Matrix Formation Research Articles

Distinctions in bone matrix nanostructure, composition, and formation between osteoblast-like cells, MG-63, and human mesenchymal stem cells, UE7T-13

Osteoblast-like cells and human mesenchymal stem cells (hMSCs) are frequently employed as osteoprogenitor cell models for evaluating novel biomaterials in bone healing and tissue engineering. In this study, the characterization of UE7T-13 hMSCs and MG-63 human osteoblast-like cells was examined. Both cells can undergo osteogenesis and produce calcium extracellular matrix; however, calcium nodules produced by MG-63 lacked a central mass and appeared flatter than UE7T-13. The absence of growing calcium nodules in MG-63 was discovered by SEM-EDX to be associated with the formation of alternating layers of cells and calcium extracellular matrix. The nanostructure and composition analysis showed that UE7T-13 had a finer nanostructure of calcium nodules with a higher calcium/phosphate ratio than MG-63. Both cells expressed high intrinsic levels of collagen type I alpha 1 chain, while only UE7T-13 expressed high levels of alkaline phosphatase, biomineralization associated (ALPL). High ALP activity in UE7T-13 was not further enhanced by osteogenic induction, but in MG-63, low intrinsic ALP activity was greatly induced by osteogenic induction. These findings highlight the differences between the two immortal osteoprogenitor cell lines, along with some technical notes that should be considered while selecting and interpreting the pertinent in vitro model.

Investigation of gasification reactivity and properties of biocarbon at high temperature in a mixture of CO/CO2

Understanding the conversion behaviors of biocarbon under conditions relevant to industrial conditions is important to ensure proper and efficient utilization of the biocarbon for a dedicated metallurgical process. The present work studied the reactivity of biocarbon by using a Macro-TGA at 1100 °C in a gas mixture of CO2 and CO to simulate the conditions in an industrial closed submerged arc manganese alloy furnace. The conversion residues from the Macro-TGA tests were collected for detailed characterization through a combination of different analytical techniques. Results showed that biocarbons produced under various conditions have different reactivities under the studied conditions. The biocarbon produced in an atmospheric fixed bed reactor with continuous purging of N2 has the highest reactivity. Its fixed carbon loss started as the gas atmosphere shifted from the inert Ar to a mixture of CO and CO2 at 1100 °C. And only 450 s was needed to reach a desired fixed-carbon loss of 20%. The high reactivity of the biocarbon is mainly related to its porous structure and high content of catalytic inorganic elements, which favor gasification reactions of the carbon matrix towards the surrounding gas atmosphere and consumption of carbon consequently. In contrast, biocarbon produced under constrained conditions and from wood pellets and steam exploded pellets have more compact appearance and dense structures. Significant fixed carbon loss for these biocarbons started 80–200 s later than that of the biocarbon produced at atmospheric conditions with purging of N2. Additionally, it took longer time, 557–1167 s, for these biocarbons to realize the desired fixed-carbon loss. SEM-EDX analyses results revealed clear accumulation and aggregation of inorganic elements, mainly Ca, on the external surface of the residues from gasification of biocarbon produced in the fixed bed reactor with purging of N2. It indicates more intensive migration and transformation of inorganic elements during gasification at this condition. This resulted in formation of a carbon matrix with more porous structure and active sites on the carbon surface, promoting the Boudouard reaction and conversion of carbon.

Critical role of transcriptome-wide m6A methylation in the aqueous humor of patients with pseudoexfoliation glaucoma

