Bone Morphogenic Protein Research Articles

The Principle of Cortical Development and Evolution.

Human's robust cognitive abilities, including creativity and language, are made possible, at least in large part, by evolutionary changes made to the cerebral cortex. This paper reviews the biology and evolution of mammalian cortical radial glial cells (primary neural stem cells) and introduces the concept that a genetically step wise process, based on a core molecular pathway already in use, is the evolutionary process that has molded cortical neurogenesis. The core mechanism, which has been identified in our recent studies, is the extracellular signal-regulated kinase (ERK)-bone morphogenic protein 7 (BMP7)-GLI3 repressor form (GLI3R)-sonic hedgehog (SHH) positive feedback loop. Additionally, I propose that the molecular basis for cortical evolutionary dwarfism, exemplified by the lissencephalic mouse which originated from a larger gyrencephalic ancestor, is an increase in SHH signaling in radial glia, that antagonizes ERK-BMP7 signaling. Finally, I propose that: (1) SHH signaling is not a key regulator of primate cortical expansion and folding; (2) human cortical radial glial cells do not generate neocortical interneurons; (3) human-specific genes may not be essential for most cortical expansion. I hope this review assists colleagues in the field, guiding research to address gaps in our understanding of cortical development and evolution.

Regenerative potential of mesoporous silica nanoparticles scaffold on dental pulp and root maturation in immature dog’s teeth: a histologic and radiographic study

ObjectiveTo evaluate histologically and radiographically the potential of dog’s immature roots with apical periodontitis to regenerate after regenerative endodontic treatment using mesoporous silica nanoparticles (MSNs) with/without bone morphogenic protein (BMP-2) as scaffolds.MethodsIn 4 mongrel dogs, 56 immature teeth with 96 roots were infected, resulting in necrotic pulps and periapical pathosis. According to the evaluation time (Group I = 30 days and Group II = 90 days), 90 roots were divided into two equal groups (45 roots each) and 6 roots used to replace any lost root during the procedure. The two main groups were further divided according to treatment protocol into 5 subgroups (9 roots each): blood clot (BC subgroup), mesoporous silica nanoparticles scaffold only (MSNs subgroup), mesoporous silica nanoparticles impregnated with BMP2 (MSNs + BMP2 subgroup), infected teeth without treatment (+ ve control subgroup) and normal untouched teeth (-ve control subgroup). All teeth surfaces were coated with Tincture iodine and calcium hydroxide was applied prior to treatment protocols. Then, teeth were restored with glass ionomer filling to seal the remaining part of the access cavity. Radiography evaluation of the increase in root length, root thickness and occurrence of apical closure were performed. Following the sacrifice of the two dogs at each time of evaluation, histopathological analysis was performed and included the inflammatory cells count, bone resorption, tissue ingrowth, deposition of hard tissue, and closure of the apical part. All data were statistically analyzed.ResultsCompared to BC subgroup, MSNs and MSNs + BMP-2 subgroups exhibited significant higher increase in root length and thickness as well as higher vital tissue in-growth and new hard tissue formation in group II (P < 0.05). MSNs + BMP-2 subgroup had significant higher increase in root length and thickness as well as significant lower inflammatory cell count than MSNs subgroup in both groups (P < 0.05). There were no significant differences between MSNs and MSNs + BMP-2 subgroups regarding new hard tissue formation in both groups and apical closure in group I (P > 0.05).ConclusionMSNs with/without BMP-2 scaffolds enabled the continuing growth of roots in immature teeth with necrotic pulps and periapical pathosis. Addition of BMP-2 to MSNs scaffold improved its outcome in regenerative endodontics.Clinical relevanceMSNs with/without BMP-2 scaffolds may alternate blood clot for regenerative endodontic treatment of immature teeth with necrotic pulps.

LRIG1 controls proliferation of adult neural stem cells by facilitating TGFβ and BMP signalling pathways

Adult Neural Stem Cells (aNSCs) in the ventricular-subventricular zone (V-SVZ) are largely quiescent. Here, we characterize the mechanism underlying the functional role of a cell-signalling inhibitory protein, LRIG1, in the control of aNSCs proliferation. Using Lrig1 knockout models, we show that Lrig1 ablation results in increased aNSCs proliferation with no change in neuronal progeny and that this hyperproliferation likely does not result solely from activation of the epidermal growth factor receptor (EGFR). Loss of LRIG1, however, also leads to impaired activation of transforming growth factor beta (TGFβ) and bone morphogenic protein (BMP) signalling. Biochemically, we show that LRIG1 binds TGFβ/BMP receptors and the TGFβ1 ligand. Finally, we show that the consequences of these interactions are to facilitate SMAD phosphorylation. Collectively, these data suggest that unlike in embryonic NSCs where EGFR may be the primary mechanism of action, in aNSCs, LRIG1 and TGFβ pathways function together to fulfill their inhibitory roles.

