Bone Morphogenic Protein Research Articles

Mapping prohormone processing by proteases in human enteroendocrine cells using genetically engineered organoid models

Enteroendocrine cells (EECs) secrete hormones in response to ingested nutrients to control physiological processes such as appetite and insulin release. EEC hormones are synthesized as large proproteins that undergo proteolytic processing to generate bioactive peptides. Mutations in EEC-enriched proteases are associated with endocrinopathies. Due to the relative rarity of EECs and a paucity of invitro models, intestinal prohormone processing remains challenging to assess. Here, human gut organoids in which EECs can efficiently be induced are subjected to CRISPR-Cas9-mediated modification of EEC-expressed endopeptidase and exopeptidase genes. We employ mass spectrometry-based analyses to monitor peptide processing and identify glucagon production in intestinal EECs, stimulated upon bone morphogenic protein (BMP) signaling. We map the substrates and products of major EECs endo- and exopeptidases. Our studies provide a comprehensive description of peptide hormones produced by human EECs and define the roles of specific proteases in their generation.

A novel application of neural networks to identify potentially effective combinations of biologic factors for enhancement of bone fusion/repair

The use of biologic adjuvants (orthobiologics) is becoming commonplace in orthopaedic surgery. Among other applications, biologics are often added to enhance fusion rates in spinal surgery and to promote bone healing in complex fracture patterns. Generally, orthopaedic surgeons use only one biomolecular agent (ie allograft with embedded bone morphogenic protein-2) rather than several agents acting in concert. Bone fusion, however, is a highly multifactorial process and it likely could be more effectively enhanced using biologic factors in combination, acting synergistically. We used artificial neural networks, trained via machine learning on experimental data on orthobiologic interventions and their outcomes, to identify combinations of orthobiologic factors that potentially would be more effective than single agents. This use of machine learning applied to orthobiologic interventions is unprecedented. Available data on the outcomes associated with various orthopaedic biologic agents, electrical stimulation, and pulsed ultrasound were curated from the literature and assembled into a form suitable for machine learning. The best among many different types of neural networks was chosen for its ability to generalize over this dataset, and that network was used to make predictions concerning the expected efficacy of 2400 medically feasible combinations of 9 different agents and treatments. The most effective combinations were high in the bone-morphogenic proteins (BMP) 2 and 7 (BMP2, 15mg; BMP7, 5mg), and in osteogenin (150ug). In some of the most effective combinations, electrical stimulation could substitute for osteogenin. Some other effective combinations also included bone marrow aspirate concentrate. BMP2 and BMP7 appear to have the strongest pairwise linkage of the factors analyzed in this study. Artificial neural networks are powerful forms of artificial intelligence that can be applied readily in the orthopaedic domain, but neural network predictions improve along with the amount of data available to train them. This study provides a starting point from which networks trained on future, expanded datasets can be developed. Yet even this initial model makes specific predictions concerning potentially effective combinatorial therapeutics that should be verified experimentally. Furthermore, our analysis provides an avenue for further research into the basic science of bone healing by demonstrating agents that appear to be linked in function.

GC-MS profiling and bioactivity prediction of compounds from Momordica charantia L. extract.

Momordica species are vegetable crops, belonging to the family of Cucurbitaceae. For the present study plant samples of Momordica charantia were collected from different parts of Barak Valley of Assam, India. Hyphenated technique Gas Chromatography-Mass Spectroscopy (GC-MS) of the chromatographically separated components of the plant extract was performed to identify some of the principles. All the isolated compounds exhibited notable interaction with the enzyme Bone morphogenic protein upon in silico screening. All the compounds were screened in silico for toxicity and then docked with the target enzyme individually. It was found that these compounds are interacting with the target enzyme and is able to inhibit the enzyme activity more efficiently as compared to the reference ligand.

Chitosan as Bone Scaffold for Craniofacial Bone Regeneration: A Systematic Review

Background: The reconstruction of bone defect in the face and head is indispensable yet one of the most challenging procedure to date. Chitosan has emerged as a promising low-cost natural biopolymer for the bone scaffold as an alternative to surgery. This study aims to review the effectiveness of chitosan as a bone scaffold for craniofacial bone regeneration.
 Methods: This systematic review used Google Scholar and PubMed as database sources. Study selection using PRISMA diagram and Boolean operator to specify the study search. The quality assessment of the study used a checklist from Joanna Briggs Institute for experimental study.
 Result: We included 18 experimental studies, both in vivo and in vitro study—the in vivo study used animal subjects such as mice, goats and rabbits. The studies mostly used chitosan combined with other biomaterials such as demineralized bone matrix (DBM), genipin (GP), sodium alginate (SA), resveratrol (Res), polycaprolactone (PCL) and collagen, growth factor and stem cells such as bone morphogenic protein-2 (BMP-2), dental pulp stem cell (DPSC), and human umbilical cord mesenchymal stem cells (hUCMSC).
 Conclusion: Chitosan is a natural polymer with promising osteoconductive, osteoinductive and osteo-integrative effects in bone regeneration. Chitosan utilization for bone scaffolds combined with other biomaterials, growth factors, or stem cells gives better bone regeneration results than chitosan alone.

