877%, 3.589%) and trochanter: 4.058% (2.791%, 5.325%) compared with the participants who received the placebo. In addition, QL1206 injection significantly decreased the serum levels of C-terminal crosslinked telopeptides of type 1 collagen (CTX): -77.352% (-87.080%, -66.844%), and N-terminal procollagen of type l collagen (P1NP): -50.867% (-57.184%, -45.217%) compared with the placebo over the period from baseline to 12 months. No new or unexpected adverse events were observed. We concluded that compared with placebo, QL1206 effectively increased the BMD of the lumbar spine, total hip, femoral neck and trochanter in postmenopausal Chinese women with osteoporosis and rapidly decreased bone turnover markers. This study demonstrated that QL1206 has beneficial effects on postmenopausal Chinese women with osteoporosis and high fracture risk.

Cardiac CIP protein regulates dystrophic cardiomyopathy.

Heart failure is a leading cause of fatality in Duchenne muscular dystrophy (DMD) patients. Previously, we discovered that cardiac and skeletal-muscle-enriched CIP proteins play important roles in cardiac function. Here, we report that CIP, a striated muscle-specific protein, participates in the regulation of dystrophic cardiomyopathy. Using a mouse model of human DMD, we found that deletion of CIP leads to dilated cardiomyopathy and heart failure in young, non-syndromic mdx mice. Conversely, transgenic overexpression of CIP reduces pathological dystrophic cardiomyopathy in old, syndromic mdx mice. Genome-wide transcriptome analyses reveal that molecular pathways involving fibrogenesis and oxidative stress are affected in CIP-mediated dystrophic cardiomyopathy. Mechanistically, we found that CIP interacts with dystrophin and calcineurin (CnA) to suppress the CnA-Nuclear Factor of Activated T cells (NFAT) pathway, which results in decreased expression of Nox4, a key component of the oxidative stress pathway. Overexpression of Nox4 accelerates the development of dystrophic cardiomyopathy in mdx mice. Our study indicates CIP is a modifier of dystrophic cardiomyopathy and a potential therapeutic target for this devastating disease.

LRTM1 promotes the differentiation of myoblast cells by negatively regulating the FGFR1 signaling pathway.

The proliferation and differentiation of myoblast cells are regulated by the fibroblast growth factor receptor (FGFR) signaling pathway. Although the regulation of FGFR signaling cascades has been widely investigated, the inhibitory mechanism that particularly function in skeletal muscle myogenesis remains obscure. In this study, we determined that LRTM1, an inhibitory regulator of the FGFR signaling pathway, negatively modulates the activation of ERK and promotes the differentiation of myoblast cells. LRTM1 is dynamically expressed during myoblast differentiation and skeletal muscle regeneration after injury. In mouse myoblast C2C12 cells, knockout (KO) of Lrtm1 significantly prevents the differentiation of myoblast cells; this effect is associated with the reduction of MyoD transcriptional activity and the overactivation of ERK kinase. Notably, further studies demonstrated that LRTM1 associates with p52Shc and inhibits the recruitment of p52Shc to FGFR1. Taken together, our findings identify a novel negative regulator of FGFR1, which plays an important role in regulating the differentiation of myoblast cells.

LSD1 negatively regulates autophagy in myoblast cells by driving PTEN degradation.

Lysine-specific demethylase 1 (LSD1) is a well characterized transcriptional regulator functioning on the chromatin to remove mono- and di-methyl groups from lysine 4 or lysine 9 of histone 3 (H3K4 or H3K9). LSD1 also has non-transcriptional activities via targeting non-histone substrates that participate in diverse biological processes. In this report, we determined that LSD1 negatively regulates autophagy in skeletal muscle cells by promoting PTEN degradation in a transcription-independent mechanism. In C2C12 cells, LSD1 inhibition or depletion significantly induced the initiation of autophagy; and autophagy resulted from LSD1 inhibition is associated with AKT/mTORC1 inactivation. Notably, the proteins of PTEN, a prominent repressive AKT modulator, are stabilized by LSD1 inhibition despite a decrease of its mRNA levels. Further data demonstrated that LSD1 interacts with PTEN protein and enhances its ubiquitination and degradation. Together, our findings identify a novel biological function of LSD1 in autophagy, mediated by regulating the stability of PTEN and the activity of AKT/mTORC1.

