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Background Angiogenesis and osteogenesis are closely interrelated. The interaction between endothelial and bone-forming cells, such as osteoblasts, is crucial for normal bone development and repair. Juxtacrine and paracrine mechanisms play key roles in cell differentiation towards the osteogenic direction, assuming the direct effect of endothelium on osteogenic differentiation. However, the mechanisms of this interplay have yet to be thoroughly studied. Methods Isolated endothelial cells (EC) from human umbilical vein and human osteoblasts (OB) from the epiphysis of the femur or tibia were cultured in direct and indirect (separated by membrane) contact in vitro under the osteogenic differentiation conditions. Osteogenic differentiation was verified by RT-PCR, and alizarin red staining. Shotgun proteomics and RNA-sequencing were used to compare both EC and OB under different co-culture conditions to assess the mechanisms of EC-OB interplay. To verify the role of Notch signaling, experiments with Notch modulation in EC were performed by EC lentiviral transduction with further co-cultivation with OB. Additionally, the effect of Notch modulation in EC was assessed by RNA-sequencing. Results EC have opposite effects on osteogenic differentiation depending on the co-culture conditions with OB. In direct contact, EC enhance osteogenic differentiation, but in indirect cultures, EC suppress it. Our proteotranscriptomic analysis revealed that the osteosuppressive effect is related to the action of paracrine factors secreted by EC, while the osteoinductive properties of EC are mediated by the Notch signaling pathway, which can be activated only upon a physical contact of EC with OB. Indeed, in the direct co-culture, the knockdown of Notch1 and Notch3 receptors in EC has an inhibitory effect on the OB osteogenic differentiation, whereas activation of Notch by intracellular domain of either Notch1 or Notch3 in EC has an inductive effect on the OB osteogenic differentiation. Conclusion The data indicate the dual role of the endothelium in regulating osteogenic differentiation and highlight the unique role of the Notch signaling pathway in inducing osteogenic differentiation during cell-to-cell interactions. The findings of the study emphasize the importance of intercellular communication in the regulation of osteoblast differentiation during bone development and maintenance. Graphical Abstract

BackgroundMicrobiological profile of pathogens causing periprosthetic joint infection (PJI) after primary total hip (THA) and knee (TKA) arthroplasty varies in different regions, clinics and even departments. The objective of this study was to analyze the pathogen structure in patients with PJI after primary THA and TKA and its influence on the effectiveness of the infection eradication after two-stage reimplantation.Materials and methodsWe collected the retrospective data of 364 patients—161 with PJI after primary TKA (113 treated in two stages 48 with failure after spacer implantation) and 203 patients with infected THA (127 after successful two-stage reimplantation and 76 with PJI recurrence after the first stage) within the time period from January 2012 to December 2017, treated with two-stage protocol in the single center. A comparative analysis of pathogen structure was performed between cohorts of patients with hip and knee PJI. A subanalysis was made between the subsets comprised from patients with successful two-stage treatment and the subsets with failure to treat the infection.ResultsStaphylococcus epidermidis was the most commonly identified pathogen in the full hip and knee cohorts: 30.1% and 32.5%, respectively. However, the percentage of methicillin-resistant Staphylococcus epidermidis (MRSE) among all S. epidermidis isolates was higher in the hip cohort—50% compared with 35% in the knee cohort (p = 0.073). Other coagulase-negative Staphylococci were more common to patients with PJI after primary TKA—10.3% compared with 5% (p < 0.02). Streptococcus sp. caused hip PJI in a larger percentage of cases than in knee PJI (p < 0.01)—7% and 2%, respectively (p < 0.01).Polymicrobial associations were significantly more common in hip PJI compared to knee PJI: 45.3% and 14% of cases, respectively (p < 0.001). The presence of polymicrobial infection significantly raised the risk of PJI recurrence [OR 2 (95% CI from 1.24 to 3.24)] in knee PJI patients and reduced the effectiveness of infection eradication from 73.9% to 47.8%.ConclusionComparative analysis showed significant differences in the structure of PJI pathogens in the hip and knee. These findings are useful when choosing treatment strategies and empirical antibiotics regimens, in the management of patients with PJIs after primary hip and knee arthroplasty.

High-strength composite hydrogels “cellulose–polyacrylamide” were synthesized by free-radical polymerization of acrylamide conducted inside the previously formed physical network of regenerated plant cellulose. Partial hydrolysis of the amide groups of these hydrogels yielded their ionic forms with a degree of hydrolysis of 0.1 and 0.25. The cylindrical hydrogel samples of three compositions were implanted in the preformed osteochondral defects of the rabbit’s femoral knee joints. No signs of migration or disintegration of the tested implants were revealed in the course of in vivo tests as long as 90 and 120 days after the implantation. The mechanical behavior of hydrogel samples-implants before implantation and after their removal from the joints of laboratory animals was studied in detail. The morphology and chemical composition of the removed implants were studied by SEM combined with the EDX method. The results showed that the mechanical characteristics of hydrogel implants remained practically unchanged after in vivo tests. The removed implants, as well as the initial hydrogels, endured cyclic compression loading at the amplitude up to 50%. Compression stresses up to 3–10 MPa were recorded in these tests, which is close to the data obtained by several authors for natural articular cartilages in the same conditions of loading. The principal differences in the chemical composition and morphology of the implant area adjacent to the subchondral bone for non-ionic and ionic types of implants have been revealed. For non-ionic implants in this area intensive mineralization with formation of calcium phosphates inside the polymeric hydrogel network is observed, while the border area of ionic implants practically does not undergo mineralization.