The Osteogenesis Mechanisms of Dental Alveolar Bone Socket Post Induction with Hydroxyapatite Bovine Tooth Graft: An Animal Experimental in Rattus norvegicus Strain Wistar.

Surgical endodontics (hemisection) commonly involves the alveolar bone socket and the periradicular tissue. In today's era, optimizing the bone healing process is updated by using bone graft induction. This study explores the mechanisms of bone healing of the alveolar bone socket post-dental extraction of Wistar rats after administration of a bovine tooth graft (hydroxyapatite bovine tooth graft [HAp-BTG]). Fifty Wistar rats were randomly selected into two groups, control and treatment, and into five subgroups on days 3, 7, 14, 21, and 28. The postextraction socket was filled with polyethylene glycol (PEG) as the control and PEG + HAp-BTG as the treatment group. On days 3, 7, 14, 21, and 28, Wistar rats were sacrificed, mandibles were taken, paraffin blocks were made, cut 4 µm thick, and made into glass preparations for microscopic examination. The variable analysis was performed by staining hematoxylin-eosin for osteoblasts (OBs) and osteoclasts (OCs) and immunohistochemistry for runt-related transcription factor 2 (RUNX2), osterix (OSX), osteocalcin (OCN), bone morphogenic protein (BMP) 2. We analyzed the expressed cell count per microscope field. In general, the number of cell expressions in the treatment group was significantly higher and faster, except for significantly lower OC. The high variables peak occurred on day 14 for RUNX2 and OCN, on day 7 for OSX, while OB significantly increased on day 21 and remained until day 28. The decrease of OC cells occurred on day 7 and remained low until 28 days. BMP2 was first dominantly induced by HAp-BTG, then the others. HAp-BTG can induce higher and faster bone healing biomarkers. BMP2 is the dominant first impacted. On the 28th day, it did not significantly express the suppression of OC by OB, which entered the bone formation and remodeling step.

Multifunctional Alendronate‐PEI Carbon Dots for the Treatment of Bone‐Destructive Diseases via Bidirectional Regulation of Osteoblast‐Osteoclast Function

AbstractBone damage results from the imbalance of bone homeostasis maintained by osteoblast‐regulated bone formation and osteoclast‐mediated bone resorption. Conventional drug therapy and gene therapy may not optimally regulate the bone remodeling process due to the unitary pharmacological mechanism of drug and the safety and targeting of gene vectors. In the present study, multifunctional fluorescent alendronate‐polyethyleneimine carbon dots (Alen‐PEI CDs) are innovatively synthesized, which have excellent biocompatibility, selective bone targeting, gene carrying capacity, and anti‐bone absorption effect. As gene vectors, bone morphogenic protein 2 (BMP2) gene‐loaded Alen‐PEI CDs greatly enhance osteoblast differentiation and bone regeneration. Notably, Alen‐PEI CDs are found to directly inhibit the formation and function of osteoclasts, effectively attenuating the LPS‐induced skull osteolysis in mice. Furthermore, their cumulative toxicity is lower than that of Alen, indicating their potential as an anti‐bone resorptive nanodrug. These results show that the bone‐targeting Alen‐PEI CDs significantly reverse the imbalance of bone homeostasis through a dual‐directional mechanism—directly suppressing bone resorption and indirectly inducing bone formation by transfection of osteogenic therapeutic genes. Overall, the study expands the application for carbon dots in biomedicine, providing new insights for the further development of novel selectively targeted multifunctional nanodrugs for the treatment of bone‐destructive diseases.

Analysis of the Anti-Inflammatory and Anti-Osteoarthritic Potential of Flonat Fast®, a Combination of Harpagophytum Procumbens DC. ex Meisn., Boswellia Serrata Roxb., Curcuma longa L., Bromelain and Escin (Aesculus hippocastanum), Evaluated in In Vitro Models of Inflammation Relevant to Osteoarthritis.