Novel unilateral C1 double screw and ipsilateral C2 pedicle screw placement combined with contralateral laminar screw-rod fixation for atlantoaxial instability.

PURPOSE: To investigate the anatomical and biomechanical feasibility of the unilateral C1 double screw [pedicle screw (PS) + lateral mass screw (LMS)] and ipsilateral C2 PS combined with contralateral C2 laminar screw (LS)-rod fixation for atlantoaxial instability by comparison with traditional posterior fixation methods. METHODS: Fifteen sets of complete dry bony specimens of atlas were used for morphometric analysis. The working length, width and thickness of the C1 PSs and LMSs were manually measured. Ten fresh-frozen cervical spines (C0-C7) were used to complete the range of motion (ROM) testing in their intact condition, under destabilization and after stabilization by the following procedures: unilateral C1-C2 PS rod fixation (Group A), bilateral C1-C2 PS rod fixation (Group B), and unilateral C1 double screw and ipsilateral C2 PS combined with contralateral C2 LS rod fixation (Group C). RESULTS: The working thickness of the C1 PS was ≤ 3.5 mm in only one (1/15 = 6.7%) specimen. The other parameters were > 3.5 mm in all specimens. In the ROM test, all fixation groups showed significantly reduced flexibility in all directions compared with both the intact and destabilization groups. Further, Groups B and C showed better stability in all directions than Group A. However, no significant differences were observed between Groups B and C. CONCLUSION: The C1 unilateral lateral mass could mostly contain two screws(PS + LMS) with diameters ≤ 3.5 mm. The novel technique of unilateral C1 double screw and ipsilateral C2 PS combined with contralateral C2 LS rod fixation provided better stability than unilateral PS rod fixation and similar as bilateral PS rod fixation. Therefore, it is a feasible salvage method that provides a new insight into atlantoaxial instability. These slides can be retrieved under Electronic Supplementary Material.

BMP9/COX-2 axial mediates high phosphate-induced calcification in vascular smooth muscle cells via Wnt/ß-catenin pathway.

Vascular calcification is a notable risk factor for cardiovascular system. High phosphate can induce calcification in vascular smooth muscle cells (VSMCs), but the detail mechanism underlying this process remains unclear. In the present study, we determined the relationship between high phosphate and bone morphogenetic protein 9 (BMP9) in VSMCs, the effect of BMP9 on calcification in VSMCs and the effect of COX-2 on BMP9 induced calcification in VSMCs, as well as the possible mechanism underlying this biological process. We found that high phosphate obviously up-regulates the expression of BMP9 in VSMCs. Over-expression of BMP9 decreases the level of alpha-smooth muscle cell actin (α-SMA) apparently, but increases the level of Runx-2, Dlx-5, and ALP in VSMCs. Meanwhile, BMP9 increases the level of OPN and OCN, promotes mineralization in VSMCs and induces calcification in thoracic aorta. High phosphate and over-expression of BMP9 increases the level of COX-2. Over-expression of COX-2 enhances the inhibitory effect of BMP9 on α-SAM and increases the level of OPN and OCN induced by BMP9. However, inhibition of COX-2 decreases the BMP9-induced calcification in VSMCs and thoracic aorta. For mechanism, we found that high phosphate or BMP9 increases the level of ß-catenin and p-GSK3ß in VSMCs, but no substantial effect on GSK3ß. However, COX-2 inhibitor decreases the expression of ß-catenin induced by BMP9. Our findings indicated that BMP9 is involved in the phosphate-induced calcification in VSMCs and COX-2 partly mediates the BMP9-induced calcification in VSMCs through activating Wnt/ß-catenin pathway.

Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression.

