Bone Morphogenic Protein Research Articles

The importance of BMPs and TGF-βs for endochondral bone repair – A longitudinal study in hip arthroplasty patients

IntroductionOsseointegration of hip implants, although a decade-long process, shows striking similarities with the four major phases of endochondral bone repair. In the current study we investigated the spatiotemporal involvement of bone morphogenic proteins (BMPs) and transforming growth factor betas (TGF-βs) throughout the process of bone repair leading to successfully osseointegrated hip implants. Materials and methodsTwenty-four patients that had undergone primary total hip arthroplasty (THA) due to one-sided osteoarthritis (OA) were investigated during a period of 18 years (Y) with repeated measurements of plasma biomarkers as well as clinical and radiological variables. All implants were clinically and radiographically well anchored throughout the follow-up. Eighty-one healthy donors divided in three gender- and age-matched groups and twenty OA patients awaiting THA, served as controls. Plasma was analyzed for BMP-1, -2, -3, -4, -6, -7 -9 and TGF-β1, -β2, -β3 by use of a high-sensitivity and wide dynamic range electrochemiluminescence technique allowing for detection of minor changes. ResultsSpatiotemporal changes during the follow-up are presented in the context of the four phases of endochondral bone repair shown in earlier studies and transposed to the current study based on similarities in biomarker responses. Phase 1: Primary proinflammatory phase lasting from surgery until day 7, Phase 2: Chondrogenic phase from day 7 until 18 months postsurgery, Phase 3: Secondary proinflammatory and cartilage remodeling phase lasting from 18 months until 7Y, Phase 4: coupled bone remodeling from 7Y until 18Y postsurgery. BMP-1 increased sharply shortly after surgery and remained significantly above healthy during the chondrocyte recruitment, proliferation, and hypertrophy phases with a subsequent return to control level at 5Y postsurgery. BMP-2 was above healthy controls before surgery and 1 day after surgery before decreasing to control level and remaining there throughout the follow-up. BMP-3 was at control level from presurgery until 6M after surgery when it increased to a peak at 2Y during the cartilage hypertrophy phase followed by a gradual decrease to control level at 10Y during the phase of bone formation. In the following, BMP-3 decreased below controls to a nadir 15Y postsurgery during coupled bone remodeling. BMP-4 was at control level from presurgery until 10Y postsurgery when it increased to a sharp peak at 15Y after surgery followed by a return to the level of healthy at 18Y. BMP-6 did not differ from healthy during the follow-up. BMP-7 was at control level from presurgery until 1Y postsurgery before gradually increasing to a peak at 10Y during the early phase of osteogenesis with a gradual return to control level at 18Y during the phase of coupled bone remodeling. BMP-9 was above OA before surgery followed by a decrease to basal level on day 1 after surgery and a renewed increase to a plateau above controls lasting from 6 W until returning to the level of healthy at 18Y postsurgery, i.e., throughout the phases of cartilage formation, cartilage hypertrophy and remodeling, bone formation and coupled bone remodeling. TGF-β1 was above controls presurgery before decreasing to baseline shortly after surgery followed by a renewed increase at 6 M to a peak at 2Y during cartilage hypertrophy/remodeling followed by a gradual return to baseline at 10Y during early osteoblastogenesis. TGF-β2 was at control level from presurgery until the phase of cartilage remodeling at 5Y when it increased sharply to a peak at 7Y with a gradual return to baseline at 18Y postsurgery. TGF-β3 remained at control level throughout the study. ConclusionThis study shows that the involvement of BMPs and TGF-βs in endochondral bone repair is a process of stepwise recruitment of individual biomarkers characterized by distinct, yet overlaping, spatiotemporal patterns that extend from the early phase of pre-chondrocyte recruitment until the late phase of coupled bone remodeling.

Repeated measurement of the novel atrial biomarker BMP10 improves risk stratification in anticoagulated patients with atrial fibrillation: Insights from the ARISTOTLE trial