Osteoarthritis (OA) is a joint disease characterized by inflammation of the synovium, angiogenesis, cartilage degradation, and osteophyte formation. Harpagophytum Procumbens DC. ex Meisn., Boswellia Serrata Roxb., Curcuma longa L., Bromelain and Escin (Aesculus hippocastanum) are plants which extracts, together to Bromelain and Escin (Aesculus hippocastanum) are traditionally used in OA. However, their mechanistic role remains unclear. We aimed to investigate whether these bioactives alone or in combination (as in Flonat Fast®) can suppress TNF-α-induced inflammation, angiogenesis, and osteophyte formation using two cell models involved in OA: endothelial cells and monocytes. Each plant extract was evaluated for its polyphenol content, antioxidant activity, and toxicity. In endothelial cells and monocytes, expression of genes involved in OA was assessed, functional assays for inflammation and angiogenesis were performed, and impairment of reactive oxygen species production (ROS) was evaluated. Exposure of cells to the bioactives alone and in combination before cytokine stimulation resulted in differential counterregulation of several gene and protein expressions, including those for cyclooxygenases-2, metalloproteinase-9, transforming growth factor β1, and bone morphogenic protein-2. We demonstrated that these bioactives modulated monocyte adhesion to endothelial cells as well as cell migration and endothelial angiogenesis. Consistent with radical scavenging activity in the cell-free system, the bioactives curbed TNF-α-stimulated intracellular ROS production. We confirmed the potential anti-inflammatory and antiangiogenic effects of the combination of Harpagophytum procumbens, Boswellia, Curcuma, Bromelain, and Escin and provided new mechanistic evidence for their use in OA. However, further clinical studies are needed to evaluate the true clinical utility of these bioactives as supportive, preventive, and therapeutic agents.

Does the Addition of Bone Marrow Aspirate Concentrate from the Pelvis Improve Fusion Rates of Hindfoot and Ankle Arthrodesis?

Category: Ankle Arthritis; Ankle; Hindfoot Introduction/Purpose: Ankle and hindfoot arthrodesis are common interventions for degenerative and inflammatory foot and ankle conditions. These include, but are not limited to osteoarthritis, inflammatory arthritis, deformity, and instability. While we know the addition of bone graft is critical for successful union, we do not fully understand how graft selection affects union rates. Demineralized bone matrix (DBM) is commonly chosen due to its ease of availability and osteoconductive and osteoinductive properties. In our study, we aim to determine if adding BMAc, and thus osteogenic properties, to a DBM allograft improves union rates following ankle and hindfoot arthrodesis. Methods: After receiving IRB approval from our institution, we collected the records of patients who underwent arthrodesis of the hindfoot (subtalar, calcaneocuboid, talonavicular) or ankle during a 10-year time period. Patients were then divided into two groups: arthrodesis performed with the use of DBM and arthrodesis performed with DMB-BMAc. Cases using other graft options such as bulk allograft, autograft, or bone morphogenic protein (BMP) were excluded. Fusion was determined by standard radiographic and clinical criteria and CT scans were utilized in cases which fusion could not be determined. Patients were followed for a minimum of 6 months. If fusion was not complete at that time clinical and radiographic exams were performed at the 9- and 12-month period. Patients were analyzed for sucussesful arthrodesis. Complications and revisions were recorded and analyzed. Results: A total of 124 patients who met the inclusion criteria were identified. In 49 patients only DBM was utilized, and 75 patients BMC was added to the DBM. At 12 months post operatively the group utilizing only DBM had a union rate of 90% while the union rate of the BMAc/DBM had a union rate of 88% (p=0.985) There was no difference in the rate of complications between the two groups and the groups were matched in terms of age, smoking status, DM, sex and BMI. Additionally, in our cohort there was a trend toward increased non-union and complication rate in smokers, although this did not reach statistical significance. Conclusion: Ankle and hindfoot arthrodesis are a reliable treatment option for patients with specific foot and ankle pathology. Our study reports a fusion rate in line with previous studies. The addition of BMAc from the pelvis, attempting to improve the biology of the fusion site, to DBM does not seem to affect fusion rates.

Biomimetic chitosan with biocomposite nanomaterials for bone tissue repair and regeneration.

Biomimetic materials for better bone graft substitutes are a thrust area of research among researchers and clinicians. Autografts, allografts, and synthetic grafts are often utilized to repair and regenerate bone defects. Autografts are still considered the gold-standard method/material to treat bone-related issues with satisfactory outcomes. It is important that the material used for bone tissue repair is simultaneously osteoconductive, osteoinductive, and osteogenic. To overcome this problem, researchers have tried several ways to develop different materials using chitosan-based nanocomposites of silver, copper, gold, zinc oxide, titanium oxide, carbon nanotubes, graphene oxide, and biosilica. The combination of materials helps in the expression of ideal bone formation genes of alkaline phosphatase, bone morphogenic protein, runt-related transcription factor-2, bone sialoprotein, and osteocalcin. In vitro and in vivo studies highlight the scientific findings of antibacterial activity, tissue integration, stiffness, mechanical strength, and degradation behaviour of composite materials for tissue engineering applications.