FBLN2 is associated with Goldenhar syndrome and is essential for cranial neural crest cell development.

Goldenhar syndrome, a rare craniofacial malformation, is characterized by developmental anomalies in the first and second pharyngeal arches. Its etiology is considered to be heterogenous, including both genetic and environmental factors that remain largely unknown. To further elucidate the genetic cause in a five-generation Goldenhar syndrome pedigree and exploit the whole-exome sequencing (WES) data of this pedigree, we generated collapsed haplotype pattern markers based on WES and employed rare variant nonparametric linkage analysis. FBLN2 was identified as a candidate gene via analysis of WES data across the significant linkage region. A fbln2 knockout zebrafish line was established by CRISPR/Cas9 to examine the gene's role in craniofacial cartilage development. fbln2 was expressed specifically in the mandible during the zebrafish early development, while fbln2 knockout zebrafish exhibited craniofacial malformations with abnormal chondrocyte morphologies. Functional studies revealed that fbln2 knockout caused abnormal chondrogenic differentiation, apoptosis, and proliferation of cranial neural crest cells (CNCCs), and downregulated the bone morphogenic protein (BMP) signaling pathway in the zebrafish model. This study demonstrates the role of FBLN2 in CNCC development and BMP pathway regulation, and highlights FBLN2 as a candidate gene for Goldenhar syndrome, which may have implications for the selection of potential screening targets and the development of treatments for conditions like microtia-atresia.

Neuregulin4-ErbB4 signalling pathway is driven by electroacupuncture stimulation to remodel brown adipose tissue innervation.

To show that electroacupuncture stimulation (ES) remodels sympathetic innervation in brown adipose tissue (BAT) via the bone morphogenic protein 8B (BMP8B)-neuregulin 4 (NRG4)-ErbB4 axis, with somatotopic dependence. We established a high-fat diet (HFD) model with C57BL/6J mice to measure the thermogenesis and metabolism of BAT. In addition, the sympathetic nerve activity (SNA) was measured with the electrophysiological technique, and the immunostaining of c-Fos was used to detect the central nervous system sources of sympathetic outflows. Finally, the key role of the BMP8B-NRG4-ErbB4 axis was verified by peripheral specific antagonism of ErbB4. ES at the forelimb and abdomen regions significantly up-regulate SNA, whereas ES at the hindlimb region has a limited regulatory effect on SNA but still partially restores HFD-induced BAT dysfunction. Mechanistically, ES at the forelimb and abdomen regions driving catecholaminergic signals in brown adipocytes depends on neural activities projected from the ventromedial nucleus of the hypothalamus (VMH) to the spinal cord intermediolateral column (IML). Notably, the peripheral suppression of ErbB4 in BAT inhibits the thermogenesis and metabolic function of BAT, as well as significantly hindering the SNA activation and metabolic benefits induced by ES. These results suggest that ES appears to be an effective approach for remodeling sympathetic innervation in BAT, which is closely related to neuronal activity in the VMH and the NRG4-ErbB4 signaling pathway.

Stimuli-Responsive Codelivery System-Embedded Polymeric Nanofibers with Synergistic Effects of Growth Factors and Low-Intensity Pulsed Ultrasound to Enhance Osteogenesis Properties.