Abstract Background Atrial fibrillation (AF) increases the risk of stroke, heart failure, and death. Biomarkers provide important prognostic information about these complications in AF. Recently, bone morphogenic protein 10 (BMP10), a protein mainly expressed in atrial tissue, was identified as a novel marker in AF for the risk of ischemic stroke. Purpose To assess the association between baseline characteristics with BMP10 concentrations, and if repeated BMP10 measurements at 2 months improve the prognostication of ischemic stroke or systemic embolism and other cardiovascular events in patients with AF. Methods BMP10 was measured in plasma samples collected at randomization and at 2 months in patients with AF who were randomized to apixaban or warfarin in the ARISTOTLE trial (n = 2878) with a median follow-up time of 1.8 years. BMP10 was analyzed with a prototype Elecsys immunoassay. The association of baseline variables with BMP10 at 2 months was evaluated with linear regression analysis. Cox-regression models adjusted for baseline characteristics and comorbidities including kidney function and BMP10-level at baseline were used in order to evaluate associations with outcomes. Results Median age was 70 (63.0-76.0) and 63% were male. The events during the follow up period were 34 ischemic strokes, 155 deaths and 99 hospitalizations for heart failure. Median concentration of BMP10 was 2.4 ng/mL at inclusion and 2.6 ng/mL at 2 months (median increase 7.8%, p<0.001). The Spearman correlation for BMP10 at baseline and 2 months was 0.71. The baseline variables most strongly associated with higher BMP10 levels at 2 months, in descending order of importance, were: higher BMI, female sex, worse kidney function, older age, AF-rhythm, diabetes, and randomized treatment. BMP10 levels at 2 months improved the risk stratification in models adjusted for clinical variables including baseline levels of BMP10. For ischemic stroke and death, the estimated associations were nonlinear (Figure) with increased risk with higher BMP10 levels (p=0.037 and p<0.001, respectively), whereas the association with HF hospitalization was linear with HR of 1.91 (p=0.012) comparing the third and first sample quartiles. Patients randomized to apixaban had approximately 2.2% lower BMP10 levels at 2 months compared to those randomized to warfarin (p<0.001). Conclusions In patients with AF, the level of the atrial biomarker BMP10 is consistently associated with the risk of stroke at baseline and after 2 months of treatment with warfarin or apixaban. Repeated compared to only one measurement of BMP10 further improves risk stratification. Apixaban as compared to warfarin is associated with a reduction in the BMP10 level, which is in accordance with the lower risk of stroke with apixaban as compared to warfarin.

Use of biosilica to improve loading and delivery of bone morphogenic protein 2

The clinical utility of bone morphogenetic protein 2 (BMP2) is limited because of the poor attraction between BMP2 and carriers, resulting in low loading efficiency and initial burst release. Here, the high binding affinity of BMP2 to the biosilica surface was utilized to overcome this limitation. Atomic force microscopy revealed that BMP2 bound nearly 8- and 2-fold more strongly to biosilica-coated hydroxyapatite than to uncoated and plain silica-coated hydroxyapatite, respectively. To achieve controlled release, collagen was introduced between the silica layers on hydroxyapatite, which was optimized by adjusting the collagen concentration and number of layers. The optimal biosilica/collagen formulation induced sustained BMP2 release without compromising loading efficiency. BMP2 combined with the mentioned formulation led to an increase in osteogenesis, as compared to the combination of BMP2 with either biosilica-coated or non-coated hydroxyapatite in vitro. In rat calvarial defect models, the biosilica/collagen-coated hydroxyapatite with 1 μg BMP2 showed 26 % more bone regeneration than the same dose of BMP2-loaded hydroxyapatite and 10.6 % more than hydroxyapatite with 2.5-fold dose of BMP2. Using BMP2 affinity carriers coated with biosilica/collagen allows for more efficacious in situ loading and delivery of BMP2, making them suitable for the clinical application of growth factors through a soaking method.

Upregulation of human GD3 synthase (hST8Sia I) gene expression during serum starvation-induced osteoblastic differentiation of MG-63 cells.

In this study, we have firstly elucidated that serum starvation augmented the levels of human GD3 synthase (hST8Sia I) gene and ganglioside GD3 expression as well as bone morphogenic protein-2 and osteocalcin expression during MG-63 cell differentiation using RT-PCR, qPCR, Western blot and immunofluorescence microscopy. To evaluate upregulation of hST8Sia I gene during MG-63 cell differentiation by serum starvation, promoter area of the hST8Sia I gene was functionally analyzed. Promoter analysis using luciferase reporter assay system harboring various constructs of the hST8Sia I gene proved that the cis-acting region at -1146/-646, which includes binding sites of the known transcription factors AP-1, CREB, c-Ets-1 and NF-κB, displays the highest level of promoter activity in response to serum starvation in MG-63 cells. The -731/-722 region, which contains the NF-κB binding site, was proved to be essential for expression of the hST8Sia I gene by serum starvation in MG-63 cells by site-directed mutagenesis, NF-κB inhibition, and chromatin immunoprecipitation (ChIP) assay. Knockdown of hST8Sia I using shRNA suggested that expressions of hST8Sia I and GD3 have no apparent effect on differentiation of MG-63 cells. Moreover, the transcriptional activation of hST8Sia I gene by serum starvation was strongly hindered by SB203580, a p38MAPK inhibitor in MG-63 cells. From these results, it has been suggested that transcription activity of hST8Sia I gene by serum starvation in human osteosarcoma MG-63 cells is regulated by p38MAPK/NF-κB signaling pathway.

Stromal BMP signaling regulates mucin production in the large intestine via interleukin-1/17.