FGF receptor inhibitor BGJ398 partially rescues osteoarthritis-like phenotype in older high molecular weight FGF2 transgenic mice via multiple mechanisms

We have used Basic Fibroblast Growth Factor (FGF2) transgenic mice as experimental models for human X-linked hypophosphatemia (XLH)-related degenerative osteoarthritis (OA) to investigate the pathogenesis of the disease and to test potential pharmacotherapies for treatment. This study tested the efficacy of BJG398, a small molecule fibroblast growth factor receptor tyrosine kinase (FGFRTK) inhibitor, to rescue the knee joint osteoarthritis phenotype in High Molecular Weight fibroblast growth factor 2 transgenic (HMWTgFGF2) mice. BJG398 was administered in vivo to 8-month-old female HMWTgFGF2 mice for six weeks. Histomorphometry, immunohistochemistry and micro-CT were used to examine the knee joints in BGJ398-treated and control mice. We assessed: Fibroblast Growth Factor 23 (FGF23) expression and FGFR1 activity; Matrix metalloproteinase 13 (MMP13) and Aggrecanase2 (ADAMTS5) expression; then signaling by SMAD1/5/8-pSMAD6, pERK1/2 and Runt-related transcription factor 2 (RUNX2). Using PrimePCR arrays, we identified a contributing role for major target genes in the TGFB/BMP2 signaling pathway that were regulated by BGJ398. BGJ398 inhibited HMWFGF2/FGF23-induced increase in bone morphogenic protein receptor-1, bone morphogenic protein-2 and 4 and Serine peptidase inhibitor, clade E, member 1. The results from Micro-CT and histology show BGJ398 treatment rescued the OA changes in subchondral bone and knee articular cartilage of HMWTgFGF2 mice. The gene expression and signal transduction results provide convincing evidence that HMWFGF2 generates OA through FGFRTK with characteristic downstream signaling that defines OA, namely: increased FGF23-FGFR1 activity with BMP-BMPR, activation of pSMAD1/5/8-RUNX2 and pERK signaling pathways, then upregulation of MMP13 and ADAMTS5 to degrade matrix. BGJ398 treatment effectively reversed these OA molecular phenotypes, providing further evidence that the OA generated by HMWFGF2 in the transgenic mice is FGFR-mediated and phenocopies the OA found in the Hyp mouse homolog of XLH with a spontaneous mutation in the Phex (phosphate regulating endopeptidase on the X chromosome) gene and human XLH-OA. Overall, the results obtained here explain how the pleotropic effects of FGF2 emanate from the different functions of HMW protein isoforms for cartilage and bone homeostasis, and the pathogenesis of XLH-degenerative osteoarthropathy. BGJ398 inhibits HMWFGF2-induced osteoarthritis via multiple mechanisms. These results provided important scientific evidence for the potential application of BGJ398 as a therapeutic agent for osteoarthritis in XLH.

PSV-A-6 Poor Maternal Nutrition During Gestation Decreases Shear Force and Alters Gene Expression Related to Fibrogenesis

Abstract To test the hypothesis that poor maternal nutrition during gestation would alter offspring muscle shear force and adipogenesis, fibrogenesis, and myogenesis mRNA expression, Dorset ewes (n=46) pregnant with twins were fed 100%, 60% or 140% of NRC requirements from d30±0.02 of gestation until parturition. Male offspring from CON (n=12), OVER (n=13), and RES (n=21) ewes were euthanized at d284±1.82 and longissimus muscle (LM) was harvested for shear force and gene expression. Vacuum-sealed LM steaks were aged for 10 d postmortem (4℃) and tenderness was measured using Warner-Bratzler shear force (WBSF) methodology. RNA was isolated from the LM to quantify mRNA expression of genes related to adipogenesis (fatty acid binding protein 4, CCAAT/enhancer-binding protein α, and peroxisome proliferator-activated receptor γ), fibrogenesis [collagen A1A (COLA1A), COL3A1, cystain-c, fibronectin (FN1), and lysyl oxidase (LOX)], and myogenesis [bone-morphogenic protein (BMP)-1, myostatin, myogenin, and myogenic differentiation 1). The WBSF decreased by 21% and 18% in OVER relative to CON (P=0.01) and RES (P=0.03), respectively, but CON and RES were not different (P=0.60). Expression of BMP1 was 1.5- and 1.8-fold greater in RES than OVER and CON, respectively (P≤0.03). Expression of FN1 was 1.6-fold greater in RES than OVER (P=0.02). Expression of COL1A1 was 2.2-fold greater in RES than CON (P=0.05) and was 1.6-fold greater in RES than OVER (P=0.07). Expression of LOX was decreased 37% in OVER compared with CON (P=0.04) and decreased 21% compared with RES (P=0.08). An effect of diet was not observed for other genes (P≥0.18). In summary, RES offspring had greater expression of genes related to fibrogenesis, but no observed difference in tenderness relative to CON. Over-feeding during gestation increased meat tenderness, which may be due to reduced collagen cross-linking by LOX.