N6-methyladenosine (m6A) modification is one of the most common types of methylation modifications in eukaryotic mRNA. However, its role in the pathogenesis of pseudoexfoliation glaucoma (PXG) has not yet been reported. To enhance understanding in this regard, we assessed the m6A methylome in the aqueous humor of patients with PXG. MeRIP-Seq and RNA-Seq analyses were performed to compare the m6A methylomes and gene expression profiles of the aqueous humor of patients with PXG with those of patients with age-related cataract (ARC). Colorimetric m6A quantification was performed to detect global m6A levels. Quantitative reverse transcription PCR confirmed the expression of m6A-related enzymes and mRNAs in both groups. Results showed significantly higher aqueous humor m6A levels in the PXG group than in the ARC group. Five m6A-related enzymes, including METTL3, YTHDC2, HNRNPA2B1, HNRNPC, and LRPPRC, were significantly up-regulated in PXG specimens. We also observed 9728 m6A-modified peaks related to 6126 gene transcripts in the PXG group, with more than 250 genes containing one m6A peak (hypomethylated or hypermethylated). The distribution of the m6A peaks was enriched in coding sequences and 3′-untranslated regions for both groups. GGAC motif structures were also significantly enriched. Bioinformatics analysis further revealed that m6A plays a critical role in extracellular matrix formation and histone deacetylation. Additionally, MMP14, ADAMTSL1, FN1, and HDAC1 showed significant changes in m6A methylation and mRNA expression in the PXG group. Therefore, m6A methylation may regulate extracellular matrix composition in PXG and METTL3 may be a pivotal regulator of this process. In the future, it would be necessary to investigate MMP14, ADAMTSL1, FN1, and HDAC1, which are potential target genes.

Comparative characterization between autologous serum and platelet lysate under different temperatures and storage times

Therapies using autologous serum and platelet lysate have shown promise among blood and biological products in the treatment of various diseases. The autologous serum has been shown to be a superior alternative to traditional eye drops in treating eye diseases in ophthalmology. Figurelet lysate (PL) has recently been considered a more interesting alternative for the treatment of multiple tissues, as it does not have the unfavorable reactions seen with traditional platelet-rich plasma (PRP), making it a valuable blood derivative for use in ocular therapy. There is no definitive comparison in veterinary medicine between PL and autologous serum in terms of the content of Transforming Growth Factor beta 1 (TGF-1), which is known to have chemotactic, mitogenic, matrix formation, and angiogenesis effects on tissues, and beneficial proteins in ocular tissue. This study aimed to estimate the concentrations of TGF-1, total protein, and albumin, as well as autologous serum and platelet lysate, in horses over an 8-day storage period at temperatures of 4 °C and 37 °C. To produce autologous serum, 63 ml of blood was collected from each animal in seven 9 ml tubes without anticoagulant. For platelet lysate, 180 ml of blood was collected in 50 tubes of 3.6 ml with 3.2% sodium citrate. The most significant findings were the positive relationship between the baseline platelet count in the blood and the final platelet concentration in PRP. Specifically, we found a correlation (R = 0.9) with a p - value of 0.005 between the average baseline platelet level of seven animals and their corresponding PRP results, both on an individual level and as a group. Additionally, there was a correlation between growth factor concentration and PRP platelets, with the highest growth factor concentration in PL. The temperature storage group exhibited higher concentrations of total protein and serum albumin, as well as the maximum amount of growth factor for both products at a temperature of 37 °C.

PECULIARITIES OF CONNECTIVE TISSUE METABOLISM IN PATIENTS WITH POST-INFARCTION REMODELING AND REPERFUSION SYNDROME

Early restoration of coronary blood flow in acute myocardial infarction with elevation helps reduce the area of myocardial necrosis, prevent dilatation of the ventricular cavity, and reduce the development of arrhythmias and mortality. However, despite modern technologies in STEMI treatment, the development of reperfusion syndrome, in particular, arrhythmias, is possible after the restoration of blood flow in the infarct-related artery, which is a consequence of acute cellular, metabolic and local electrophysiological changes in the myocardium.
 The aim: is to investigate the long-term effects of reperfusion syndrome on the catabolic/anabolic activity of connective tissue and the processes of connective tissue framework formation in the post-infarction period in patients with myocardial infarction.
 Materials and methods. The study involved 156 patients with acute myocardial infarction with ST segment elevation (STEMI), and 15 apparently healthy individuals (control of laboratory parameters). The average age of the patients was (57.8±1.2) years. The percent of male patients prevailed (76 %).
 All MI patients were divided into two groups: Group I (98 people) – MI patients without clinical signs of reperfusion syndrome during revascularization, Group II (58 people) – MI patients with the signs of reperfusion syndrome in the form of rhythm and conductivity disturbances during revascularization of coronary arteries by percutaneous coronarography intervention (PCI) with prolonged stenting of the infarct-dependent coronary artery.
 Peculiarities of connective tissue metabolism were studied by the content of free and bound oxyproline, glycosaminoglycans, and antibodies to atypical collagens (Ig M and Ig G) in blood serum.
 Results. The recovery period after myocardial infarction showed an increase in collagen catabolism over collagen synthesizing processes in patients with MI who had reperfusion syndrome. The described patterns were also established by changes in glycosaminoglycans. The study showed an increase in the level of antibodies to atypical collagens. Particularly, the level of Ig M, which was within the norm (0.43±0.18) ng/ml, in patients with MI without reperfusion manifestations was three times higher (p<0.001), and in cases of combination of MI and reperfusion syndrome – the highest (p<0.001).
 Conclusions:
 