Bone morphogenic protein (BMP) signaling is critical for intestinal development, homeostasis, and function performance. Although the function of BMP signaling in the intestinal epithelium is well appreciated, the direct effect of BMP on intestinal stromal cells is poorly understood. Here, we show that disruption of BMP signaling by genetic ablation of Alk3 or Smad4 expands the stromal cell pool, the mucosa tumefaction, and colonic polyposis in the large intestine. Interleukin (IL) secretion by stromal cells is notably increased, including IL-1, IL-11, and IL-17. Specifically, IL-1 and IL-17a hyperactivate the mucin production by goblet cells through nuclear factor κB signaling, and abnormal mucin accumulation results in the morphological changes, epithelial barrier destruction, and polyposis development. Together, our results provide an insight into the role of BMP signaling in intestinal stromal cells to regulate epithelium function. This study further highlights the role of mucin-producing goblet cells in intestinal homeostasis and colitis development.

Regeneration of Pancreatic Cells Using Optimized Nanoparticles and l-Glutamic Acid-Gelatin Scaffolds with Controlled Topography and Grafted Activin A/BMP4.

Regeneration of insulin-producing cells (IPCs) from induced pluripotent stem cells (iPSCs) under controlled conditions has a lot of promise to emulate the pancreatic mechanism in vivo as a foundation of cell-based diabetic therapy. l-Glutamic acid-gelatin scaffolds with orderly pore sizes of 160 and 200 μm were grafted with activin A and bone morphogenic proteins 4 (BMP4) to differentiate iPSCs into definitive endoderm (DE) cells, which were then guided with fibroblast growth factor 7 (FGF7)-grafted retinoic acid (RA)-loaded solid lipid nanoparticles (FR-SLNs) to harvest IPCs. Response surface methodology was adopted to optimize the l-glutamic acid-to-gelatin ratio of scaffolds and to optimize surfactant concentration and lipid proportion in FR-SLNs. Experimental results of immunofluorescence, flow cytometry, and western blots revealed that activin A (100 ng/mL)-BMP4 (50 ng/mL)-l-glutamic acid (5%)-gelatin (95%) scaffolds provoked the largest number of SOX17-positive DE cells from iPSCs. Treatment with FGF7 (50 ng/mL)-RA (600 ng/mL)-SLNs elicited the highest number of PDX1-positive β-cells from differentiated DE cells. To imitate the natural pancreas, the scaffolds with controlled topography were appropriate for IPC production with sufficient insulin secretion. Hence, the current scheme using FR-SLNs and activin A-BMP4-l-glutamic acid-gelatin scaffolds in the two-stage differentiation of iPSCs can be promising for replacing impaired β-cells in diabetic management.

Efficacy of rhBMP-2 and Cellular Bone Matrix in the Revision of Alveolar Bone Grafting

Objective: This study presents a novel approach that utilizes recombinant human bone morphogenic protein 2 (rhBMP-2) and cellular bone matrix (CBM) for revision alveolar bone graft (ABG) in unilateral cleft lip and palate (UCLP) and bilateral cleft lip and palate (BCLP) patients following failed autologous ABG. Methods: A case series of 14 UCLP and 4 BCLP patients who had failed autologous ABG with revision ABG with rhBMP-2 and CBM. Review of demographics, operative data, Bergland score, and alveolar cleft gap volume was performed. Results: Revision ABG had short operative times and low blood loss in both UCLP and BCLP. No postoperative complications were found in either revision group. Repeat ABG surgery was required in one patient. Bergland score improved from 3.86 ± 0.53to 1.21 ± 0.80 in the UCLP group and from 3.75 ± 0.50to 1.00 ± 0.00 in the BCLP group. The cleft volume decreased by 83.62 ± 9.78% in UCLP and by 86.73 ± 13.65% in the BCLP group. Conclusions: Revision ABG with rhBMP-2 and CBM is a successful and reliable approach. This method has decreased operative time and no postoperative complications. Most patients achieved clinically successful grafting with canine eruption. Both UCLP and BCLP groups saw a significant decrease in alveolar cleft gap volume.

Synergistic effect of FGF-2 and TGF-β1 on the mineralization of human umbilical cord perivascular cells