The present work aims to develop optimized scaffolds for bone repair by incorporating mesoporous nanoparticles into them, thereby combining bioactive factors for cell growth and preventing rapid release or loss of effectiveness. We synthesized biocompatible and biodegradable scaffolds designed for the controlled codelivery of curcumin (CUR) and recombinant human bone morphogenic protein-2 (rhBMP-2). Active agents in dendritic silica/titania mesoporous nanoparticles (DSTNs) were incorporated at different weight percentages (0, 2, 5, 7, 9, and 10 wt %) into a matrix of polycaprolactone (PCL) and polyethylene glycol (PEG) nanofibers, forming the CUR-BMP-2@DSTNs/PCL-PEG delivery system (S0, S2, S5, S7, S9, and S10, respectively, with the number showing the weight percentage). To enhance the formation process, the system was treated using low-intensity pulsed ultrasound (LIPUS). Different advanced methods were employed to assess the physical, chemical, and mechanical characteristics of the fabricated scaffolds, all confirming that incorporating the nanoparticles improves their mechanical and structural properties. Their hydrophilicity increased by approximately 25%, leading to ca. 53% enhancement in their water absorption capacity. Furthermore, we observed a sustained release of approximately 97% for CUR and 70% for BMP-2 for the S7 (scaffold with 7 wt % DSTNs) over 28 days, which was further enhanced using ultrasound. In vitro studies demonstrated accelerated scaffold biodegradation, with the highest level observed in S7 scaffolds, approximately three times higher than the control group. Moreover, the cell viability and proliferation on DSTNs-containing scaffolds increased when compared to the control group. Overall, our study presents a promising nanocomposite scaffold design with notable improvements in structural, mechanical, and biological properties compared to the control group, along with controlled and sustained drug release capabilities. This makes the scaffold a compelling candidate for advanced bone tissue engineering and regenerative therapies.

Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage.

Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

Complement factor I knockdown inhibits colon cancer development by affecting Wnt/β-catenin/c-Myc signaling pathway and glycolysis.

Colon cancer (CC) occurrence and progression are considerably influenced by the tumor microenvironment. However, the exact underlying regulatory mechanisms remain unclear. To investigate immune infiltration-related differentially expressed genes (DEGs) in CC and specifically explored the role and potential molecular mechanisms of complement factor I (CFI). Immune infiltration-associated DEGs were screened for CC using bioinformatics. Quantitative reverse transcription polymerase chain reaction was used to examine hub DEGs expression in the CC cell lines. Stable CFI-knockdown HT29 and HCT116 cell lines were constructed, and the diverse roles of CFI in vitro were assessed using CCK-8, 5-ethynyl-2'-deoxyuridine, wound healing, and transwell assays. Hematoxylin and eosin staining and immunohistochemistry staining were employed to evaluate the influence of CFI on the tumorigenesis of CC xenograft models constructed using BALB/c male nude mice. Key proteins associated with glycolysis and the Wnt pathway were measured using western blotting. Six key immune infiltration-related DEGs were screened, among which the expression of CFI, complement factor B, lymphoid enhancer binding factor 1, and SRY-related high-mobility-group box 4 was upregulated, whereas that of fatty acid-binding protein 1, and bone morphogenic protein-2 was downregulated. Furthermore, CFI could be used as a diagnostic biomarker for CC. Functionally, CFI silencing inhibited CC cell proliferation, migration, invasion, and tumor growth. Mechanistically, CFI knockdown downregulated the expression of key glycolysis-related proteins (glucose transporter type 1, hexokinase 2, lactate dehydrogenase A, and pyruvate kinase M2) and the Wnt pathway-related proteins (β-catenin and c-Myc). Further investigation indicated that CFI knockdown inhibited glycolysis in CC by blocking the Wnt/β-catenin/c-Myc pathway. The findings of the present study demonstrate that CFI plays a crucial role in CC development by influencing glycolysis and the Wnt/β-catenin/c-Myc pathway, indicating that it could serve as a promising target for therapeutic intervention in CC.

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential before implantation.

Bone morphogenic protein (BMP) signaling plays an essential and highly conserved role in embryo axial patterning in animal species. However, in mammalian embryos, which develop inside the mother, early development includes a preimplantation stage, which does not occur in externally developing embryos. During preimplantation, the epiblast is segregated from extra-embryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling is imprecisely defined in mouse early embryos. Here, we show that, in contrast to previous reports, BMP signaling (SMAD1/5/9 phosphorylation) is not detectable until implantation when it is detected in the primitive endoderm - an extra-embryonic lineage. Moreover, preimplantation development appears to be normal following deletion of maternal and zygotic Smad4, an essential effector of canonical BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extra-embryonic cell types drives epiblast morphogenesis postimplantation.

