Matrix-Bound Zolzoledronate Enhances the Biofilm Colonization of Hydroxyapatite: Effects on Osteonecrosis

(1) Background: The aim of this study was to test whether matrix-bound zoledronate (zol) molecules enhanced the oral biofilm colonization of a mineralized matrix, rendering the alveolar bone more susceptible to medication-related osteonecrosis of the jaw (MRONJ) following invasive dental procedures. (2) Methods: We tested the effect of matrix-bound zol on the growth and attachment of Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn) and Actinomyces israelii (Ai), and whether the nitrogen-containing component of zol contributed to such effect. The role of oral bacteria in the induction of osteonecrosis was then tested using an extra-oral bone defect model. (3) Results: The attachment of biofilm to hydroxyapatite discs increased when the discs were pre-treated with zol. Bacterial proliferation was not affected. Matrix-bound zol was more potent than non-nitrogen-containing etidronate in enhancing the colonization. Stimulation was dampened by pre-treating the bacteria with histidine. The delivery of oral biofilm to a tibial defect caused osteonecrosis in zol-treated rats. (4) Conclusions: We conclude that matrix-bound zol enhances the oral biofilm colonization of hydroxyapatite. This enhancement depended on the presence of the nitrogen-containing group. The oral biofilm rendered the extra-oral bone susceptible to medication-related osteonecrosis, suggesting that it has an important role in the induction of MRONJ.

Technical note: Unresolved aspects of the direct vapor equilibration method for stable isotope analysis (<i>δ</i>¹⁸O, <i>δ</i>²H) of matrix-bound water: unifying protocols through empirical and mathematical scrutiny

The direct vapor equilibration laser spectrometry (DVE-LS) method has been developed for obtaining matrix-bound water stable isotope data in soils, the critical zone, and bedrock, deriving therefrom subsurface water flow and transport processes and, ultimately, characterizing, for example, groundwater recharge and vulnerability. Recently, DVE-LS has been increasingly adopted due to its possible high sample throughput, relative simplicity, and cost-efficiency. However, this has come at the cost of a non-unified standard operation protocol (SOP), and several contradictory suggestions regarding protocol details do exist which have not been resolved to date. Particularly, sample container material and equilibration times have not yet been agreed upon. Beside practical constraints, this often limits DVE-LS applicability to interpreting relative isotope dynamics instead of absolute values. It also prevents data comparability among studies or laboratories, and several previous comparisons of DVE-LS with other, more traditional approaches of water extraction and subsequent stable isotope analysis yielded significant discrepancies for various sample matrices and physical states. In a series of empirical tests, we scrutinized the controversial DVE-LS protocol details. Specifically, we tested 10 different easily available and cost-efficient inflatable bags previously employed or potentially suitable for DVE-LS sample collection and equilibration. In storage tests similar to the DVE-LS equilibration process but lasting several weeks, we quickly found heat-sealed bags made of laminated aluminum (Al) sheets to be superior by several orders of magnitude over more frequently used freezer bags in terms of evaporation safety and accompanying adverse isotope effects. For the first time, Al-laminated bags allow the applied equilibration time to be adapted exclusively to sample requirements instead of accepting reduced data quality in a trade-off with material shortcomings. Based on detailed physical considerations, we further describe how to calculate the minimum available container headspace and sample-contained liquid water volume and how their ratio affects analytical precision and accuracy. We are confident that these guidelines will expand DVE-LS applicability and improve data quality and comparability among studies and laboratories by contributing to a more unified, physically well-founded SOP based on more appropriate components.

TIMP3 involvement and potentiality in the diagnosis, prognosis and treatment of diabetic nephropathy

Diabetic kidney disease, one of the most severe complications associated with diabetes, is characterized by albuminuria, glomerulosclerosis and progressive loss of renal function. Loss of TIMP3, an Extracellular matrix-bound protein, is a hallmark of diabetic nephropathy in human and mouse models, suggesting its pivotal role in renal diseases associated to diabetes. There is currently no specific therapy for diabetic nephropathy, and the ability to restore high TIMP3 activity specifically in the kidney may represent a potential therapeutic strategy for the amelioration of renal injury under conditions in which its reduction is directly related to the disease. Increasing evidence shows that diabetic nephropathy is also regulated by epigenetic mechanisms, including noncoding RNA. This review recapitulates the pathological, diagnostic and therapeutic potential roles of TIMP3 and the noncoding RNA (microRNA, long noncoding RNA) related to its expression, in the progression of diabetic nephropathy.