ObjectiveHuman umbilical cord perivascular cells (HUCPVCs) are derived from the human umbilical cord perivascular tissue and are expected to replace mesenchymal stromal cells in the future. We investigated the synergistic effects of fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 1 (TGF-β1) on HUCPVC mineralization. DesignWe prepared HUCPVCs with (FGF(+)HUCPVCs) or without FGF-2 (FGF(-)HUCPVCs) in the presence of activated vitamin D3, a bone morphogenic protein inhibitor, and TGF-β1. We examined the cell proliferative capacity, expression of various hard tissue-forming cell gene markers, and mineralization induction ability and identified the crystalline phases of the mineralized nodules. ResultsFGF(+)HUCPVCs exhibited higher intracellular alkaline phosphatase (ALP) gene expression and ALP activity, and their cell proliferation rate was higher than that of FGF(-)HUCPVCs. The expression levels of osteoblast marker genes increased in FGF(+)HUCPVCs, whereas those of elastic fiber and muscle cell markers increased in FGF(-)HUCPVCs. The expression of genes related to matrix vesicle-mediated mineralization was increased in FGF(+)HUCPVCs. While FGF(-)HUCPVCs displayed myofibroblast-like properties and could not induce mineralization, FGF(+)HUCPVCs demonstrated the ability to produce mineralized nodules. The resulting mineralized nodules consisted of hydroxyapatite as the major phase and minor amounts of octacalcium phosphate. The mineralized nodules exhibited the morphological characteristics of bone hydroxyapatite, composed of fibrous hydroxyapatite nanorods and polycrystalline sheets. ConclusionWe found that FGF-2 synergizes with TGF-β1 and is a key factor in the differentiation of HUCPVCs into osteoblast-like cells. Thus, HUCPVCs can potentially serve as a new stem cell source for future bone regeneration and dental treatments.

Effect of Mixture of Recombinant Human Bone Morphogenic Protein-2 and Demineralized Bone Matrix in Lateral Lumbar Interbody Fusion.

This study aims to determine the optimal dose of recombinant-human bone morphogenic protein-2 (rhBMP-2) for successful bone fusion in minimally invasive lateral lumbar interbody fusion (MIS LLIF). Previous studies show that rhBMP is an effective alternative to autologous iliac crest bone graft, but the optimal dose remains uncertain. The study analyzes the fusion rates associated with different rhBMP doses to provide a recommendation for the optimal dose in MIS LLIF. Ninety-three patients underwent MIS LLIF using demineralized bone matrix (DBM) or a mixture of rhBMP-2 and DBM as fusion material. The group was divided into the following three groups according to the rhBMP-2 usage : group A, only DBM was used (n=27); group B, 1 mg of rhBMP-2 per 5 mL of DBM paste (n=41); and group C, 2 mg of rhBMP-2 per 5 mL of DBM paste (n=25). Demographic data, clinical outcomes, postoperative complication and fusion were assessed. At 12 months post-surgery, the overall fusion rate was 92.3% according to Bridwell fusion grading system. Groups B and C, who received rhBMP-2, had significantly higher fusion rates than group A, who received only DBM. However, there was no significant increase in fusion rate when the rhBMP-2 dosage was increased from group B to group C. The groups B and C showed significant improvement in back pain and Oswestry disability index compared to the group A. The incidence of screw loosening was decreased in groups B and C, but there was no significant difference in the occurrence of other complications. Usage of rhBMP-2 in LLIF surgery leads to early and increased final fusion rates, which can result in faster pain relief and return to daily activities for patients. The benefits of using rhBMP-2 were not significantly different between the groups that received 1 mg/5 mL and 2 mg/5 mL of rhBMP-2. Therefore, it is recommended to use 1 mg of rhBMP-2 with 5 mL of DBM, taking both economic and clinical aspects into consideration.

OR2803 Corticotroph Fate Weighted Cell Differentiation Paradigm For Human Induced Pluripotent Stem Cells

Abstract Disclosure: M.E. Moore: None. K.G. Chen: None. K. Park: None. D. Asuzu: None. N. Ramavenkat: None. S. Walbridge: None. D. Mullaney: None. M. Arhin: None. D. Maric: None. D. Mandal: None. P. Chittiboina: None. Introduction: The vertebrate anterior pituitary gland is a highly conserved ‘master’ organ that controls hormonal milieu in real-time. Pituitary adenomas and their treatments (surgery, chemotherapy or radiation) can result in pituitary organ failure, specifically hypocortisolism, a challenging condition to manage. Here, we created an optimized paradigm (anterior pituitary corticotroph or APcor) to preferentially drive human induced pluripotent stem cells (hiPSCs) towards a pituitary corticotroph fate. Methods: We directed U112I hiPSCs towards terminally differentiated cell types using a combination of rho-associated kinase inhibitor (Y-27632), bone morphogenic protein 4 (BMP4), transforming growth factor β inhibitor (SB431542), sonic hedgehog (SHH), fibroblast growth factor 8b (FGF8b), fibroblast growth factor 10 (FGF10) and leukemia inhibitory factor (LIF). Markers for pluripotent stem cells, cranial placode (CP), Rathke’s Pouch (RP), anterior pituitary progenitors and mature cell types were assessed at 10-day intervals using quantitative qRT-PCR and multiplex immunocytochemistry (mICC) or immunohistochemistry (IHC). ACTH activity was measured using ELISA (MD Bio). Results: We tested initial differentiation conditions and found optimal CP initiation in presence of SB431542. We observed decrease in embryonic stem cell markers OCT4 and NANOG by day 10, with concomitant increase in CP/RP markers SIX1, PAX6 and SIX6 using mICC/IHC and qRT-PCR. Continuation of differentiation paradigm with LIF showed strong expression of anterior pituitary progenitor markers PITX1, TBX19 and POU1F1 at day 20. pRT-PCR analysis at this time point confirms differentiation towards pituitary cell types without needing enrichment for SIX1. At day 30, POMC expression was observed by IHC in 10-15% of cells as measured by quantitative analysis (Qupath 0.2.0), indicating mature corticotrophs. Corticotroph cell fate was confirmed with corticotropin releasing hormone (CRH) stimulation which led to up to 40x-fold increase in ACTH expression detected by ELISA (D30 w/CRH vs Pluripotent w/CRH). Conclusions: We found with the APcor paradigm, we can reliably drive towards corticotroph cell fate. We successfully differentiated hiPSCs into mature functioning corticotrophs with ability to recapitulate pituitary organ function. Our method is amenable to 3D organoid formation and pre-clinical implantation in animal models of hypophysectomy as a novel hormone replacement therapy. Presentation: Sunday, June 18, 2023