 Changes in collagen-synthesizing processes are observed in patients with MI, with a tendency to increase the processes of catabolism of the connective tissue matrix.
 The presence of reperfusion syndrome manifestations in MI patients after revascularization aggravates the processes of catabolism, even in the recovery period after MI.
 Increase in the activity of catabolic processes stimulates fibrosis and formation of rigid matrix and atypical collagen, to which antibodies are synthesized, thus blocking the pathological circuit of restructuring of the heart connective tissue framework, and accordingly its pathological remodeling.
 The obtained study results contribute to the identification of more sophisticated approaches to revascularization techniques to avoid negative effects of reperfusion syndrome, as well as new effective medications for collagen synthesis correction, which can increase the effectiveness of rehabilitative management of such

Inhibition of GSK-3β Enhances Osteoblast Differentiation of Human Mesenchymal Stem Cells through Wnt Signalling Overexpressing Runx2.

Small-molecule-inhibitor-based bone differentiation has been recently exploited as a novel approach to regulating osteogenesis-related signaling pathways. In this study, we identified 1-Azakenpaullone, a highly selective inhibitor of glycogen synthase kinase-3β (GSK-3β), as a powerful inducer of osteoblastic differentiation and mineralization of human mesenchymal stem cells (MSCs). GSK-3β is a serine-threonine protein kinase that plays a major role in different disease development. GSK-3β is a key regulator of Runx2 activity in osteoblastic formation. We evaluated alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin Red staining to assess the mineralization of cultured human MSCs. Gene expression profiling was assessed using an Agilent microarray platform, and bioinformatics were performed using Ingenuity Pathway Analysis software. Human MSCs treated with 1-Azakenpaullone showed higher ALP activity, increased in vitro mineralized matrix formation, and the upregulation of osteoblast-specific marker gene expression. Global gene expression profiling of 1-Azakenpaullone-treated human MSCs identified 1750 upregulated and 2171 downregulated mRNA transcripts compared to control cells. It also suggested possible changes in various signaling pathways, including Wnt, TGFβ, and Hedgehog. Further bioinformatics analysis employing Ingenuity Pathway Analysis recognized significant enrichment in the 1-Azakenpaullone-treated cells of genetic networks involved in CAMP, PI3K (Complex), P38 MAPK, and HIF1A signaling and functional categories associated with connective tissue development. Our results suggest that 1-Azakenpaullone significantly induced the osteoblastic differentiation and mineralization of human MSCs mediated by the activation of Wnt signaling and the nuclear accumulation of β-catenin, leading to the upregulation of Runx2, a key transcription factor that ultimately promotes the expression of osteoblast-specific genes. Thus, 1-Azakenpaullone could be used as an osteo-promotor factor in bone tissue engineering.