BMP9 attenuates IL-33 signaling and prevents endothelial-to-mesenchymal transition in pulmonary arterial hypertension

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Cardiovascular Alliance - PHAEDRA IMPACT Background Pulmonary Arterial hypertension (PAH) is a rare and progressive disease characterized by increased vascular resistance and remodeling of the pulmonary arteries. Disturbed Transforming growth factor (TGF)-β signaling, often associated with mutations in the gene encoding for the bone morphogenic protein receptor type II (BMPR2) and endothelial-to-mesenchymal transition (EndMT), emerges as a central culprit in the pathogenesis of PAH. Interleukin-33 (IL-33) has been identified as an inducer of EndMT, and its soluble receptor ST2 (sST2) has emerged as a potential biomarker for PAH. Bone morphogenic protein (BMP) 9, a BMPR2 ligand, showing a high affinity for endothelial cells, is being explored as a therapeutic approach. Purpose We hypothesize that a crosstalk between the TGF-β pathway and IL-33, a known promoter of vascular remodeling, occurs in PAH. This study aims to unravel the protective role of BMP9 in PAH, with a specific focus on the involvement of EndMT, a key player in the pathogenesis of vascular diseases. Methods The expression of IL-33 protein levels was assessed in Pulmonary Arterial Endothelial Cells (PAEC) and in lung tissues from experimentally induced PH (Sugen/Hypoxia (SuHx)) or control mice. Signal transduction and EndMT were investigated in PAECs exposed to IL-33, BMP9 and sST2, by gene expression analysis (qPCR), western blotting, ELISA and immunostaining. BMP9 and sST2 levels were measured in plasma from PAH patients by ELISA. Results Elevated IL-33 expression was observed in PAEC from PAH patients and lung tissues from SuHx mice compared to healthy controls. In vitro, BMP9 significantly induced the expression of the IL-33 soluble receptor ST2, while concurrently reducing IL-33 target gene expression. IL-33-induced EndoMT in PAEC was effectively mitigated by pre-incubation with BMP9, proving more efficacious than ST2 neutralizing antibody. Furthermore, a correlation between BMP9 and sST2 levels in plasma from PAH patients was identified. Conclusion Our findings demonstrate that BMP9 offers superior protections against IL-33-induced EndMT in PAECs compared to a ST2 neutralizing antibody. BMP9 induces expression of sST2, leading to IL-33 neutralization and subsequent EndMT inhibition. Therefore, targeting the IL-33 pathway in PAH patients presents a promising therapeutic option for preventing EndMT. This study provides novel insights into the pathogenesis of PAH, revealing a potential therapeutic target for this rare disease.BMP9 inhibits IL-33 downstream signalingBMP9 protects from IL-33-induced EndMT

Iron and bones: effects of iron overload, deficiency and anemia treatments on bone.

Iron is a vital trace element and exerts opposing effects on bone in both iron overload and iron deficiency situations. Remarkably, iron supplementation through intravenous infusion in patients with iron deficiency can also have detrimental effects on bone in special cases. The diverse mechanisms underlying these effects and their manifestations contribute to the complexity of this relationship. Iron overload impacts both bone resorption and formation, accelerating bone resorption while reducing bone formation. These effects primarily result from the direct action of reactive oxygen species (ROS), which influence the proliferation, differentiation, and activity of both osteoclasts and osteoblasts differently. This imbalance favors osteoclasts and inhibits the osteoblasts. Simultaneously, multiple pathways, including bone morphogenic proteins, RANK ligand, and others, contribute to these actions, leading to a reduction in bone mass and an increased susceptibility to fractures. In contrast, iron deficiency induces low bone turnover due to energy and co-factor deficiency, both of which require iron. Anemia increases the risk of fractures in both men and women. This effect occurs at various levels, reducing muscular performance and, on the bone-specific level, decreasing bone mineral density. Crucially, anemia increases the synthesis of the phosphaturic hormone iFGF23, which is subsequently inactivated by cleavage under physiological conditions. Thus, iFGF23 levels and phosphate excretion are not increased. However, in specific cases where anemia has to be managed with intravenous iron treatment, constituents-particularly maltoses-of the iron infusion suppress the cleavage of iFGF23. As a result, patients can experience severe phosphate wasting and, consequently, hypophosphatemic osteomalacia. This condition is often overlooked in clinical practice and is often caused by ferric carboxymaltose. Ending iron infusions or changing the agent, along with phosphate and vitamin D supplementation, can be effective in addressing this issue.