BACKGROUND/AIMS: Bone morphogenetic proteins (BMPs) and BMP receptors widely participate in osteolytic metastasis of breast cancer, while their role in tumor-stromal interaction is largely unknown. In this study, we investigated whether BMP receptor type 1a (BMPR1a) can alter the interaction between metastatic cancer cells and osteoclast precursors. METHODS: Adenovirus-mediated RNA interference was used to interrupt target genes of human breast cancer cell lines and nude mice were injected intratibially with the cancer cells. Tumor-bearing mice were examined by bioluminescence imaging and microCT. Sections of metastatic legs were measured by a series of staining methods. Murine bone marrow mononuclear cells or RAW264.7 cells were cultured with conditioned media of breast cancer cells. RT-PCR, Western blotting and ELISA were used to test mRNA and protein expressions of target molecules. RESULTS: Expression of BMPR1a of MDA-MB-231-luc cells at tumor-bone interface was apparently stronger than that of cancer cells distant from the interface. Mice injected with BMPR1a-knockdown MDA-MB-231-luc cells showed reduced tumor growth and bone destruction compared with control groups. Knockdown (KD) of BMPR1a of MDA-MB-231-luc cells or MCF-7 cells decreased the level of receptor activator for NF-κB ligand (RANKL). Level of RANKL in MDA-MB-231-luc cells or MCF-7 cells was reduced by p38 inhibitor. Compared with control group, knockdown of p38 of breast cancer cells decreased cancer-induced osteoclastogenesis. CONCLUSION: Knockdown of BMPR1a of breast cancer cells suppresses their production of RANKL via p38 pathway and inhibits cancer-induced osteoclastogenesis, which indicates that BMPR1a might be a possible target in breast cancer-induced osteolytic metastasis.

Adenovirus-mediated expression of vascular endothelial growth factor-a potentiates bone morphogenetic protein9-induced osteogenic differentiation and bone formation.

Mesenchymal stem cells (MSCs) are suitable seed cells for bone tissue engineering because they can self-renew and undergo differentiation into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Vascular endothelial growth factor-a (VEGF-a), an angiogenic factor, is also involved in osteogenesis and bone repair. However, the effects of VEGF-a on osteogenic MSCs differentiation remain unknown. It was previously reported that bone morphogenetic protein9 (BMP9) is one of the most important osteogenic BMPs. Here, we investigated the effects of VEGF-a on BMP9-induced osteogenesis with mouse embryo fibroblasts (MEFs). We found that endogenous VEGF-a expression was undetectable in MSCs. Adenovirus-mediated expression of VEGF-a in MEFs potentiated BMP9-induced early and late osteogenic markers, including alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In stem cell implantation assays, VEGF-a augmented BMP9-induced ectopic bone formation. VEGF-a in combination with BMP9 effectively increased the bone volume and osteogenic activity. However, the synergistic effect was efficiently abolished by the phosphoinositide 3-kinase (PI3K)/AKT inhibitor LY294002. These results demonstrated that BMP9 may crosstalk with VEGF-a through the PI3K/AKT signaling pathway to induce osteogenic differentiation in MEFs. Thus, our findings demonstrate the effects of VEGF-a on BMP9-induced bone formation and provide a new potential strategy for treating nonunion fractures, large segmental bony defects, and/or osteoporotic fractures.

A Novel Organ Culture Model of Mouse Intervertebral Disc Tissues.

The intervertebral disc (IVD) is a fibrocartilaginous joint between two vertebral bodies. An IVD unit consists of a gelatinous central nucleus pulposus, encased by the annulus fibrosus, which is sandwiched between cartilaginous endplates (EPs). The IVD homeostasis can be disrupted by injuries, ageing and/or genetic predispositions, leading to degenerative disc disorders and subsequent lower back pain. The complex structure and distinct characteristics of IVDs warrant the establishment of robust in vitro IVD organ culture for studying the etiology and treatment of disc degeneration. Here, we isolate mouse lumbar IVDs and culture the minimal IVD units in submersion or suspension medium supplemented with 2% bovine serum or 10% fetal bovine serum (FBS). We find the minimal IVD units remain healthy for up to 14 days when cultured in submersion culture supplemented with 10% FBS. New bone formation in the EPs of the cultured IVDs can be assessed with calcein labeling. Furthermore, the cultured IVDs can be effectively transduced by recombinant adenovirus, and transgene expression lasts for 2 weeks. Thus, our findings demonstrate that the optimized IVD organ culture system can be used to study IVD biology and screen for biological factors that may prevent, alleviate and/or treat disc degeneration.