Self-fuelled nickel oxide encapsulated sodium alginate-grafted-polypyrrole for potentiometric sensing of lead ions

Precise determination of lead ions in edible and non-edible items is essential due to high toxicity and existing technical challenges viz; requirement of external energy source, portability, sensing range, and other parameters despite tremendous technological and materials advancement. The present study demonstrates the portable potentiometric sensing of lead ions over the in-situ prepared and chemically functionalized self-fuelled ternary biopolymer nanocomposite of nickel oxide, sodium alginate, and polypyrrole (NiO/SA-g-PPY). The prepared bio-nanocomposite's chemical structure, physical, and morphological properties were studied by FT-IR, XRD, SEM, and other relevant standard techniques. The analytical results revealed the formation of a porous hybrid composite matrix with improved electrical conductivity in the range of 1.13 × 10−3 S cm−1, along with chemical interactive nature and self-fuelled behavior. Further, a film of the prepared composite was cast on ITO-coated glass by spin coating methods and has been demonstrated as a sensing electrode for effective potentiometric sensing of Pb2+ ions in natural and artificial samples after using laboratory fabricated potentiometric setup. The proposed electrode exhibits the potential response for Pb2+ ion on the broader concentration range of 10−8 M to 10−3 M at pH 7.0 and temperature 298 K, along with sensitivity 83.2 mV mM−1cm−2, recovery time 10 s, response time 50 s, and stability for 70 days. Further, the sensing mechanism was also explained based on redox surface interaction between Pb2+ and NiO/SA-g-PPy-based electrodes in terms of induced structural changes and generated potential.

Relaxin in fibrotic ligament diseases: Its regulatory role and mechanism.

Fibrotic ligament diseases (FLDs) are diseases caused by the pathological accumulation of periarticular fibrotic tissue, leading to functional disability around joint and poor life quality. Relaxin (RLX) has been reported to be involved in the development of fibrotic lung and liver diseases. Previous studies have shown that RLX can block pro-fibrotic process by reducing the excess extracellular matrix (ECM) formation and accelerating collagen degradation in vitro and in vivo. Recent studies have shown that RLX can attenuate connective tissue fibrosis by suppressing TGF-β/Smads signaling pathways to inhibit the activation of myofibroblasts. However, the specific roles and mechanisms of RLX in FLDs remain unclear. Therefore, in this review, we confirmed the protective effect of RLX in FLDs and summarized its mechanism including cells, key cytokines and signaling pathways involved. In this article, we outline the potential therapeutic role of RLX and look forward to the application of RLX in the clinical translation of FLDs.

Modulating osteoclasts with nanoparticles: A path for osteoporosis management?

Osteoclasts are the cells responsible for the bone resorption process during bone remodeling. In a healthy situation, this process results from an equilibrium between new matrix formation by osteoblast and matrix resorption by osteoclast. Osteoporosis (OP) is a systemic bone disease characterized by a decreased bone mass density and alterations in bone microarchitecture, increasing fracture predisposition. Despite the variety of available therapies for OP management there is a growing gap in its treatment associated to the low patients' adherence owing to concerns related with long-term efficacy or safety. This makes the development of new and safe treatments necessary. Among the newly developed strategies, the use of synthetic and natural nanoparticles to modulate osteoclasts differentiation, activity, apoptosis or crosstalk with osteoblasts have arisen. Synthetic nanoparticles exert their therapeutic effect either by loading antiresorptive drugs or including molecules for osteoclasts gene regulation. Moreover, this control over osteoclasts can be improved by their targeting to bone extracellular matrix or osteoclast membranes. Furthermore, natural nanoparticles, also known as extracellular vesicles, have been identified to play a key role in bone homeostasis. Consequently, these systems have been widely studied to control osteoblasts and osteoclasts under variable environments. Additionally, the ability to bioengineer extracellular vesicles has allowed to obtain biomimetic systems with desirable characteristics as drug carriers for osteoclasts. The analyzed information reveals the possibility of modulating osteoclasts by different mechanisms through nanoparticles decreasing bone resorption. These findings suggest that controlling osteoclast activity using nanoparticles has the potential to improve osteoporosis management. This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Pathophysiological aspects of the mechanisms of action of osteotropic drugs in an in vitro experiment on the blood mononuclear cells of patients with frontal bone injuries