PSXIII-B-20 Variants within Genes Tspan18 and NRG3 Associated with Angular Limb Deformity in Rambouillet Rams

Abstract Incidence of angular limb deformities (ALD) have been described in many species of livestock and companion animals. Young, rapidly growing animals are at an increased risk for developing ALD. Although work has been done to identify the casual genetic basis of ALD, bone deformities are likely influenced by the environment, making clear genetic answers challenging to discover. Rambouillet rams from Wyoming, North Dakota and South Dakota annual ram performance tests have been observed to develop limb abnormalities consistent with ALD. Historical incidence of ALD in test rams has ranged from 3 to 18%. A genome-wide association study (GWAS) was conducted with 342 ram-test animals, including 40 ALD-affected rams and 302 unaffected rams collected over three consecutive years. Significant markers were identified within the genes tetraspanin 18 (TSPAN18; p-value = 4.87e-07) and neuregulin 3 (NRG3; p-value = 8.06e-07). In mice, Tspan18 has been proposed as a regulator of endothelial cell Orai1/Ca2+ signaling. Orai1 has also been shown to mediate osteogenic differentiation via the bone morphogenic protein signaling pathway, which suggests the possibility for TSPAN18 involvement in differentiation of chondrocytes and osteocytes. The gene NRG3 is a member of the neuregulin family, which encodes ligands for epidermal growth factor receptors ERBB3 and ERBB4. The epidermal growth factor receptor system has described roles in the regulation of proliferation and differentiation of osteoblast, chondrocyte and osteoclast cells. Identifying genetic risk factors for ALD has the potential to improve animal welfare and animal production in at-risk breeds. This study proposes TSPAN18 and NRG3 as targets for further research towards identifying genetic risk factors for ALD in Rambouillet sheep of the NW United States.

Initial upsurge of BMPs enhances long-term osteogenesis in in-vitro bone regeneration

Recent challenges in repair of critical size bone defects necessitates the need for scaffolds with accelerated bone defect repair. The higher surface area, complex porous architecture, and appropriate mechanical properties offered by engineered interlocking block scaffolds enables their use as bone grafts for critical size defect repair. Here, an interlocking scaffold system is designed by incorporating the bone morphogenic proteins (BMP-2 and BMP-7) and co-culturing with human osteoblasts (hFOB) and human mesenchymal stem cells (hMSCs). The complete release of BMPs from the porous scaffolds is observed by day 16. We evaluate the long-term effect of BMPs on in vitro bone regeneration at 9 weeks. A significant increase in the bone-related proteins and osteogenesis-related Wnt-factors with BMPs coated samples is observed as compared to uncoated samples, indicating that BMPs played an essential role in initial osteogenesis and ECM formation. We also report significant increase in the mineralized bone nodules with BMPs coated samples compared to without BMPs samples, suggesting that BMPs play a crucial role in mineralized ECM formation. Furthermore, nanoindentation results demonstrate a 120% increase in the elastic modulus at nine weeks with BMPs coated scaffolds indicating enhanced ECM formation. Thus, BMPs play a crucial role in osteogenesis at the initial stages and the effect of BMPs persists beyond the timescale of complete release at 16 days. This study provides valuable insight into the mechanisms of the BMPs association with bone tissue formation and demonstrates the feasibility of the interlocking scaffold system as a bone graft for bone defect repair application. Statement of SignificanceBMP-2 and BMP-7 are highly regarded as potent growth factors used for large bone defect repair and can be used with porous scaffolds. It is unclear how and what role BMPs play over a long period of time for bone defect therapies. Here, for the first time, we examined the change in the nanomechanical properties of the newly formed bony tissue over nine weeks and report a 120% increase in the elastic modulus at nine weeks with BMP coated scaffolds. In addition, alizarin Red S staining images and gene expression results suggest that BMPs play a significant role in mineralized ECM formation and influence osteogenesis initiation. This work provides a deeper understanding of the mechanisms of the BMPs association with bone tissue formation.

