Computed microtomography visualization and quantification of mouse ischemic brain lesion by nonionic radio contrast agents

Aim To explore the possibility of brain imaging by microcomputed tomography (microCT) using x-ray contrasting methods to visualize mouse brain ischemic lesions after middle cerebral artery occlusion (MCAO). Methods Isolated brains were immersed in ionic or nonionic radio contrast agent (RCA) for 5 days and subsequently scanned using microCT scanner. To verify whether ex-vivo microCT brain images can be used to characterize ischemic lesions, they were compared to Nissl stained serial histological sections of the same brains. To verify if brains immersed in RCA may be used afterwards for other methods, subsequent immunofluorescent labeling with anti-NeuN was performed. Results Nonionic RCA showed better gray to white matter contrast in the brain, and therefore was selected for further studies. MicroCT measurement of ischemic lesion size and cerebral edema significantly correlated with the values determined by Nissl staining (ischemic lesion size: P=0.0005; cerebral edema: P=0.0002). Brain immersion in nonionic RCA did not affect subsequent immunofluorescent analysis and NeuN immunoreactivity. Conclusion MicroCT method was proven to be suitable for delineation of the ischemic lesion from the non-infarcted tissue, and quantification of lesion volume and cerebral edema

Effect of Bone Morphogenetic Protein-7 on Gene Expression of Bone Morphogenetic Protein-4, Dentin Matrix Protein-1, Insulin-like Growth Factor-I and -II in Cementoblasts In Vitro

Formation of root cementum is a crucial moment in the development of the periodontium. Cells that produce the cementum are named cementoblasts and they posses some unique characteristics, which differentiates them from osteoblasts. Bone morphogenetic proteins (BMPs) are crucial regulators of both bone and tooth formation. In animal studies BMPs have shown to induce periodontal regeneration, however the molecular mechanism as how BMP-7 induces cementogenesis is largely unknown. We have investigated how BMP-7 regulates gene expression of BMP-4, Dentin matrix protein- 1 (DMP-1), Insulin-like growth factor-I (IGF-I) and –II (IGF-II) in cementoblasts. BMP-7 induced proliferation, and mineralized nodule formation of cementoblasts. Our results show that gene expression was influenced by the BMP-7 concentration used, with 75 ng/mL generally down regulating gene expression at 6 hours and then up-regulating after 24 hours. The 300 ng/mL concentration had an opposite effect while the 150 ng/mL concentration generally up-regulated gene expression after 6 hours and then after 24 hours maintained this up-regulation or had no effect compared to control, depending on the examined gene. The results show that BMP-7 down-regulated BMP-4 expression in cementoblasts but still up-regulated DMP-1 gene expression suggesting that BMP-7 can, in a paracrine manner, functionally substitute for BMP-4. Furthermore, it seems that BMP-7 exerts its effect more through the IGF-II than the IGF-I pathway as shown by an up-regulation of IGF-II and down-regulation of IGF-I. These results suggest that a combination of BMP-7/IGF-II could have a potential therapeutical significance in inducing cementogenesis and periodontal regeneration

Constitutively elevated blood serotonin is associated with bone loss and type 2 diabetes in rats

Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, β-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)2D3 decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin

Influence of epinephrine and medetomidine on systemic absorption of lidocaine applied epidurally in anesthetized swine

Epinephrine and alpha 2 agonist drugs are often used with epidural anesthesia to minimize local anesthetic systemic absorption, as well as to prolong the duration of the block. The aim of the current study was to determine by which extent epinephrine and medetomidine influenced lidocaine systemic absorption rate following epidural application. This was achieved by monitoring the serum lidocaine concentration in a porcine model. During general anesthesia, the first group received epidurally plane lidocaine, the second received lidocaine containing epinephrine (1 : 80.000), and the third lidocaine with medetomidine (15 μg/kg). Venous blood samples were taken before and 5, 10, 20, 30, 45, 60 and 90 minutes following epidural administration of the anesthetic. The effects of epinephrine and medetomidine were comparable. They both failed to cause a significant decrease in serum lidocaine concentration (p>0.05). In these settings we were unable to demonstrate a greater capacity of these two adrenergic agonists for reducing lidocaine systemic uptake and, accordingly, its systemic toxicity potential.Epinefrin i alfa 2 agonisti se uvek koriste u epiduralnoj anesteziji da umanje sistemsku apsorpciju lokalnog anestetika i da produže trajanje epiduralnog bloka. U serumu svinja se određivala koncentracija lidokaina, stoga je cilj ove studije da se odredi do koje mere epinefrin i medetomidin utiču na sistemsku apsorpciju lidokaina nakon epiduralne primene. Tokom opšte anestezije prvoj grupi je epiduralno aplikovan lidokain, drugoj lidokain s epinefrinom (1: 80000), a trećoj lidokain sa medetomidinom (15 μg/ kg). Krv iz vene je uzorkovana pre, 5, 10, 20, 30, 45, 60 i 90 minuta nakon epiduralne primene anestetika. Poređeni su efekti epinefrina i medetomidina, ali ni jedan nije signifi kantno smanjio serumsku koncentraciju lidokaina (p>0,05). Ove kombinacije adrenergičnih agonista nisu smanjile sistemsku resorpciju lidokaina. U skladu s tim, potencijalno je moguća sistemska toksičnost lidokaina