Adenovirus-mediated siRNA targeting CXCR2 attenuates titanium particle-induced osteolysis by suppressing osteoclast formation.

BACKGROUND Wear particle-induced peri-implant loosening is the most common complication affecting long-term outcomes in patients who undergo total joint arthroplasty. Wear particles and by-products from joint replacements may cause chronic local inflammation and foreign body reactions, which can in turn lead to osteolysis. Thus, inhibiting the formation and activity of osteoclasts may improve the functionality and long-term success of total joint arthroplasty. The aim of this study was to interfere with CXC chemokine receptor type 2 (CXCR2) to explore its role in wear particle-induced osteolysis. MATERIAL AND METHODS Morphological and biochemical assays were used to assess osteoclastogenesis in vivo and in vitro. CXCR2 was upregulated in osteoclast formation. RESULTS Local injection with adenovirus-mediated siRNA targeting CXCR2 inhibited titanium-induced osteolysis in a mouse calvarial model in vivo. Furthermore, siCXCR2 suppressed osteoclast formation both directly by acting on osteoclasts themselves and indirectly by altering RANKL and OPG expression in osteoblasts in vitro. CONCLUSIONS CXCR2 plays a critical role in particle-induced osteolysis, and siCXCR2 may be a novel treatment for aseptic loosening.

Clinical Observation and Cause Analysis of Perioperative Superior Cluneal Neuralgia in Vertebral Augmentation.

BACKGROUND: In our clinical practice, we observed that some osteoporotic vertebral compression fracture patients undergoing vertebral augmentation exhibited pain in the iliac crest region. This pain aligned with the diagnostic criteria for superior cluneal neuralgia (SCN) and affected treatment satisfaction. OBJECTIVE: This study aims to clinically observe patients undergoing vertebral augmentation in a hospital setting and analyze the etiology and risk factors associated with SCN. STUDY DESIGN: Retrospective cohort study. SETTING: Inpatient population of a single center. METHODS: We retrospectively analyzed clinical data from 630 patients who underwent vertebral augmentation in our hospital from March 2022 to March 2023. Fifty-two patients enrolled in the study experienced pain that met the diagnostic criteria for superior cluneal neuralgia during the perioperative period of the vertebral augmentation procedures. Those patients were divided into 2 subgroups according to the conditions involved in the occurrence of SCN: Group A (26 patients) had either no preoperative SCN but developed it postoperatively, or had preoperative SCN that worsened or did not alleviate postoperatively. Group B (26 patients) had preoperative SCN that was relieved postoperatively. Additionally, 52 consecutive patients in March 2022 to March 2023. who did not experience SCN during the perioperative period were selected as the control group (Group C). Variables such as surgical segment, age, height, weight, body mass index, duration of hospitalization, chronic low back pain (CLBP), duration of pain, anesthesia, surgical approach, fracture pattern, preoperative visual analog scale (pre-op VAS) score, intraoperative VAS score, one-day VAS score, one-month VAS score, lumbar sacral angle, and sacral tilt angle were statistically described and analyzed. RESULTS: In our hospital, the incidence of SCN during the perioperative period of vertebral augmentation procedures is 8.25% (52/630). Among all the segments of patients who developed SCN during the perioperative period, the L1 segment had the highest proportion, which was 29.03% and 35.14% in Groups A and B, respectively. Group B and Group C showed significant differences in duration of hospitalization (P = 0.012), pre-op VAS scores (P = 0.026), and CLBP (P < 0.001). Group A had significantly higher VAS scores preoperatively (P = 0.026) and intraoperatively (P = 0.004) and in CLBP (P = 0.001) than did Group C. LIMITATIONS: This is a retrospective study. Single-center noncontrolled studies may introduce selection bias. The small sample size in each group might have also led to bias. CONCLUSION: Perioperative SCN associated with vertebral augmentation is significantly correlated with preoperative VAS scores and CLBP. In addition, intraoperative VAS scores might be a factor contributing to the nonalleviation or exacerbation of postoperative SCN.