Antagonizing CD105 and androgen receptor to target stromal-epithelial interactions for clinical benefit

Androgen receptor signaling inhibitors (ARSIs) are standard of care for advanced prostate cancer (PCa) patients. Eventual resistance to ARSIs can include the expression of androgen receptor (AR) splice variant, AR-V7, expression as a recognized means of ligand-independent androgen signaling. We demonstrated that interleukin (IL)-6-mediated AR-V7 expression requires bone morphogenic protein (BMP) and CD105 receptor activity in both PCa and associated fibroblasts. Chromatin immunoprecipitation supported CD105-dependent ID1- and E2F-mediated expression of RBM38. Further, RNA immune precipitation demonstrated RBM38 binds the AR-cryptic exon 3 to enable AR-V7 generation. The forced expression of AR-V7 by primary prostatic fibroblasts diminished PCa sensitivity to ARSI. Conversely, downregulation of AR-V7 expression in cancer epithelia and associated fibroblasts was achieved by a CD105-neutralizing antibody, carotuximab. These compelling pre-clinical findings initiated an interventional study in PCa patients developing ARSI resistance. The combination of carotuximab and ARSI (i.e., enzalutamide or abiraterone) provided disease stabilization in four of nine assessable ARSI-refractory patients. Circulating tumor cell evaluation showed AR-V7 downregulation in the responsive subjects on combination treatment and revealed a three-gene panel that was predictive of response. The systemic antagonism of BMP/CD105 signaling can support ARSI re-sensitization in pre-clinical models and subjects that have otherwise developed resistance due to AR-V7 expression.

Tailoring of TiAl6V4 Surface Nanostructure for Enhanced In Vitro Osteoblast Response via Gas/Solid (Non-Line-of-Sight) Oxidation/Reduction Reactions.

An aging global population is accelerating the need for better, longer-lasting orthopaedic and dental implants. Additive manufacturing can provide patient-specific, titanium-alloy-based implants with tailored, three-dimensional, bone-like architecture. Studies using two-dimensional substrates have demonstrated that osteoblastic differentiation of bone marrow stromal cells (MSCs) is enhanced on surfaces possessing hierarchical macro/micro/nano-scale roughness that mimics the topography of osteoclast resorption pits on the bone surface. Conventional machined implants with these surfaces exhibit successful osseointegration, but the complex architectures produced by 3D printing make consistent nanoscale surface texturing difficult to achieve, and current line-of-sight methods used to roughen titanium alloy surfaces cannot reach all internal surfaces. Here, we demonstrate a new, non-line-of-sight, gas/solid-reaction-based process capable of generating well-controlled nanotopographies on all open (gas-exposed) surfaces of titanium alloy implants. Dense 3D-printed titanium-aluminum-vanadium (TiAl6V4) substrates were used to evaluate the evolution of surface nanostructure for development of this process. Substrates were either polished to be smooth (for easier evaluation of surface nanostructure evolution) or grit-blasted and acid-etched to present a microrough biomimetic topography. An ultrathin (90 ± 16 nm) conformal, titania-based surface layer was first formed by thermal oxidation (600 °C, 6 h, air). A calciothermic reduction (CaR) reaction (700 °C, 1 h) was then used to convert the surface titania (TiO2) into thin layers of calcia (CaO, 77 ± 16 nm) and titanium (Ti, 51 ± 20 nm). Selective dissolution of the CaO layer (3 M acetic acid, 40 min) then yielded a thin nanoporous/nanorough Ti-based surface layer. The changes in surface nanostructure/chemistry after each step were confirmed by scanning and transmission electron microscopies with energy-dispersive X-ray analysis, X-ray diffraction, selected area electron diffraction, atomic force microscopy, and mass change analyses. In vitro studies indicated that human MSCs on CaR-modified microrough surfaces exhibited increased protein expression associated with osteoblast differentiation and promoted osteogenesis compared to unmodified microrough surfaces (increases of 387% in osteopontin, 210% in osteocalcin, 282% in bone morphogenic protein 2, 150% in bone morphogenic protein 4, 265% in osteoprotegerin, and 191% in vascular endothelial growth factor). This work suggests that this CaR-based technique can provide biomimetic topography on all biologically facing surfaces of complex, porous, additively manufactured TiAl6V4 implants.

Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways.