Therapeutic potential and impact of nanoengineered patient‐derived mesenchymal stem cells in a murine resection and recurrence model of human glioblastoma

AbstractConfounding results of engineered mesenchymal stem cells (MSCs) used as cellular vehicles has plagued technologies whereby success or failure of novel approaches may be dismissed or inaccurately ascribed solely to the biotechnology platform rather than suitability of the human donor. Polymeric materials were screened for non‐viral engineering of MSCs from multiple human donors to deliver bone morphogenic protein‐4 (BMP4), a protein previously investigated in clinical trials for glioblastoma (GBM) to combat a subpopulation of highly invasive and tumorigenic clones. A “smart technology” that target the migratory and stem‐like nature of GBM will require: (1) a cellular vehicle (MSC) which can scavenge and target residual cells left behind after surgical debulking and deliver; (2) anti‐glioma cargo (BMP4). Multiple MSC donors are safely engineered, though varied in susceptibility to accept BMP4 due to intrinsic characteristics revealed by their molecular signatures. Efficiency is compared via secretion, downstream signaling, differentiation, and anti‐proliferative properties across all donors. In a clinically relevant resection and recurrence model of patient‐derived human GBM, we demonstrate that nanoengineered MSCs are not “donor agnostic” and efficacy is influenced by the inherent suitability of the MSC to the cargo. Therefore, donor profiles hold greater influence in determining downstream outcomes than the technical capabilities of the engineering technology.

Abstract PO4-13-10: The anti-metastatic role of BMP4 through cholesterol biosynthesis inhibition and consequent interaction with statin benefit in breast cancer

Abstract We have previously identified a role for bone morphogenic protein 4 (BMP4) in reducing distant relapse risk after early breast cancer (BrCa) (1). We therefore set out to identify mechanisms underlying the protective effect of BMP4, looking to translate this finding for patient benefit. A highly metastatic variant of the triple negative MDA-MB-231 human BrCa line (231-HM-turboGFP) was transduced with BMP4, leading to sustained BMP4 secretion. These modified tumours were established in NSG mice, resected at a given size and turboGFP-positive cancer cells were recovered from primary tumours for RNA sequencing analysis. Gene set enrichment analysis (GSEA) was completed to identify signalling pathways that were significantly modulated by BMP4. The four most significantly downregulated gene sets were associated with cholesterol synthesis, with the overwhelming majority of genes in this pathway being downregulated. BMP4 downregulation of 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1, involved in cholesterol biosynthesis) was confirmed at the protein level by western blotting on whole tumour lysates. Forced expression of BMP4 also led to a significant reduction in the levels of free and total cholesterol. In a confirmatory analysis of the METABRIC human BrCa dataset, the expression of cholesterol biosynthesis-related genes inversely correlated with the expression of BMP4. Further, for the majority of these genes, expression was elevated in high-grade breast tumours, and predicted worse overall survival of patients. Finally, we tested the effect of a lipophilic statin, lovastatin, on the growth and metastasis of 231-HM-turboGFP tumours. Treatment did not affect the growth of primary tumours but significantly less metastases were observed in the lungs of treated mice, at least partially replicating the anti-metastatic effect of BMP4. Having identified inhibition of cholesterol biosynthesis as a potential mechanism of protective BMP4 action we then asked if BMP4 status interacted with any protective effect of co-administered statins. We therefore returned to the cohort initially employed to demonstrate BMP4 benefit (1) and looked at the interaction between BMP4 protein levels and statin usage with regards to recurrence. BMP4 protein and statin usage data were available on a cohort of 417 patients with early BrCa. Statin use compared to none led to a reduced risk of distant relapse (8 v 22%, 0.0029) at 15 years. BMP4 led to borderline reduction in distant relapse (16 v 24%, p=0.052). In BMP4 negative tumours, statin protected against any relapse (p=0.0025) whereas in BMP4 positive disease no significant risk reduction was not significant (p=0.074). Absence of BMP4 expression may be a biomarker of both higher relapse risk and statin benefit in early BrCa. 1. Eckhardt et al, Cancer Res. 2020;80(6):1304-1315 Citation Format: Lap Hing Chi, Andrew Redfern, Allan Burrows, Suraya Roslan, Leone Oh, Lisa Spalding, Robin Anderson. The anti-metastatic role of BMP4 through cholesterol biosynthesis inhibition and consequent interaction with statin benefit in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-13-10.