Unilateral Biplanar Screw-Rod Fixation Technique for the Treatment of Odontoid Fractures in Patients with Atlantoaxial Bone or Vascular Abnormalities.

OBJETIVE: This study aims to introduce the unilateral biplanar screw-rod fixation (UBSF) technique (a hybrid fixation technique: 2 sets of atlantoaxial screws were placed on the same side), which serves as a salvage method for traditional posterior atlantoaxial fixation. To summarize the indications of this technique and to assess its safety, feasibility, and clinical effectiveness in the treatment of odontoid fractures. METHODS: Patients with odontoid fractures were enrolled according to special criteria. Surgical duration and intraoperative blood loss were documented. Patients were followed up for a minimum of 12 months. X-ray and computerized tomography scans were conducted and reviewed at 1 day, and patients were asked to return for computerized tomography reviews at 3, 6, 9, and 12 months after surgery until fracture union. Recorded and compared the Neck Visual Analog Scale and Neck Disability Index presurgery and at 1 week and 12 months postsurgery. RESULTS: Between January 2016 and December 2022, our study enrolled 7 patients who were diagnosed with odontoid fractures accompanied by atlantoaxial bone or vascular abnormalities. All 7 patients underwent successful UBSF surgery, and no neurovascular injuries were recorded during surgery. Fracture union was observed in all patients, and the Neck Visual Analog Scale and Neck Disability Index scores improved significantly at 1 week and 12 months postoperative (P < 0.01). CONCLUSIONS: The UBSF technique has been demonstrated to be safe, feasible, and effective in treating odontoid fractures. In cases where the atlantoaxial bone or vascular structure exhibits abnormalities, it can function as a supplementary or alternative approach to the conventional posterior C1-2 fixation.

Leptin attenuates the osteogenic induction potential of BMP9 by increasing ß-catenin malonylation modification via Sirt5 down-regulation.

BMP9 has demonstrated significant osteogenic potential. In this study, we investigated the effect of Leptin on BMP9-induced osteogenic differentiation. Firstly, we found Leptin was decreased during BMP9-induced osteogenic differentiation and serum Leptin concentrations were increased in the ovariectomized (OVX) rats. Both in vitro and in vivo, exogenous expression of Leptin inhibited the process of osteogenic differentiation, whereas silencing Leptin enhanced. Exogenous Leptin could increase the malonylation of ß-catenin. However, BMP9 could increase the level of Sirt5 and subsequently decrease the malonylation of ß-catenin; the BMP9-induced osteogenic differentiation was inhibited by silencing Sirt5. These data suggested that Leptin can inhibit the BMP9-induced osteogenic differentiation, which may be mediated through reducing the activity of Wnt/ß-catenin signalling via down-regulating Sirt5 to increase the malonylation level of ß-catenin partly.

Inhibitory effects of recombinant IL-4 and recombinant IL-13 on UHMWPE-induced bone destruction in the murine air pouch model.

PURPOSE: We administered recombinant interleukin (IL)-4 and recombinant IL-13 locally into the air pouch of mice to improve bone resorption induced by ultra-high-molecular-weight polyethylene (UHMWPE) particles. METHODS: Air pouches were established on the back of BALB/c mice, followed by the surgical introduction of a section of calvaria from a syngeneic mouse donor. We stimulated the bone-implanted pouches with the UHMWPE suspension. We divided UHMWPE-containing mice into four study groups to receive injections of phosphate-buffered saline (control), IL-4 alone, IL-13 alone, or IL-4 and IL-13 into the pouches. We harvested the tissues at 14 d after treatment for molecular and histological analyses. RESULTS: The inhibitory effect of IL-4 was stronger than that of IL-13 toward osteoclast differentiation and osteoblast for the induction of osteoprotegerin production and down-regulation of receptor for activation of nuclear factor-κB ligand production. Furthermore, the combined treatment with both IL-4 and 1L-13 had a more important role in inhibiting bone resorption in these pouches with UHMWPE stimulation, compared with IL-4 or IL-13 treatment alone. CONCLUSIONS: Local administration of recombinant IL-4 and IL-13 may be a feasible and effective therapeutic candidate to treat or prevent wear debris-associated osteolysis.