Abstract P-41: Contribution of Matrix-bound Vesicles Produced by Mesenchymal Stromal Cells in the Differentiation of Multipotent Stem Cells in vitro

Background: According to the current view on the extracellular matrix (ECM) composition and functions, it includes not only structural proteins and components of cell adhesion, but also various deposited components, including enzymes involved in ECM remodeling, growth factors, and matrix-bound vesicles (MBV). MBV can presumably participate in the formation of a specific microenvironment for stem cells and regulate their differentiation. However, the contribution of MBV to these processes remains poorly understood. In our work, we evaluated the effects of MBV within native ECM produced by mesenchymal stromal cells (MSCs) cultured in cell sheet on multipotent stem cell differentiation. Methods: We isolated MBV from decellularized MSC-produced ECM by treatment with the following enzymes: collagenase, hyaluronidase, or trypsin, and centrifugation on 1000 kDa filters. The nanostructure and relative size in each sample were observed using TEM. The particle size and concentration were also studied with NTA. In addition, the obtained MBV were examined for the presence of key exosome markers using Western blot. Then we investigated the effect of MBV on the formation of capillary-like structures by endothelial cells (in vitro model of angiogenesis) as well as on the differentiation of primary MSCs isolated from human adipose tissue in the adipogenic, osteogenic, and chondrogenic directions. Results: As a result of comparative analysis of isolation protocols, it was shown that all MBV samples had the characteristics of extracellular vesicles (EV), but differed in size and representation of exosomal markers. The MBV isolated from ECM did not stimulate the formation of capillary-like structures by endothelial cells, in contrast to EV secreted by MSCs to the conditioned medium, but maintained the viability of the endothelium. Isolated MBV stimulated osteogenic and adipogenic differentiation of MSCs similar to secreted EV. On the other hand, preincubation of MSCs with MBV leads to reorganization of cell monolayer to spheroid-like structures during chondrogenic differentiation. Conclusion: Here, we developed the protocol of isolation of MBV from ECM that have distinguished characteristics and functional activity.

Technical note: Controversial aspects of the direct vapor equilibration method for stable isotope analysis (δ¹⁸O, δ²H) of matrix-bound water: Unifying protocols through empirical and mathematical scrutiny

The direct vapor equilibration laser spectrometry (DVE-LS) method has been developed for obtaining matrix-bound water stable isotope data in soils, the critical zone and bedrock, deriving therefrom subsurface water flow and transport processes and, ultimately, characterising e.g. groundwater recharge and vulnerability. Recently, DVE-LS has been increasingly adopted due to its possible high sample throughput, relative simplicity and cost-efficiency. However, this has come at the cost of a non-unified standard operation protocol (SOP) and several contradictory suggestions regarding protocol details do exist which have not been resolved to date. Particularly, sample container material and equilibration times have not yet been agreed upon. Beside practical constraints, this often limits DVE-LS applicability to interpreting relative isotope dynamics instead of absolute values. It also prevents data comparability among studies or laboratories and several previous comparisons of DVE-LS with other, more traditional approaches of water extraction and subsequent stable isotope analysis yielded significant discrepancies for various sample matrices and physical states. In a series of empirical tests, we scrutinized the controversial DVE-LS protocol details. Specifically, we tested ten different easily available and cost-efficient inflatable bags previously employed or potentially suitable for DVE-LS sample collection and equilibration. In storage tests similar to the DVE-LS equilibration process but lasting several weeks, we quickly found heat-sealed bags made of laminated Aluminum (Al) sheets to be superior by several orders of magnitude over more frequently used freezer bags in terms of evaporation-safety and accompanying adverse isotope effects. For the first time, Al-laminated bags allow the applied equilibration time to be adapted exclusively to sample requirements instead of accepting reduced data quality in a trade-off with material shortcomings. Based on detailed physical considerations, we further describe how to calculate the minimum available container headspace and sample-contained liquid water volume and how their ratio affects analytical precision and accuracy. We are confident, that these guidelines will expand DVE-LS applicability and improve data quality and comparability among studies and laboratories by contributing to a more unified, physically well-founded SOP based on more appropriate components.

Aggregation Susceptibility of Low-Density Lipoproteins—A Novel Modifiable Biomarker of Cardiovascular Risk

Circulating low-density lipoprotein (LDL) particles enter the arterial intima where they bind to the extracellular matrix and become modified by lipases, proteases, and oxidizing enzymes and agents. The modified LDL particles aggregate and fuse into larger matrix-bound lipid droplets and, upon generation of unesterified cholesterol, cholesterol crystals are also formed. Uptake of the aggregated/fused particles and cholesterol crystals by macrophages and smooth muscle cells induces their inflammatory activation and conversion into foam cells. In this review, we summarize the causes and consequences of LDL aggregation and describe the development and applications of an assay capable of determining the susceptibility of isolated LDL particles to aggregate when exposed to human recombinant sphingomyelinase enzyme ex vivo. Significant person-to-person differences in the aggregation susceptibility of LDL particles were observed, and such individual differences largely depended on particle lipid composition. The presence of aggregation-prone LDL in the circulation predicted future cardiovascular events in patients with atherosclerotic cardiovascular disease. We also discuss means capable of reducing LDL particles’ aggregation susceptibility that could potentially inhibit LDL aggregation in the arterial wall. Whether reductions in LDL aggregation susceptibility are associated with attenuated atherogenesis and a reduced risk of atherosclerotic cardiovascular diseases remains to be studied.