Pathobiology of Pulmonary Arterial Hypertension

Abstract: Pulmonary Arterial Hypertension (PAH) is a progressive disease associated with occlusive pulmonary arterial remodeling of vessels &lt; 500 μm for which there is no cure. Even in the era of PAH-specific combination therapies, aberrant lung pathology and progressive right ventricular (RV) dysfunction occur, culminating in a median survival of 6.2 years, according to the latest data in the treatment era. While better than a median survival from symptom onset of 2.8 years prior to PAH-specific therapies, it is still poor. Thus, there is an urgent need to move the opportunities forward for meaningful treatment strategies. Clearly, a better understanding of the highly complex pathobiology of PAH is needed if we are to achieve new and novel treatment strategies. This is especially so if we are to pursue a more personalized treatment approach to PAH in light of the multitude of pathobiological abnormalities described in PAH, which likely culminate in a final common pathway for PAH development. : In this State-of-the-Art review, we provide comprehensive insights into the complex pathobiology of PAH to provide understanding and insights for the practicing clinician. We review the pathology of PAH and the cells involved and their impact in driving pathological abnormalities (pulmonary artery endothelial cells, smooth muscle cells, fibroblasts and pericytes) as well as the role of the extracellular matrix. Inflammation and immune dysfunction are considered important drivers of PAH and are comprehensively discussed. Another pathway relates to TGFβ/ bone morphogenic protein (BMP) imbalance, which is highlighted, as well as a new novel agent, sotatercept that impacts this imbalance. Genetic factors underlying heritable PAH (HPAH) are addressed, as well as epigenetic influences. Other important pathways highlighted include growth factor signaling, ion channels/channelopathy, hypoxia signaling pathways, and altered metabolism and mitochondrial dysfunction. We also address the “estrogen paradox”, whereby PAH is more common in women but more severe in men. The basis for drug-induced PAH is discussed, including the new methamphetamine epidemic. We briefly provide insights into DNA damage and senescence factors in pathobiology and highlight commonalities between PAH and cancer pathobiology. Furthermore, we provide concluding insights for the treating physician. In conclusion, we need to pose the right questions to motivate novel and effective treatment strategies for the management of PAH based on pathobiological principles and understanding.

Elucidating the Benefit of Perforated vs Non-Perforated Membranes in Guided Bone Regeneration: An in Vivo Histologic Evaluation and Histomorphometric Analysis.

Non-perforated Polytetrafluoroethylene (PTFE) membranes are effectively utilized in guided bone regeneration (GBR) but may hinder cell migration due to limited interaction with the periosteum. This study compared bone regeneration using occlusive or perforated membranes combined with acellular collagen sponge (ACS) and recombinant human bone morphogenic protein-2 (rhBMP-2) in a canine mandibular model. Male beagle dogs (n&#61;3) received two mandibular defects each to compare ACS/rhBMP-2 with experimental (perforated group) and control (non-perforated group) membranes (n&#61;3 defects/group). Tissue healing was assessed histomorphologically, histomorphometrically and through volumetric reconstruction using microcomputed tomography. The perforated group showed increased bone formation and reduced soft tissue formation compared to the non-perforated group. For the primary outcome, histomorphometric analysis revealed significantly greater total regenerated bone in the perforated group (67.08 ± 6.86%) relative to the nonperforated group (25.18 ± 22.44%) (p &#61; 0.036). Perforated membranes had less soft tissue infiltration (32.91 ± 6.86%) compared to non-perforated membranes (74.82 ± 22.44%) (p &#61; 0.036). The increased permeability of membranes in the perforated group potentially enabled periosteal precursor cells greater accessibility to rhBMP-2. The availability may have accelerated their differentiation into mature bone-forming cells, contributing to the stimulation of new bone production, relative to the non-perforated group.