FRI116 Inhibition Of Mitochondrial Fission And Restoration Of Mitochondrial Function Attenuates Vascular Calcification

Abstract Disclosure: S. Kwon: None. M. Kim: None. S. Zerwa: None. S. Kim: None. B. Jueun: None. J. Jeon: None. I. Lee: None. Objectives: Vascular calcification is associated with cardiovascular disease mortality, especially in patients with diabetes mellitus and chronic kidney disease. One of proposed mechanisms for development of vascular calcification suggested mitochondrial dysfunction of vascular smooth muscle cells (VMSCs), especially excessive mitochondrial fission that led to apoptosis and tissue calcium deposit formation. In existing literature, Vitamin B1 inhibited mitochondrial fission and improved mitochondrial function. This study investigated whether inhibiting mitochondrial fission prevents vascular calcification in vitro and in vivo by using inorganic phosphate and cholecalciferol-induced vascular calcification models, respectively. Methods: VMSCs from thoracic aorta of 4-week-old rats were treated by 2.6mM inorganic phosphate and 2mM calcium chloride to induce calcification. 6-week-old male C57BL/6J mice were given vitamin B1 analogue solution or water with daily oral gavage for 13 days, then aortic calcification was induced by subcutaneous injection of 5.5×10-5 IU/kg cholecalciferol for 3 days before sacrifice. Cells and tissues were stained for calcium deposition, (van Kossa stain), apoptosis (TUNEL) and mitochondrial morphology (Mitotracker). Western blotting and quantitative real-time PCR were used to assess calcification pathways and mitochondrial function. Tissue calcium content, normalized with total protein contents, was measured to quantify the extent of calcification. Mitochondrial oxygen consumption ratio (OCR) and mitochondrial membrane potential (JC-1 dye staining) were assessed to analyze quantified mitochondrial function. Results: Calcium deposition in both in vivo and in vitro models were alleviated by vitamin B1 analogue through inhibition of mitochondrial fission and restoration of mitochondrial function. In addition, vitamin B1 analogue downregulated expression of genes related to calcification and osteogenesis, such as bone morphogenic protein 2, osteocalcin, osteopontin, and runt-related transcription factor 2. Conclusion: Prevention of mitochondrial dysfunction in VSMCs may be a viable therapeutic strategy for treatment of vascular calcification. Presentation: Friday, June 16, 2023

Bone Morphogenic Proteins and Their Antagonists in the Lower Airways of Stable COPD Patients.

Bone morphogenic proteins (BMPs) and their antagonists are involved in the tissue development and homeostasis of various organs. To determine transcriptomic and protein expression of BMPs and their antagonists in stable COPD. We measured the expression and localization of BMPs and some relevant antagonists in bronchial biopsies of stable mild/moderate COPD (MCOPD) (n = 18), severe/very severe COPD (SCOPD) (n = 16), control smokers (CS) (n = 13), and control non-smokers (CNS) (n = 11), and in lung parenchyma of MCOPD (n = 9), CS (n = 11), and CNS (n = 9) using immunohistochemistry and transcriptome analysis, in vitro after the stimulation of the 16HBE cells. In bronchial biopsies, BMP4 antagonists CRIM1 and chordin were increased in the bronchial epithelium and lamina propria of COPD patients. BMP4 expression was decreased in the bronchial epithelium of SCOPD and MCOPD compared to CNS. Lung transcriptomic data showed non-significant changes between groups. CRIM1 and chordin were significantly decreased in the alveolar macrophages and alveolar septa in COPD patients. External 16HBE treatment with BMP4 protein reduced the bronchial epithelial cell proliferation. These data show an imbalance between BMP proteins and their antagonists in the lungs of stable COPD. This imbalance may play a role in the remodeling of the airways, altering the regenerative-reparative responses of the diseased bronchioles and lung parenchyma.