The anatomic study and surgical technique for canal decompression with "pedicle-plasty" strategy in lumbar burst fractures with pedicle rupture.

In the treatment of lumbar burst fractures with nerve injury, fusion is often required to rebuild spinal stability, but it can lead to the loss of motor units and increase the occurrence of adjacent segment diseases. Thus, a novel approach of lumbar canal decompression with "pedicle-plasty" strategy (DDP) was needed in clincal treatment. Firstly, image measurement analysis, the images of 60 patients with lumbar spine CT examinations were selected to measure osteotomy angle (OA), distance from the intersection of osteotomy plane and skin to the posterior midline (DM),transverse length of the osteotomy plane (TLOP), and sagittal diameter of the outer edge of superior articular process (SD). Secondary, cadaver study, distance between the intermuscular space and midline (DMSM), anterior and posterior diameters of the decompression (APDD), and lateral traction distance of the lumbosacral plexus (TDLP) were measured on 10 cadaveric specimens. Finally, procedure of DDP was demonstrated on cadaver specimens. OA ranged from 27.68°+4.59° to 38.34°+5.97°, DM ranged from 43.44+6.29 to 68.33+12.06 mm, TLOP ranged from 16.84+2.19 to 19.64+2.36 mm, and SD ranged from 22.49+1.74 to 25.53+2.21 mm. DMSM ranged from 45.53+5.73 to 65.46+6.43 mm. APDD were between 10.51+3.59 and 12.12+4.54 mm, and TDLP were between 3.28+0.81 and 6.27+0.62 mm.DDP was successfully performed on cadaveric specimens. DDP, as a novel approach of decompression of burst fractures with pedicle rupture, can fully relieve the occupation and at the same time preserve the spinal motor unit because of no resection of intervertebral discs and no destruction of facet joints,and has certain developmental significance.

Inhibitory effect of adenovirus-mediated siRNA-targeting BMPR-IB on UHMWPE-induced bone destruction in the murine air pouch model.

OBJECTIVE: Adenovirus expressing small interfering RNA (siRNA)-targeting BMPR-IB was locally administered into the air pouch of mice to improve bone resorption induced by ultra-high molecular weight polyethylene (UHMWPE) particles. METHOD: Air pouches were established on the back of BALB/c mice, followed by the surgical introduction of a section of calvaria from a syngeneic mouse donor. The bone-implanted pouches were stimulated with the UHMWPE suspension. UHMWPE-containing mice were divided into three study groups to receive injections of adenovirus expressing BMPR-IB siRNA (BMPR-IB group), adenovirus expressing missense siRNA, and virus-free culture medium (control group) into the pouches, respectively. The tissues were harvested at 14 days after the treatment for molecular and histological analyses. RESULTS: Adenovirus-mediated BMPR-IB siRNA treatment significantly improved UHMWPE particle-induced bone resorption, reduced TRAP and RANK gene and protein expression levels, and diminished the number of TRAP-positive cells. Furthermore, the BMPR-IB siRNA inhibited osteoclast differentiation by targeting osteoblast for the induction of osteoprotegerin formation and downregulation of receptor for activation of nuclear factor-κB ligand production. CONCLUSIONS: This study suggested that loss of bone morphogenetic protein signaling by BMPR-IB siRNA directs osteoblasts to decrease bone destruction in part by downregulating osteoclastogenesis through the receptor for activation of nuclear factor-κB ligand-osteoprotegerin pathway. Local administration of adenovirus expressing siRNA-targeting BMPR-IB may be a feasible and effective therapeutic candidate to treat or prevent wear debris-associated osteolysis.

The SIRT1 activator SRT2104 promotes BMP9-induced osteogenic and angiogenic differentiation in mesenchymal stem cells.