Nonoperative and Operative Soft-Tissue, Cartilage, and Bony Regeneration and Orthopaedic Biologics of the Elbow and Upper Extremity: An Orthoregeneration Network Foundation Review

Orthoregeneration is defined as a solution for orthopaedic conditions that harnesses the benefits of biology to improve healing, reduce pain, improve function, and, optimally, provide an environment for tissue regeneration. Options include drugs, surgical intervention, scaffolds, biologics as a product of cells, and physical and electromagnetic stimuli. The goal of regenerative medicine is to enhance the healing of tissue after musculoskeletal injuries as both isolated treatment and adjunct to surgical management, using novel therapies to improve recovery and outcomes. Various orthopaedic biologics (orthobiologics) have been investigated for the treatment of pathology involving the elbow and upper extremity, including the tendons (lateral epicondylitis, medial epicondylitis, biceps tendonitis, triceps tendonitis), articular cartilage (osteoarthritis, osteochondral lesions), and bone (fractures, nonunions, avascular necrosis, osteonecrosis). Promising and established treatment modalities include hyaluronic acid; botulinum toxin; corticosteroids; leukocyte-rich and leukocyte-poor platelet-rich plasma; autologous blood; bone marrow aspirate comprising mesenchymal stromal cells (alternatively termed medicinal signaling cells and frequently mesenchymal stem cells [MSCs]) and bone marrow aspirate concentrate; MSCs harvested from adipose and skin (dermis) sources; vascularized bone grafts; bone morphogenic protein scaffold made from osteoinductive and conductive β-tricalcium phosphate and poly-ε-caprolactone with hydrogels, human MSCs, and matrix metalloproteinases; and collagen sponge. Autologous blood preparations such as autologous blood injections and platelet-rich plasma show positive outcomes for nonresponsive tendinopathy. In addition, cellular therapies such as tissue-derived tenocyte-like cells and MSCs show a promising ability to regulate degenerative processes by modulating tissue response to inflammation and preventing continuous degradation and support tissue restoration.

Mechanistic Insights into Nonylphenol Stress on BMP2 and BMP4 Gene Expression in Red Crucian Carp (Carassius auratus Red var.)

Nonylphenol (NP) is a known endocrine-disrupting chemical (EDC) that has been shown to affect bone development in mammals. However, the detrimental impacts of NP on the skeletal growth and development of aquatic species, especially bony fish, remain poorly understood. Bone morphogenic proteins (BMPs), essential for bone formation and osteoblast differentiation, act through the BMP-Smad signaling pathway. In this study, two BMP genes, BMP2 and BMP4, were cloned and characterized in the red crucian carp (Carassius auratus red var.). The full-length cDNAs of BMP2 and BMP4 were 2029 bp and 2095 bp, respectively, encoding polypeptides of 411 and 433 amino acids, and share a typical TGF-β domain with other BMPs. The tissue expression patterns of both genes were identified, showing ubiquitous expression across all studied tissues. Additionally, the exposure of embryos or adult fish to NP stress resulted in a downregulation of BMP2, BMP4, and other genes associated with the BMP-Smad signaling pathway. Moreover, the combined treatment of adult fish with NP and the specific BMP receptor inhibitor significantly reduced these genes’ expression. These findings elucidate the mechanism of NP stress on BMP2 and BMP4, suggesting a role for the BMP-Smad signaling pathway in the response to endocrine-disrupting chemicals in fish.

Osteogenic Effects of the Diospyros lotus L. Leaf Extract on MC3T3-E1 Pre-Osteoblasts and Ovariectomized Mice via BMP2/4 and TGF β Pathways.

Osteoporosis, a disease defined by the primary bone strength due to a low bone mineral density, is a bone disorder associated with increased mortality in the older adult population. Osteoporosis is mainly treated via hormone replacement therapy, bisphosphates, and anti-bone resorption agents. However, these agents exert severe side effects, necessitating the development of novel therapeutic agents. Many studies are focusing on osteogenic agents as they increase the bone density, which is essential for osteoporosis treatment. Here, we aimed to investigate the effects of Diospyros lotus L. leaf extract (DLE) and its components on osteoporosis in MC3T3-E1 pre-osteoblasts and ovariectomized mice and to elucidate the underlying related pathways. DLE enhanced the differentiation of MC3T3-E1 pre-osteoblasts, with a 1.5-fold elevation in ALP activity, and increased the levels of osteogenic molecules, RUNX family transcription factor 2, and osterix. This alteration resulted from the activation of bone morphogenic protein 2/4 (BMP2/4) and transformation of growth factor β (TGF β) pathways. In ovariectomized mice, DLE suppressed the decrease in bone mineral density by 50% and improved the expression of other bone markers, which was confirmed by the 3~40-fold increase in osteogenic proteins and mRNA expression levels in bone marrow cells. The three major compounds identified in DLE exhibited osteogenic and estrogenic activities with their aglycones, as previously reported. Among the major compounds, myricitrin alone was not as strong as whole DLE with all its constituents. The osteogenic activity of DLE was partially suppressed by the inhibitor of estrogen signaling, indicating that the estrogenic activity of DLE participated in its osteogenic activity. Overall, DLE suppresses osteoporosis by inducing osteoblast differentiation.