Relevance: Pathogenetic treatment of head injuries should be based on the development of post-traumat ic disease, including the role of immune reactions in bone matrix regeneration processes. Repair of bone structures depends on activating stimuli from cells of the immune system. Currently, there is research on the importance of vitamin D in metabolic processes in mesenchymal tissue that affect the formation of the bone matrix and general mineralization of bones, as well as the drug osteogenon, the basis of which are ossein-hydroxyapatite compounds, which stimulate local regulators of bone tissue remodelling and calcium-phosphorus metabolism. Taking into account these data, it will be appropriate to determine the effect of drugs on the processes of bone tissue regeneration in patients with injuries of the frontal bone. Purpose: to determine the effect of various drugs used in the treatment of frontal skull injuries on the pathophysiological mechanisms of inflammation (interleukin-1β, interleukin-10, γ-interferon), stabilization of cell membranes, and the level of regenerative cytokines. Materials and methods: The authors conducted immunological studies on the determination of cytokine factors of inflammation and regeneration in 29 patients with frontal bone injury under the influence of various osteotropic drugs: ossein-hydroxyapatite compounds, osteogenon and vitamin D3 (water-soluble form – aquadetrim), both individually and in combination with each other. The research was carried out in an in vitro experiment in RPMI-1640 medium (Serva, Germany) with blood mononuclear cells from patients. γ-interferon), anti-inflammatory regeneration factor (transforming growth factor-TGF-1β). In the conditions of the experiment, the membrane-protective properties of the above-mentioned osteotropic drugs were investigated on cultures of peritoneal tissue basophils under the conditions of using protamine sulfate as a damaging factor. Results and discussion: it was established that osteogenon and aquadetrim (vit. D3) have a positive effect on the production of cytokines by mononuclear cells in the blood of patients with TLC: they reduce the level of in vitro pro-inflammatory production and increase the production of anti-inflammatory cytokines, especially when used together. Protamine sulphate (liberator) was able to destroy 61% of tissue basophils, SK and osteogenon reduced this percentage, but not reliably. Reliable data were obtained under the action of vitamin, and combinations of ossein-hydroxyapatite compounds + vitamin and osteogenon + vitamin (р=0.01). The use of a general mixture of all drugs did not cancel the effect of liberation of TB. It can be assumed that the main membrane protector is vitamin D3 in the form of a water-soluble form – aquadetrim, which has various directions of pathophysiological influence on the mechanisms of inflammation and regeneration. Conclusions: The growth factor was better stimulated with a mixture of all drugs (~1.7 μg/ml) in combination with vitamin D3. Membrane-protective properties were better expressed under the action of vitamin D3 and combinations of osseinhydroxyapatite compounds + vitamin D3 and osteogenon + vitamin D3. The most effective in activating immunocompetent blood cells were combinations of osteotropic drugs that reduced the level of proinflammatory cytokines and increased the content of anti-inflammatory interleukin-10 in the culture fluid.

Developmental growth plate cartilage formation suppressed by artificial light at night via inhibiting BMAL1-driven collagen hydroxylation.

Exposure to artificial light at night (LAN) can induce obesity, depressive disorder and osteoporosis, but the pernicious effects of excessive LAN exposure on tissue structure are poorly understood. Here, we demonstrated that artificial LAN can impair developmental growth plate cartilage extracellular matrix (ECM) formation and cause endoplasmic reticulum (ER) dilation, which in turn compromises bone formation. Excessive LAN exposure induces downregulation of the core circadian clock protein BMAL1, which leads to collagen accumulation in the ER. Further investigations suggest that BMAL1 is the direct transcriptional activator of prolyl 4-hydroxylase subunit alpha 1 (P4ha1) in chondrocytes, which orchestrates collagen prolyl hydroxylation and secretion. BMAL1 downregulation induced by LAN markedly inhibits proline hydroxylation and transport of collagen from ER to golgi, thereby inducing ER stress in chondrocytes. Restoration of BMAL1/P4HA1 signaling can effectively rescue the dysregulation of cartilage formation within the developmental growth plate induced by artificial LAN exposure. In summary, our investigations suggested that LAN is a significant risk factor in bone growth and development, and a proposed novel strategy targeting enhancement of BMAL1-mediated collagen hydroxylation could be a potential therapeutic approach to facilitate bone growth.

Electrical Stimulation in Cartilage Tissue Engineering.