Bone defects resulting from trauma, bone tumors, infections and skeletal abnormalities are a common osteoporotic condition with respect to clinical treatment. Of the known bone morphogenetic proteins (BMPs), BMP9 has the strongest osteogenic differentiation potential, which could be beneficial in the construction of tissue-engineered bone. Silent mating type information regulator 2 homolog-1 (SIRT1) is a highly conserved nicotinamide adenine dinucleotide-dependent deacetylase that deacetylates and modulates histone or non-histone substrates. However, the role of SIRT1 in BMP9-induced osteogenic differentiation of stem cells has not been studied. Furthermore, it is unclear whether SIRT1 interacts with the BMP/Smad and BMP/MAPK pathways in stem cells. We found that SIRT1 expression decreased gradually in a time-dependent manner during BMP9-induced osteogenic differentiation of MSCs. Interactions between SIRT1 and Smad7 promoted degradation of Smad7 and increased Smad1/5/8 phosphorylation. SRT2104, an activator of SIRT, enhanced the expression of osteogenic- and angiogenic-related proteins in BMP9-induced MSCs. In addition, we found that activation of the BMP/MAPK pathway led to osteogenic and angiogenic differentiation of MSCs. Our study demonstrated that SIRT1 expression decreased during BMP9-induced differentiation. The SIRT1 activator SRT2104 promoted BMP9-induced osteogenic and angiogenic differentiation of MSCs through the BMP/Smad and BMP/MAPK signaling pathways.

TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.

Intra-articular Injection of Kartogenin-Enhanced Bone Marrow-Derived Mesenchymal Stem Cells in the Treatment of Knee Osteoarthritis in a Rat Model.

BACKGROUND: In this study, we investigated the in vitro and in vivo chondrogenic capacity of kartogenin (KGN)-enhanced bone marrow-derived mesenchymal stem cells (BMSCs) for cartilage regeneration. PURPOSE: To determine (1) whether functionalized nanographene oxide (NGO) can effectively deliver KGN into BMSCs and (2) whether KGN would enhance BMSCs during chondrogenesis in vitro and in vivo in an animal model. STUDY DESIGN: Controlled laboratory study. METHODS: Functionalized NGO with line chain amine-terminated polyethylene glycol (PEG) and branched polyethylenimine (BPEI) were used to synthesize biocompatible NGO-PEG-BPEI (PPG) and for loading hydrophobic KGN molecules noncovalently via π-π stacking and hydrophobic interactions (PPG-KGN). Then, PPG-KGN was used for the intracellular delivery of hydrophobic KGN by simple mixing and co-incubation with BMSCs to acquire KGN-enhanced BMSCs. The chondrogenic efficacy of KGN-enhanced BMSCs was evaluated in vitro. In vivo, osteoarthritis (OA) was induced by anterior cruciate ligament transection in rats. A total of 5 groups were established: normal (OA treated with nothing), phosphate-buffered saline (PBS; intra-articular injection of PBS), PPG-KGN (intra-articular injection of PPG-KGN), BMSCs (intra-articular injection of BMSCs), and BMSCs + PPG-KGN (intra-articular injection of PPG-KGN-preconditioned BMSCs). At 6 and 9 weeks after the surgical induction of OA, the rats received intra-articular injections of PPG-KGN, BMSCs, or KGN-enhanced BMSCs. At 14 weeks after the surgical induction of OA, radiographic and behavioral evaluations as well as histological analysis of the knee joints were performed. RESULTS: The in vitro study showed that PPG could be rapidly uptaken in the first 4 hours after incubation, reaching saturation at 12 hours and accumulating in the lysosome and cytoplasm of BMSCs. Thus, PPG-KGN could enhance the efficiency of the intracellular delivery of KGN, which showed a remarkably high chondrogenic differentiation capacity of BMSCs. When applied to an OA model of cartilage injuries in rats, PPG-KGN-preconditioned BMSCs contributed to protection from joint space narrowing, pathological mineralization, OA development, and OA-induced pain, as well as improved tissue regeneration, as evidenced by radiographic, weightbearing, and histological findings. CONCLUSION: Our results demonstrate that KGN-enhanced BMSCs showed markedly improved capacities for chondrogenesis and articular cartilage repair. We believe that this work demonstrates that a multifunctional nanoparticle-based drug delivery system could be beneficial for stem cell therapy. Our results present an opportunity to reverse the symptoms and pathophysiology of OA. CLINICAL RELEVANCE: The intracellular delivery of KGN to produce BMSCs with enhanced chondrogenic potential may offer a new approach for the treatment of OA.