Degenerative disc disease (DDD) is an important cause of low back pain. Repetitive tensile stress from the daily motion of the spine predisposes it to injury of the annulus fibrosus (AF) which causes IVD degeneration. This study aims to determine the causal relationship between mechanical stretch and osteogenesis in the AF cells of IVD as affected by bone morphogenic proteins (BMPs), specifically BMP-2/6 heterodimers. Our results found that 15% tensile stress (high cyclic stretching, HCS) may induce the expression of osteogenesis-related markers (Runx2, osterix) by upregulating BMP-2/6 heterodimeric ligands and their receptors on the human AF cell line. HCS also induced transient phosphorylation of p38 mitogen-activated protein (MAP) kinase and SMAD1/5/8. Neutralizing antibodies to the BMP-2/6 receptor (ALK3) blocked the expression of Runx2 and osterix, as well as the phosphorylation of p38 and SMAD1/5/8. In addition, treatment with a p38 MAPK inhibitor (SB203580) or siRNA to neutralize the effects of SMAD1/5/8 suppressed tensile stress-induced Runx2 and osterix expression. Mechanical stretching induces activation of p38 MAP kinase and SMAD1/5/8 signaling pathways, followed by the upregulation of BMP-2/6 heterodimer expression, thereby stimulating osteogenic Runx2 and osterix expression on AF cells. HCS may accelerate the progression of IVD degeneration by promoting an osteogenic response.

173. Anterior lumbar spinal fusion perioperative morbidity and mortality utilizing the National Inpatient Sample (NIS): 2005-2013

<h3>BACKGROUND CONTEXT</h3> Minimally invasive approaches to the lumbar spine have become increasingly popular for the treatment of degenerative conditions and deformity correction. Anterior lumbar interbody fusion (ALIF) is one type of approach to the lumbar spine that has seen a massive increase in utilization over the past two decades. However, large scale studies evaluating the overall morbidity and mortality of this procedure are scarce. The National Inpatient Sample (NIS) serves as the single largest all-payer inpatient database in the United States and provides weights for nationally representative estimates. For this reason, it can be used to provide an approximation of the overall perioperative morbidity and mortality statistics for procedures such as ALIF. <h3>PURPOSE</h3> To evaluate the perioperative morbidity and mortality, as well as demographics, of patients undergoing lumbar spinal fusion via an anterior approach from 2005 to 2013. <h3>STUDY DESIGN/SETTING</h3> National Inpatient Sample Database Study <h3>PATIENT SAMPLE</h3> A total of 27,912 patients who underwent ALIF from 2005-2013 identified using the NIS database. <h3>OUTCOME MEASURES</h3> (1) Demographics (age, gender, hospital region and teaching status, insurance status, race/ethnicity, household income and Charlson Comorbidity Index [CCI] score); 2) frequencies of complications (pulmonary embolism [PE], deep venous thrombus [DVT], infection, cardiac, hematoma, durotomy); and 3) mortality rate. <h3>METHODS</h3> The NIS database was reviewed from 2005 to 2013. Patients undergoing elective lumbar fusion surgery via an anterior approach were identified by ICD-9 code. Indications specific to the lumbar spine, such as lumbar degenerative disc disease, herniated disc, stenosis or radiculopathy were also identified by their respective ICD-9 codes. Demographics including age, sex, hospital region and teaching status, insurance status, race/ethnicity, median household income and CCI score were assessed. Frequencies of complications including PE, DVT, infection, cardiac, hematoma, durotomy and mortality were also analyzed. Statistical analysis involved T tests, χ 2 analysis and binary logistic regression with p<0.001 denoting significance. <h3>RESULTS</h3> We identified 27,912 patients which represented an estimated 137,928 of weighted patients hospitalized for primary anterior lumbar spine fusion. Patients undergoing anterior lumbar fusion had a mean age of 55.4 (SD±14.3) with 55.3% female patients. The majority of patients were white (82.2%), privately insured (50.4%), had a median household income of $48,000-62,999 (27.1%) and had a CCI score of 0.44 (SD±0.8). The majority of procedures were performed in the South region (39.5%), in nonteaching hospitals (54.5%), in hospitals that were private or not-for-profit (70.4%). Bone morphogenic protein (BMP) was used in 43.4% of cases. The most common complication was durotomy (1.2%) followed by cardiac complications and hematoma (0.7%) with the least common being PE (0.1%). The mortality rate was 0.2% for this procedure. <h3>CONCLUSIONS</h3> This study provides valuable data on the patient demographics and complications of ALIF across the United States. ALIF has a low complication and mortality rate. We expect, with the advancements in instrumentation, success rate of fusions, and an aging population, that anterior fusion will continue to see an increase in utilization by spine surgeons across the country. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Aberrant activation of KRAS in mouse theca-interstitial cells results in female infertility.