Electrical stimulation (ES) has been frequently used in different biomedical applications both in vitro and in vivo. Numerous studies have demonstrated positive effects of ES on cellular functions, including metabolism, proliferation, and differentiation. The application of ES to cartilage tissue for increasing extracellular matrix formation is of interest, as cartilage is not able to restore its lesions owing to its avascular nature and lack of cells. Various ES approaches have been used to stimulate chondrogenic differentiation in chondrocytes and stem cells; however, there is a huge gap in systematizing ES protocols used for chondrogenic differentiation of cells. This review focuses on the application of ES for chondrocyte and mesenchymal stem cell chondrogenesis for cartilage tissue regeneration. The effects of different types of ES on cellular functions and chondrogenic differentiation are reviewed, systematically providing ES protocols and their advantageous effects. Moreover, cartilage 3D modeling using cells in scaffolds/hydrogels under ES are observed, and recommendations on reporting about the use of ES in different studies are provided to ensure adequate consolidation of knowledge in the area of ES. This review brings novel insights into the further application of ES in in vitro studies, which are promising for further cartilage repair techniques.

Mechanical stretching determines the orientation of osteoblast migration and cell division.

Osteoblasts alignment and migration are involved in the directional formation of bone matrix and bone remodeling. Many studies have demonstrated that mechanical stretching controls osteoblast morphology and alignment. However, little is known about its effects on osteoblast migration. Here, we investigated changes in the morphology and migration of preosteoblastic MC3T3-E1 cells after the removal of continuous or cyclic stretching. Actin staining and time-lapse recording were performed after stretching removal. The continuous and cyclic groups showed parallel and perpendicular alignment to the stretch direction, respectively. A more elongated cell morphology was observed in the cyclic group than in the continuous group. In both stretch groups, the cells migrated in a direction roughly consistent with the cell alignment. Compared to the other groups, the cells in the cyclic group showed an increased migration velocity and were almost divided in the same direction as the alignment. To summarize, our study showed that mechanical stretching changed cell alignment and morphology in osteoblasts, which affected the direction of migration and cell division, and velocity of migration. These results suggest that mechanical stimulation may modulate the direction of bone tissue formation by inducing the directional migration and cell division of osteoblasts.

Development of serum substitute medium for bone tissue engineering.

In tissue engineering, cells are grown often on scaffolds and subjected to chemical/mechanical stimuli. Most such cultures still use fetal bovine serum (FBS) despite its known disadvantages including ethical concerns, safety issues, and variability in composition, which greatly influences the experimental outcomes. To overcome the disadvantages of using FBS, chemically defined serum substitute medium needs to be developed. Development of such medium depends on cell type and application-which makes it impossible to define one universal serum substitute medium for all cells in any application. Here, we developed a serum substitute medium for bone tissue engineering (BTE) in a step-by-step process. Essential components were added to the medium while human bone marrow mesenchymal stromal cells (hBMSCs, osteoblast progenitor cells) were cultured in two-dimensional and three-dimensional substrates. In a 3-week culture, the developed serum substitute medium worked equally well as FBS containing medium in term of cell attachment to the substrate, cell survival, osteoblast differentiation, and deposition of extracellular matrix. In the next step, the use of serum substitute medium was evaluated when culturing cells under mechanical loading in the form of shear stress. The outcomes showed that the application of shear stress is essential to improve extracellular matrix formation while using serum substitute medium. The developed serum substitute medium could pave the way in replacing FBS for BTE studies eliminating the use of controversial FBS and providing a better-defined chemical environment for BTE studies.

C1q/TNF-Related Proteins 1, 6 and 8 Are Involved in Corneal Epithelial Wound Closure by Targeting Relaxin Receptor RXFP1 In Vitro.