The Effect of C2 Muscular Exposure Technique on Radiographic and Clinical Outcomes After C2-T2 Posterior Cervical Fusion.

Retrospective cohort analysis. To determine whether the C2 exposure technique was a predictor of change in cervical alignment and patient-reported outcomes measures (PROMs) after posterior cervical decompression and fusion (PCDF) for degenerative indications. In PCDF handling of the C2 posterior paraspinal musculature during the operative approach varies by surgeon technique. To date, no studies have investigated whether maintenance of the upper cervical semispinalis cervicis attachments as compared with complete reflection of upper cervical paraspinal musculature from the posterior bony elements is associated with superior radiographic and clinical outcomes after PCDF. All adult patients who underwent C2-T2 PCDF for myelopathy or myeloradiculopathy at multi-institutional academic centers between 2013 and 2020 were retrospectively identified. Patients were dichotomized by the C2 exposure technique into semispinalis preservation or midline muscular reflection groups. Preoperative and short and long-term postoperative radiographic outcomes (upper cervical alignment, global alignment, and fusion status) and PROMs (Visual Analog Scale-Neck, Neck Disability Index, and Short Form-12) were collected. Univariate analysis compared patient factors, radiographic measures, and PROMs across C2 exposure groups. A total of 129 patients met the inclusion/exclusion criteria (73 muscle preservation and 56 muscle reflection). Patients in the muscular preservation group were on average younger (P= 0.005) and more likely to have bone morphogenic protein (P< 0.001) and C2 pars screws (P= 0.006) used during surgery. Preoperative to postoperative changes in C2 slope, C2 tilt, C2-C3 segmental lordosis, C2-C3 listhesis, C0-C2 Cobb angle, proximal junctional kyphosis, ADI, C1 lamina-occiput distance, C2 sagittal vertical axis, C2-C7 lordosis, and PROMs at all follow-up intervals did not vary significantly by C2 exposure technique. Likewise, there were no significant differences in fusion status, C2-C3 pseudoarthrosis, C2 screw loosening, and complication and revision rates between C2 exposure groups. Preservation of C2 semispinalis attachments versus muscular reflection did not significantly impact cervical alignment, clinical outcomes, or proximal junction complications in long-segment PCDF. Level III.

The effect of C2 screw type on perioperative outcomes and long-term stability after C2-T2 posterior cervical decompression and fusion.

To determine if C2 pedicle versus pars screw type predicts change in fusion status, C2 screw loosening, cervical alignment, and patient-reported outcomes measures (PROMs) after C2-T2 posterior cervical decompression and fusion (PDCF). All adult patients who underwent C2-T2 PCDF for myelopathy or myeloradiculopathy between 2013-2020 were retrospectively identified. Patients were dichotomized by C2 screw type into bilateral C2 pedicle and bilateral C2 pars screw groups. Preoperative and short- and long-term postoperative radiographic outcomes and PROMs were collected. Univariate and multivariate analysis compared patient factors, fusion status, radiographic measures, and PROMs across groups. A total of 159 patients met the inclusion/exclusion criteria (76 bilateral pedicle screws, 83 bilateral pars screws). Patients in the C2 pars relative to C2 pedicle screw group were on average more likely to have bone morphogenic protein (p = 0.001) and four-millimeter diameter rods utilized intraoperatively (p = 0.033). There were no significant differences in total construct and C2-3 fusion rate, C2 screw loosening, or complication and revision rates between C2 screw groups in univariate and regression analysis. Changes in C2 tilt, C2-3 segmental lordosis, C0-2 Cobb angle, proximal junctional kyphosis, atlanto-dens interval, C1 lamina-occiput distance, C2 sagittal vertical axis, C2-7 lordosis, and PROMs at all follow-up intervals did not vary significantly by C2 screw type. There were no significant differences in fusion status, hardware complications, and radiographic and clinical outcomes based on C2 screw type following C2-T2 PCDF. Accordingly, intraoperative usage criteria can be flexible based on patient vertebral artery positioning and surgeon comfort level.