State-of-the-art of the Bone Morphogenetic Protein research field: 13th International BMP Conference, Dubrovnik 2022

The 13th International BMP Conference was held in October 2022 in Dubrovnik. The conference was attended by more than 240 participants from North America, Europe, Asia, and Australia who got an insight into the latest achievements in basic, translational, and clinical research of BMP mol- ecules through 75 lectures categorized into several scientific sections. This review paper provides the most important novel findings on the structure, function, and signaling of BMPs, the role of BMPs in patterning and organoids as well as the role of BMP in metabolism. Moreover, we discussed the role of BMPs in various diseases including cancer pathogenesis, pulmonary arterial hyperten- sion, and fibrodysplasia ossificans progressiva (FOP). Finally, we provided an overview of the new BMP-based therapies in regenerative medicine that are currently in different stages of preclinical and clinical trials

Becoming adults: Exploring the late ontogeny of the human talus

Introduction: The talus plays an important role in receiving and dissipating the forces and linking the leg and the foot. As such, it is of paramount importance to analyze how its morphology, internal and external, changes during late ontogeny and through adolescence. Method: To explore both the external shape and the internal architecture of the talus, Geometric Morphometrics and trabecular analysis have been applied to a sample of 35 tali from modern human juveniles aged between 5 and 15 years old (Middle Neolithic (4800-4500 BCE) to mid-20th century). Results: Results show that, as the overall size of the talus increases, the shape and orientation of talar facets also change. The youngest individuals exhibit a functional talus that is still characterized by a relatively immature shape (e.g., subtly expressed margins of articular surfaces) with articular facets only minimally rotated towards an adult configuration. In adolescents, talar shape has achieved adult form after the age of 11, with all the articular facets and posterior processes well-developed. Considering internal morphology, trabecular bone varies between age classes. While Bone Volume Fraction shifts during the age 5-15 range, Degree of Anisotropy is relatively more stable over the developmental period examined in the study since it exhibits smaller variations between age classes. Discussion: This study examined the late ontogeny of the human talus by considering both internal and external morphology. Results suggest that, although the locomotion has already assumed an adult-like pattern, the exploration of late talar growth may help understand how the talus adapts to changes in locomotor activity and how it responds to the increase in weight. Present results can be used to a better understanding of talar plasticity, improving interpretations of adult human talar form

Human talar ontogeny: Insights from morphological and trabecular changes during postnatal growth

The study of the development of human bipedalism can provide a unique perspective on the evolution of morphology and behavior across species. To generate new knowledge of these mechanisms, we analyze changes in both internal and external morphology of the growing human talus in a sample of modern human juveniles using an innovative approach. The sample consists of high‐resolution microCT scans of 70 modern juvenile tali, aged between 8 postnatal weeks and 10 years old, from a broad chronological range from Middle/Late Neolithic, that is, between 4800 and 4500 BCE, to the 20th century. We applied geometric morphometric and whole‐bone trabecular analysis (bone volume fraction, degree of anisotropy, trabecular number, thickness, and spacing) to all specimens to identify changes in the external and internal morphology during growth. Morphometric maps were also generated. During the first year of life, the talus has an immature and globular shape, with a dense, compact, and rather isotropic trabecular architecture, with numerous trabeculae packed closely together. This pattern changes while children acquire a more mature gait, and the talus tends to have a lower bone volume fraction, a higher anisotropy, and a more mature shape. The changes in talar internal and external morphologies reflect the different loading patterns experienced during growth, gradually shifting from an “unspecialized” morphology to a more complex one, following the development of bipedal gait. Our research shows that talar plasticity, even though genetically driven, may show mechanical influences and contribute to tracking the main locomotor milestones

Autologous blood coagulum containing rhBMP6 induces new bone formation to promote anterior lumbar interbody fusion (ALIF) and posterolateral lumbar fusion (PLF) of spine in sheep

resent study, we evaluated an autologous bone graft substitute (ABGS) composed of recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) used as a physiological carrier for new bone formation in spine fusion sheep models. The application of ABGS included cervical cage for use in the anterior lumbar interbody fusion (ALIF), while for the posterolateral lumbar fusion (PLF) sheep model allograft devitalized bone particles (ALLO) were applied with and without use of instrumentation. In the ALIF model, ABGS (rhBMP6/ABC/cage) implants fused significantly when placed in between the denuded L4- L5 vertebrae as compared to control (ABC/cage) which appears to have a fibrocartilaginous gap, as examined by histology and micro CT analysis at 16 weeks following surgery. In the PLF model, ABGS implants with or without ALLO showed a complete fusion when placed ectopically in the gutter bilaterally between two decorticated L4-L5 transverse processes at a success rate of 88% without instrumentation and at 80% with instrumentation ; however the bone volume was 50% lower in the instrumentation group than without, as examined by histology, radiographs, micro CT analyses and biomechanical testing at 27 weeks following surgery. The newly formed bone was uniform within ABGS implants resulting in a biomechanically competent and histologically qualified fusion with an optimum dose in the range of 100 g rhBMP6 per mL ABC, while in the implants that contained ALLO, the mineralized bone particles were substituted by the newly formed remodeling bone via creeping substitution. These findings demonstrate for the first time that ABGS (rhBMP6/ABC) without and with ALLO particles induced a robust bone formation with a successful fusion in sheep models of ALIF and PLF, and that autologous blood coagulum (ABC) serves as a preferred physiological native carrier to induce new bone at low doses of rhBMP6 and to achieve a successful spinal fusion