Inadequate wound healing of ocular surface injuries can lead to permanent visual impairment. The relaxin ligand-receptor system has been demonstrated to promote corneal wound healing through increased cell migration and modulation of extracellular matrix formation. Recently, C1q/tumor necrosis factor-related protein (CTRP) 8 was identified as a novel interaction partner of relaxin receptor RXFP1. Additional data also suggest a role for CTRP1 and CTRP6 in RXFP1-mediated cAMP signaling. However, the role of CTRP1, CTRP6 and CTRP8 at the ocular surface remains unclear. In this study, we investigated the effects of CTRP1, CTRP6, and CTRP8 on epithelial ocular surface wound closure and their dependence on the RXFP1 receptor pathway. CTRP1, CTRP6, and CTRP8 expression was analyzed by RT-PCR and immunohistochemistry in human tissues and cell lines derived from the ocular surface and lacrimal apparatus. In vitro ocular surface wound modeling was performed using scratch assays. We analyzed the effects of recombinant CTRP1, CTRP6, and CTRP8 on cell proliferation and migration in human corneal and conjunctival epithelial cell lines. Dependence on RXFP1 signaling was established by inhibiting ligand binding to RXFP1 using a specific anti-RXFP1 antibody. We detected the expression of CTRP1, CTRP6, and CTRP8 in human tissue samples of the cornea, conjunctiva, meibomian gland, efferent tear ducts, and lacrimal gland, as well as in human corneal, conjunctival, and meibomian gland epithelial cell lines. Scratch assays revealed a dose-dependent increase in the closure rate of surface defects in human corneal epithelial cells after treatment with CTRP1, CTRP6, and CTRP8, but not in conjunctival epithelial cells. Inhibition of RXFP1 fully attenuated the effect of CTRP8 on the closure rate of surface defects in human corneal epithelial cells, whereas the CTRP1 and CTRP6 effects were not completely suppressed. Conclusions: Our findings demonstrate a novel role for CTRP1, CTRP6, and CTRP8 in corneal epithelial wound closure and suggest an involvement of the relaxin receptor RXFP1 signaling pathway. This could be a first step toward new approaches for pharmacological and therapeutic intervention.

Abstract 2260: Characterizing tumor stromal evolution after neoadjuvant anti-PD-1 therapy in Merkel cell carcinoma

Abstract Background: Neoadjuvant anti-PD-1-based therapies have shown promise in several cancers, including Merkel cell carcinoma (MCC), a rare and aggressive skin cancer. We have shown that on-treatment specimens from patients responding to anti-PD-1 are characterized histologically by exuberant proliferative fibrosis, neovascularization, and robust inflammation including diverse immune cell types. Understanding how the stroma participates in the process of tumor regression may open therapeutic opportunities by redirecting stromal remodeling in treatment-refractory tumors. Methods: To evaluate stromal changes in neoadjuvant treated MCC, 13 on-treatment FFPE tumor specimens from patients with resectable MCC who received anti-PD-1 for ~4 weeks before surgery (NCT02488759) were subjected to laser capture microdissection to collect stromal tissue and isolate RNA. We conducted multiplex qRT-PCR targeting 125 unique mRNAs representing TME-relevant immune cell subsets, immune checkpoint pathways, and immune-modulating transcription factors and cytokines, followed by whole transcriptome RNA sequencing. Differential gene expression was considered significant if the magnitude of the signed fold-change was ≥2 and the p-value was ≤0.1 (Benjamini-Hochberg adjusted for RNAseq). Gene set enrichment analysis (GSEA) was performed with R Topper. Pathologic response was scored on H&E staining as pathologic complete response (pCR, n=6), major pathologic response (MPR, ≤10% residual viable tumor [RVT]; n=4), or non-MPR/pCR (>10% RVT; n=3). Results: Stratifying by response status (6 pCR vs 3 non-MPR/pCR) demonstrated that pCR stroma in MCC is distinguished by upregulation of molecules associated with neovascularization (VEGFA, EGR2) and M1 and M2 macrophages (IL1A, ARG1) via qRT-PCR, and mesenchymal cells (fibroblasts, myocytes, adipocytes) and extracellular matrix formation via RNAseq. GSEA demonstrated a strong association of pCR with both myocyte and fibroblast differentiation and functional pathways, suggesting a role for myofibroblasts in tissue remodeling during anti-PD-1-mediated tumor regression. In contrast, the stroma of non-MPR/pCR tumors showed evidence for ongoing IFNg-mediated inflammation, as evidenced by upregulation of IFNg, IL-27, STAT1, CXCL10 and LAG-3 via qRT-PCR; these findings were supported by RNAseq, which also showed upregulation of genes associated with inflammation (e.g., PLXNC1, TRPV2, LYZ, LAIR1, HLA-A, B2M, CD4). Similar results were obtained when MPRs were grouped with pCRs (n=10) vs non-MPR/pCR (n=3). Conclusion: These findings demonstrate that improved response to anti-PD-1 therapy is associated with a shift in the tumor stroma from an inflammatory to a proliferative myofibroblastic phenotype, recapitulating the stages of normal wound healing. This suggests a potential functional role for myofibroblasts in that critical transition. Citation Format: Joel C. Sunshine, Tracee L. McMiller, Alyza Skaist, Yan Zhang, Alan Berger, Kornel Schuebel, Jennifer Meyers, Julie S. Deutsch, Aleksandra Ogurtsova, Elizabeth L. Engle, Leslie Cope, Janis M. Taube, Suzanne L. Topalian. Characterizing tumor stromal evolution after neoadjuvant anti-PD-1 therapy in Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2260.