Minimally Invasive Transforaminal Lumbar Interbody Fusion (MI-TLIF): A Video Technique Guide.

Minimally invasive transforaminal lumbar interbody fusion (MI-TLIF) has been established as an excellent alternative to the traditional open approach for the treatment of degenerative conditions of the lumbar spine1-3. The procedure is performed with the patient under general anesthesia and on a radiolucent table in order to allow for intraoperative fluoroscopy. The procedure is performed through small incisions made over the vertebral levels of interest, typically utilizing either a fixed or expandable type of tubular dilator, which is eventually seated against the facet joint complex4. A laminectomy and/or facetectomy is performed in order to expose the disc space, and the ipsilateral neural elements are visualized5. The end plates are prepared, and an interbody device is placed after the disc is removed. Pedicle screws and rods are then placed for posterior fixation. Nonoperative alternatives include physical therapy and corticosteroid injections. Other operative techniques include open TLIF or other types of lumbar fusion approaches, such as posterior lumbar interbody fusion (PLIF), anterior lumbar interbody fusion, lateral or extreme lateral interbody fusion, or oblique lumbar interbody fusion. Open TLIF was developed in order to obtain a more lateral approach to the lumbar disc space than was previously possible with PLIF. The goal of this was to minimize the amount of thecal-sac and nerve-root retraction required during PLIF4. Additionally, as the number of patients who required revision after PLIF increased, the need arose for an approach to the lumbar spine that circumvented the posterior midline scarring from previous PLIF surgical sites6. MI-TLIF was introduced to reduce the approach-related paraspinal muscle damage of open TLIF5. Indications for MI-TLIF include most degenerative pathology of the lumbar spine, including disc herniation, low-grade spondylolisthesis, and spinal and foraminal stenosis7. However, MI-TLIF allows for less robust correction of deformity than other minimally invasive approaches; therefore, MI-TLIF may not be as effective in cases of substantial spinal deformity or high-grade spondylolisthesis8. MI-TLIF results in significantly less blood loss, postoperative pain, and hospital length of stay compared with open TLIF1-3. Although some studies have suggested increased operative time for MI-TLIF9,10, meta-analyses have shown comparable operative times between the 2 techniques1-3. It is thought that the discrepancy in reported operative times is the result of a learning curve and that, once that is overcome, the difference in operative time between the 2 techniques becomes minimal11,12. One disadvantage of MI-TLIF that has remained constant in the literature is its increased intraoperative fluoroscopy time compared with open TLIF3,13. The complication rate has largely been found to be equivalent between open and MI-TLIF1-3 or slightly lower with MI-TLIF14, especially in the hands of an experienced surgeon15. Finally, the fusion rate and improvement in patient outcome scores have also been found to be largely equivalent1-3. We suggest placing the ipsilateral pedicle screw after the interbody cage has been inserted.Fully visualize the Kambin triangle16 prior to performing the facetectomy. Protect the exiting and traversing nerve roots by placing small cottonoids around them and retracting delicately.Bone removed during facetectomy can be utilized as autograft for the interbody cage.Avoid removing pedicle bone during decompression.If central stenosis is present, the neural decompression should be extended medial to the epidural fat so that the dura mater can be visualized all of the way to the contralateral pedicle.Perform an adequate end plate preparation prior to interbody insertion while being mindful to avoid injuring the end plate, to minimize the risk of future cage subsidence.Confirm correct placement of the interbody device on intraoperative fluoroscopy.If bone morphogenic protein is utilized, be careful not to pack too much posteriorly as this may cause nerve irritation. TLIF = transforaminal lumbar interbody fusionMI-TLIF = minimally invasive TLIFPLIF = posterior lumbar interbody fusionALIF = anterior lumbar interbody fusionLLIF = lateral lumbar interbody fusionXLIF = extreme lateral interbody fusionOLIF = oblique lumbar interbody fusionDLIF = direct lateral interbody fusionMRI = magnetic resonance imagingA/P = anteroposteriorEMG = electromyographicBMP = bone morphogenic proteinXR = x-ray (radiograph)OTC = over the counterDVT = deep vein thrombosisPE = pulmonary embolismMI = myocardial infarctionMIS = minimally invasive surgeryOR = operating roomLOS = length of stayVAS = visual analog scaleODI = Oswestry Disability IndexM-H = Mantel-HaenszelRR = risk ratioCI = confidence intervalNSAIDs = nonsteroidal anti-inflammatory drugs.