KRAS plays critical roles in regulating a range of normal cellular events as well as pathological processes in many tissues mediated through a variety of signaling pathways, including ERK1/2 and AKT signaling, in a cell-, context- and development-dependent manner. The in vivo function of KRAS and its downstream targets in gonadal steroidogenic cells for the development and homeostasis of reproductive functions remain to be determined. To understand the functions of KRAS signaling in gonadal theca and interstitial cells, we generated a Kras mutant (tKrasMT) mouse line that selectively expressed a constitutively active Kras G12D in these cells. Kras G12D expression in ovarian theca cells did not block follicle development to the preovulatory stage. However, tKrasMT females failed to ovulate and thus were infertile. The phosphorylated ERK1/2 and forkhead box O1 (FOXO1) and total FOXO1 protein levels were markedly reduced in tKrasMT theca cells. Kras G12D expression in theca cells also curtailed the phosphorylation of ERK1/2 and altered the expression of several ovulation-related genes in gonadotropin-primed granulosa cells. To uncover downstream targets of KRAS/FOXO1 signaling in theca cells, we found that the expression of bone morphogenic protein 7 (Bmp7), a theca-specific factor involved in ovulation, was significantly elevated in tKrasMT theca cells. Chromosome immunoprecipitation assays demonstrated that FOXO1 interacted with the Bmp7 promoter containing forkhead response elements and that the binding activity was attenuated in tKrasMT theca cells. Moreover, Foxo1 knockdown caused an elevation, whereas Foxo1 overexpression resulted in an inhibition of Bmp7 expression, suggesting that KRAS signaling regulates FOXO1 protein levels to control Bmp7 expression in theca cells. Thus, the anovulation phenotype observed in tKrasMT mice may be attributed to aberrant KRAS/FOXO1/BMP7 signaling in theca cells. Our work provides the first in vivo evidence that maintaining normal KRAS activity in ovarian theca cells is crucial for ovulation and female fertility.

Cyclophilin A Promotes Osteoblast Differentiation by Regulating Runx2.

Cyclophilin A (CypA) is a ubiquitously expressed and highly conserved protein with peptidyl-prolyl cis-trans isomerase activity that is involved in various biological activities by regulating protein folding and trafficking. Although CypA has been reported to positively regulate osteoblast differentiation, the mechanistic details remain largely unknown. In this study, we aimed to elucidate the mechanism of CypA-mediated regulation of osteoblast differentiation. Overexpression of CypA promoted osteoblast differentiation in bone morphogenic protein 4 (BMP4)-treated C2C12 cells, while knockdown of CypA inhibited osteoblast differentiation in BMP4-treated C2C12. CypA and Runx2 were shown to interact based on immunoprecipitation experiments and CypA increased Runx2 transcriptional activity in a dose-dependent manner. Our results indicate that this may be because CypA can increase the DNA binding affinity of Runx2 to Runx2 binding sites such as osteoblast-specific cis-acting element 2. Furthermore, to identify factors upstream of CypA in the regulation of osteoblast differentiation, various kinase inhibitors known to affect osteoblast differentiation were applied during osteogenesis. Akt inhibition resulted in the most significant suppression of osteogenesis in BMP4-induced C2C12 cells overexpressing CypA. Taken together, our results show that CypA positively regulates osteoblast differentiation by increasing the DNA binding affinity of Runx2, and Akt signaling is upstream of CypA.

Reduced bone morphogenic protein signaling along the gut-neuron axis by heat shock factor promotes longevity.

Aging is a complex and highly regulated process of interwoven signaling mechanisms. As an ancient transcriptional regulator of thermal adaptation and protein homeostasis, the Heat Shock Factor, HSF‐1, has evolved functions within the nervous system to control age progression; however, the molecular details and signaling dynamics by which HSF‐1 modulates age across tissues remain unclear. Herein, we report a nonautonomous mode of age regulation by HSF‐1 in the Caenorhabditis elegans nervous system that works through the bone morphogenic protein, BMP, signaling pathway to modulate membrane trafficking in peripheral tissues. In particular, HSF‐1 represses the expression of the neuron‐specific BMP ligand, DBL‐1, and initiates a complementary negative feedback loop within the intestine. By reducing receipt of DBL‐1 in the periphery, the SMAD transcriptional coactivator, SMA‐3, represses the expression of critical membrane trafficking regulators including Rab GTPases involved in early (RAB‐5), late (RAB‐7), and recycling (RAB‐11.1) endosomal dynamics and the BMP receptor binding protein, SMA‐10. This reduces cell surface residency and steady‐state levels of the type I BMP receptor, SMA‐6, in the intestine and further dampens signal transmission to the periphery. Thus, the ability of HSF‐1 to coordinate BMP signaling along the gut–brain axis is an important determinate in age progression.