A group of new hydrogel materials combining high physical properties and pronounced antibacterial activity has been developed. These are composite hydrogels “cellulose-polyacrylamide” based on cellulose matrices of two types: bacterial or regenerated plant cellulose. To form biologically active materials, a method of introducing cerium oxide nanoparticles with sizes less than 5 nm was elaborated. The developed technology allows to obtain hydrogels with the content of cerium oxide (in swollen material) up to 0.4–0.5 wt%. Variations of the ratio of gel components concentrations, type of matrix cellulose and synthesis conditions allow to change the complex of mechanical properties of the material within a wide range, in particular, to obtain both soft, low-modular nanocomposites and hydrogels with record high rigidity. Significant differences in mechanical properties of hydrogels based on different types of cellulose fully correlate with the difference in morphological characteristics of these two groups of materials, revealed by SEM. No palpable effect of nanoparticles on the morphological characteristics of the material was revealed. Both cerium oxide nanoparticles and hydrogels containing cerium oxide showed antibacterial activity against S. aureus ATCC 29213, S. aureus ATCC 43300, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 33495. Different intensity of growth depression of the bacterial cells was determined depending on the samples composition and of the bacteria species.

Targeting virulence determinants is a promising approach to controlling S. aureus infections in the face of the global spread of antibiotic resistance. S. aureus-induced peritonitis often occurs in dialysis, implant and trauma patients. To develop novel prevention and treatment options for peritoneal infection, we investigated the oligopeptide sortase A inhibitor LPRDA as a non-conventional antibacterial that does not affect staphylococcal survival. Administration of LPRDA prior to S. aureus challenge reduced the bacterial load of internal organs and bacterial colonization of the abdominal cavity in animals. In addition, LPRDA inhibited α-hemolysin production in 80% of the 35 reference and clinical S. aureus strains tested. Consequent research of LPRDA interactions with cefazolin and vancomycin has demonstrated the potential for combined application of the antivirulent and antibiotic agents under study.

Prevention of bacterial adhesion is one of the most important antivirulence strategies for meeting the global challenge posed by antimicrobial resistance. We aimed to investigate the influence of a peptidic S. aureus sortase A inhibitor on bacterial adhesion to eukaryotic cells and biofilm formation as a potential method for reducing S. aureus virulence. The pentapeptide LPRDA was synthesized and characterized as a pure individual organic compound. Incubation of MSSA and MRSA strains with LPRDA induced a subsequent reduction in staphylococcal adhesion to Vero cells and biofilm formation, as visualized by microscopic and spectrophotometric methods, respectively. LPRDA did not have a cytotoxic effect on eukaryotic or bacterial cells. The pentapeptide LPRDA deserves further investigation using in vitro and in vivo models of Gram-positive bacteriemia as a potential antibacterial agent with an antiadhesive mechanism of action.

The culture of osteoblasts (OB) of human origin is a useful experimental model in studying bone biology, osteogenic differentiation, functions of bone proteins, oncological processes in bone tissue, testing drugs against bone desires, and many other fields. The purpose of the present study is to share a workflow that has established the conditions to efficiently isolate and grow OB cells obtained from surgically removed bones from human donors. The protocol described here also shows how to determine cell phenotype. Here we provide characteristics of cells isolated by this protocol that might help researchers to decide if such OB are suitable for the purposes of their study. Osteoblasts isolated from collagenase-treated explants of adult bones are able to proliferate and keep their phenotype in culture. OB cells have high synthetic properties. They express osteomarkers, such as RUNX2, osteocalcin, BMP2, and osteopontin both in control conditions and in an osteogenic medium that could be estimated by qPCR and immunocytochemical staining and by Western blotting. Induction of osteogenic differentiation does not dramatically influence the synthetic properties of OB cells, while the cells gain the ability to extracellular mineralization only in an osteogenic medium.

The use of mesenchymal stromal cells (MSCs) for tissue engineering of hyaline cartilage is a topical area of regenerative medicine that has already entered clinical practice. The key stage of this procedure is to create conditions for chondrogenic differentiation of MSCs, increase the synthesis of hyaline cartilage extracellular matrix proteins by these cells and activate their proliferation. The first such works consisted in the indirect modification of cells, namely, in changing the conditions in which they are located, including microfracturing of the subchondral bone and the use of 3D biodegradable scaffolds. The most effective methods for modifying the cell culture of MSCs are protein and physical, which have already been partially introduced into clinical practice. Genetic methods for modifying MSCs, despite their effectiveness, have significant limitations. Techniques have not yet been developed that allow studying the effectiveness of their application even in limited groups of patients. The use of MSC modification methods allows precise regulation of cell culture proliferation, and in combination with the use of a 3D biodegradable scaffold, it allows obtaining a hyaline-like regenerate in the damaged area. This review is devoted to the consideration and comparison of various methods used to modify the cell culture of MSCs for their use in regenerative medicine of cartilage tissue.

Damage to the hyaline layer of the articular surface is an urgent problem for millions of people around the world. At present, a large number of experimental methods are being developed to address this problem, including the transplantation of a cell-engineered construct (CEC) composed of a biodegradable scaffold with a premixed cell culture into the damaged area of the articular surface. However, current methods for analyzing the effectiveness of such CECs have significant limitations. This study aimed to compare the SEM technique, classical histology, and cryosectioning for the analysis of CECs transplanted to hyaline cartilage.