A Novel Application of Neural Networks to Identify Potentially Effective Combinations of Biologic Factors for Enhancement of Bone Fusion/Repair

Category: Basic Sciences/Biologics; Other Introduction/Purpose: The use of biologic adjuvants (orthobiologics) is becoming commonplace in orthopaedic surgery. Amongst 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 to identify combinations of orthobiologic factors that potentially would be more effective than single agents. Methods: 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. Results: The most effective combinations were high in the bone-morphogenic proteins (BMP) 2 and 7 (BMP2, 15mg; BMP7, 5mg), and in osteogenin (150ug). Some of the most effective combinations included bone marrow aspirate concentrate. In some others, electrical stimulation could substitute for osteogenin. BMP2 and BMP7 appear to have the strongest pairwise linkage of the factors analyzed in this study. Conclusion: Artificial neural networks are powerful forms of artificial intelligence that can be applied readily in the orthopeadic 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.

Endogenous Release of Bone Morphogenic Protein 4 from Dentin Scaffolds in Response to Different EDTA Surface Treatment Durations

Endogenous Release of Bone Morphogenic Protein 4 from Dentin Scaffolds in Response to Different EDTA Surface Treatment Durations

Omega-3 polyunsaturated fatty acids and its metabolite 12-HEPE rescue busulfan disrupted spermatogenesis via target to GPR120.

Busulfan is an antineoplastic, which is always accompanied with the abnormal of spermatogonia self-renewal and differentiation. It has been demonstrated that the omega-3 polyunsaturated fatty acids (PUFAs) benefits mature spermatozoa. However, whether omega-3 can protect endogenous spermatogonia and the detailed mechanisms are still unclear. Evaluate of spermatogenesis function (in vivo) were examined by histopathological analysis, immunofluorescence staining, and western blotting. The levels of lipid metabolites in testicular tissue were determined via liquid chromatography. We investigated the effect of lipid metabolites on Sertoli cells provided paracrine factors to regulate spermatogonia proliferation and differentiation using co-culture system. In our study, we showed that omega-3 PUFAs significantly improved the process of sperm production and elevated the quantity of both undifferentiated Lin28+ spermatogonia and differentiated c-kit+ spermatogonia in a mouse model where spermatogenic function was disrupted by busulfan. Mass spectrometry revealed an increase in the levels of several omega-3 metabolites in the testes of mice fed with omega-3 PUFAs. The eicosapentaenoic acid metabolite 12-hydroxyeicosapentaenoic acid (12-HEPE) up-regulated bone morphogenic protein 4 (BMP4) expression through GPR120-ERK1/2 pathway activation in Sertoli cells and restored spermatogonia proliferation and differentiation. Our study provides evidence that omega-3 PUFAs metabolite 12-HEPE effectively protects spermatogonia and reveals that GPR120 might be a tractable pharmacological target for fertility in men received chemotherapy or severe spermatogenesis dysfunction.

SMAD4 maintains the fluid shear stress set point to protect against arterial-venous malformations.

Vascular networks form, remodel, and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with bone morphogenic proteins 9 and 10 (BMP9 and BMP10). Conversely, mutation of the BMP receptors activin-like kinase 1 (ALK1), endoglin (ENG), or the downstream effector, SMAD family member 4 (SMAD4) leads to hereditary hemorrhagic telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). How endothelial cells (ECs) integrate FSS and BMP signals in vascular development and homeostasis and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between FSS and SMAD signaling in vascular stability and how disruption of this synergy leads to AVMs. We found that loss of Smad4 increased the sensitivity of ECs to flow by lowering the FSS set point, with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of SMAD4 disinhibits flow-mediated KLF4-TIE2-PI3K/Akt signaling, leading to cell cycle progression-mediated loss of arterial identity due to KLF4-mediated repression of cyclin dependent Kinase (CDK) inhibitors CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.

Accumulation of advanced glycation end products promotes atrophic nonunion incidence in mice through a CtBP1/2-dependent mechanism

Atrophic nonunion (AN) is a complex and poorly understood pathological condition resulting from impaired fracture healing. Advanced glycation end products (AGEs) have been implicated in the pathogenesis of several bone disorders, including osteoporosis and osteoarthritis. However, the role of AGEs in the development of AN remains unclear. This study found that mice fed a high-AGE diet had a higher incidence of atrophic nonunion (AN) compared to mice fed a normal diet following tibial fractures. AGEs induced two C-terminal binding proteins (CtBPs), CtBP1 and CtBP2, which were necessary for the development of AN in response to AGE accumulation. Feeding a high-AGE diet after fracture surgery in CtBP1/2−/− and RAGE−/− (receptor of AGE) mice did not result in a significant occurrence of AN. Molecular investigation revealed that CtBP1 and CtBP2 formed a heterodimer that was recruited by histone deacetylase 1 (HDAC1) and runt-related transcription factor 2 (Runx2) to assemble a complex. The CtBP1/2-HDAC1-Runx2 complex was responsible for the downregulation of two classes of bone development and differentiation genes, including bone morphogenic proteins (BMPs) and matrix metalloproteinases (MMPs). These findings demonstrate that AGE accumulation promotes the incidence of AN in a CtBP1/2-dependent manner, possibly by modulating genes related to bone development and fracture healing. These results provide new insights into the pathogenesis of AN and suggest new therapeutic targets for its prevention and treatment.