Koštani morfogenetski proteini (BMP): Od otkrića do razvoja nove autologne koštane naprave koja se sastoji od rekombinantnog humanog BMP6 u autolognom krvnom ugrušku kao nosaču

Bone Morphogenetic Proteins (BMPs) are growth and differentiation factors within the TGFβ superfam- ily of proteins. They induce ectopic and orthotopic endochondral bone formation and are involved in the regulation of cell proliferation, differentiation, apoptosis and mesenchymal-epithelial interactions in critical morphogenetic processes of tissues beyond bone. BMP2 and BMP7 osteogenic devices have been approved for enhancing healing in patients with long bone defects and anterior spinal fusion proce- dures. However, due to a high price and various serious adverse events including heterotopic ossifica- tion, retrograde ejaculation and pain their clinical use have been limited. In this review we discuss the BMP discovery, biology and their use in clinical studies with particular reference to the newly developed BMP6 based autologous bone graft substitute (ABGS). A novel ABGS consisting of an autologous bone coagulum (ABC) carrier with dispersed BMP6 to initiate the differentiation of mesenchymal cells into endochondral bone. The ABC met the conditions for an optimal delivery system for BMP6 due to han- dling simplicity, without an immunogenic and inflammatory response at the implantation site. Addition of allograft or synthetic ceramics to ABGS demonstrated in animal models significantly increased volume and better microarchitecture of the newly formed bone. The first clinical study was conducted in patients with distal radial fractures (Phase I study) and the second in patients undergoing high tibial osteotomy (Phase I/II study) and no serious adverse events have been observed. Finally, in the ongoing OSTEO- proSPINE study ABGS enforced with allograft bone is evaluated in patients with chronic back pain due to degenerative disc diseases. The novel ABGS bone mimetic is a major breakthrough and contribution to bone biology and regenerative medicine of skeletal repair.Koštani morfogenetski proteini (BMP) čine grupu čimbenika rasta i diferencijacije unutar TGFβ nado- bitelji. Oni induciraju stvaranje ektopične i ortotopične endohondralne kosti te su uključeni u regulaciju stanične proliferacije, diferencijacije, apoptoze i mezenhimalno-epitelne interakcije u važnim tkivnim morfogenetskim procesima izvan koštanog sustava. Koštane naprave koje sadrže BMP2 i BMP7 pro- tein odobrene su za poboljšanje koštanog cijeljenja kod pacijenata s defektima dugih cjevastih kostiju i kod prednje spinalne fuzije kralježnice. Međutim, zbog visoke cijene i mnogobrojnih nuspojava koje su uključivale pojavu heterotopičnih osifikacija, retrogradnu ejakulaciju i bol, njihova je klinička prim- jena ograničena. U ovom smo preglednom radu raspravili otkriće BMP molekula, njihovu biologiju i primjenu u kliničkim studijama s posebnim osvrtom na nedavno otkrivenu novu autolognu koštanu napravu (ABGS) koja sadrži BMP6. Novi ABGS sastoji se od nosača autolognog koaguluma (ABC) s otopljenim BMP6 koji je ključan za pokretanje diferencijacije mezenhimalnih stanica u smjeru stvaranja endohondralne kosti. ABC je ispunio sve potrebne uvjete za formulaciju optimalnog nosača za BMP6 isključivo zbog jednostavnosti priprave i primjene te odsustva imunogenog i upalnog odgovora na mjestu implantacije. Uz dodatak alografta ili sintetičke keramike što je potvrđeno na životinjskim modelima došlo je do značajnog povećanja volumena te poboljšanja mikroarhitekture novonastale kosti. Prvo kliničko ispitivanje provedeno je na pacijentima s distalnim prijelomima radijusa (faza I studije), a drugo na pacijentima koji su podvrgnuti visokoj osteotomiji tibije (faza I/II studije) bez uočenih ozbiljnih nuspojava. Trenutno je u tijeku studija OSTEOproSPINE u kojoj se testira učinkovitost ABGS u kom- binaciji s koštanim alograftom u bolesnika s kroničnim bolovima u leđima uzrokovanim degenerativnim promjenama intervertebralnog diska. Nova ABGS koštana naprava značajna je prekretnica i napredak u području koštane biologije te regenerativne medicine koštanog sustava