Lipopolysaccharide (LPS) extracted from Bacteroides vulgatus effectively prevents LPS extracted from Escherichia coli from inducing epithelial‑mesenchymal transition.

Pathological epithelial‑mesenchymal transition (EMT) has been shown to fulfill a key role in the development and progression of a variety of lung diseases. It has been demonstrated that the inflammatory microenvironment is a decisive factor in inducing pathological EMT. Hexacylated lipopolysaccharide (LPS) [or proacylated lipopolysaccharide (P‑LPS), which functions as proinflammatory lipopolysaccharide] is one of the most effective Toll‑like receptor 4 (TLR4) agonists. Furthermore, the pentacylated and tetracylated form of lipopolysaccharide (or A‑LPS, which functions as anti‑inflammatory lipopolysaccharide) has been shown to elicit competitive antagonistic effects against the pro‑inflammatory activity of P‑LPS. At present, it remains unclear whether LPS extracted from Bacteroides vulgatus (BV‑LPS) can prevent LPS extracted from Escherichiacoli (EC‑LPS) from inducing pathological EMT. In the present study, A549 cells and C57BL/6 mice lung tissue were both induced by EC‑LPS (P‑LPS) and BV‑LPS (A‑LPS), either alone or in combination. The anticipated anti‑inflammatory effects of BV‑LPS were analyzed by examining the lung coefficient, lung pathology, A549 cell morphology and expression levels both of the inflammatory cytokines, IL‑1β, IL‑6 and TNF‑α and of the EMT signature proteins, epithelial cadherin (E‑cadherin), α‑smooth muscle actin (α‑SMA) and vimentin. In addition, the expression levels of TLR4, bone morphogenic protein and activin membrane‑bound inhibitor (BAMBI) and Snail were detected and the possible mechanism underlying how BV‑LPS may prevent EC‑LPS‑induced EMT was analyzed. The results obtained showed that the morphology of the A549 cells was significantly polarized, the lung index was significantly increased, the alveolar structure was collapsed and the expression levels of IL‑1β, IL‑6, TNF‑α, α‑SMA, vimentin, TLR4 and Snail in both lung tissue and A549 cells were significantly increased, whereas those of E‑cadherin and BAMBI were significantly decreased. Treatment with BV‑LPS in combination with EC‑LPS was found to reverse these changes. In conclusion, the present study demonstrated that BV‑LPS is able to effectively prevent EC‑LPS‑induced EMT in A549 cells and in mouse lung tissue and furthermore, the underlying mechanism may be associated with inhibition of the TLR4/BAMBI/Snail signaling pathway.

Induction of Osteogenesis After BMP-2 Injection in Rabbit Shoulders: A Novel Strategy for Managing Glenoid Bone Defect Associated with Anterior Shoulder Instability

Background: Bone morphogenic protein-2 (BMP-2) has been shown to promote bone formation. The purpose of this research was to determine if an injectable form of BMP-2 could induce bone formation in anteroinferior glenoid bone defects, which is a critical factor of anterior shoulder instability. The purpose of this study was to investigate bone formation in rabbit shoulders following BMP-2 injection into the anterior capsulolabral complex. We hypothesized that the amount of new bone would be greater in rabbit shoulders injected with BMP-2 than in those injected with normal saline. Methods: An experimental study was conducted on 40 shoulders of 20 mature rabbits. Twenty right shoulders were allocated to four groups according to injection dose (5 µg or 10 µg) and follow-up period after injection (4 or 8 weeks). Twenty left shoulders were injected with normal saline as controls. At 4 or 8 weeks after injection, all rabbits were sacrificed, and bone volumes, densities, and locations of bone formation were evaluated using micro-computed tomography (micro-CT). Histologic analysis was performed on all 40 shoulders. Results: Shoulders injected with BMP-2 showed significantly more bone formation than control shoulders in all treated groups (58.6 ± 60.5 mm3 vs. 25.6 ± 32.6 mm3, p = 0.001). Moreover, bone formation increased with dose and follow-up. Locations of bone formation were the lesser tuberosity, greater tuberosity, and anterior capsulolabral complex. Histologic analysis confirmed the micro-CT-detected bone formation by revealing the presence of activated osteocytes, abundant osteoblastic rimming, and woven bone matrix. Conclusion: The study confirms that BMP-2 injection into the anterior capsulolabral complex of rabbit shoulders promotes new bone formation in adjacent soft tissue areas. Study Design: Controlled laboratory study.