Rapid Inactivation of mixed biofilms of Candida albicans and Candida tropicalis using antibacterial photodynamic therapy: Based on PAD™ Plus

BackgroundTo investigate the sterilizing effect of antimicrobial photodynamic therapy (aPDT) based on PAD™ Plus on mixed biofilms of Candida albicans and Candida tropicalis. MethodsA mature mixed biofilm model of C. albicans and C. tropicalis was constructed in vitro. FITC-concanavalin A staining was conducted to observe the formation of the extracellular matrix. MTT assay was performed to determine biofilm viability. The chromogenic medium was used to examine the Candida composition of the mixed biofilms. For aPDT treatment, based on PAD™ Plus, the biofilms were incubated with 1 mg/mL TBO for 1, 5, or 10 min, followed by 500 or 750 mW LED illumination for 1 or 2 min. The live/dead fungi were detected by SYTO9/propidium iodide staining. A multivariate factorial design was conducted to analyze the correlations of parameters with the inactivation effect of the mixed biofilms. ResultsMature mixed biofilms formed at 24 h after seeding. Compared with untreated biofilms, following 1-min TBO incubation, 500 mW LED illumination for 1 min inactivated more than 90% of the fungi. Extending the incubation time did not significantly improve the inactivation effect. Application of 750 mW output power or 2 min LED illumination inactivated more than 99% of the fungi without increasing other parameters. ConclusionsPAD™ Plus combined with 1 mg/mL TBO can rapidly inactivate the mature mixed biofilms of C. albicans and C. tropicalis, serving as a robust platform for the treatment of mixed infections of C. albicans and C. tropicalis.

A comprehensive molecular profiling approach reveals metabolic alterations that steer bone tissue regeneration

Bone regeneration after fracture is a complex process with high and dynamic energy demands. The impact of metabolism on bone healing progression and outcome, however, is so far understudied. Our comprehensive molecular profiling reveals that central metabolic pathways, such as glycolysis and the citric acid cycle, are differentially activated between rats with successful or compromised bone regeneration (young versus aged female Sprague-Dawley rats) early in the inflammatory phase of bone healing. We also found that the citric acid cycle intermediate succinate mediates individual cellular responses and plays a central role in successful bone healing. Succinate induces IL-1β in macrophages, enhances vessel formation, increases mesenchymal stromal cell migration, and potentiates osteogenic differentiation and matrix formation in vitro. Taken together, metabolites—here particularly succinate—are shown to play central roles as signaling molecules during the onset of healing and in steering bone tissue regeneration.

3D bioprinting of ultrashort self-assembling peptides to engineer scaffolds with different matrix stiffness for chondrogenesis.

62Articular cartilage is a nonvascularized and poorly cellularized tissue with a low self-repair capacity. Therefore, damage to this tissue due to trauma or degenerative joint diseases such as osteoarthritis needs a high-end medical intervention. However, such interventions are costly, have limited healing capacity, and could impair patients' quality of life. In this regard, tissue engineering and three-dimensional (3D) bioprinting hold great potential. However, identifying suitable bioinks that are biocompatible, with the desired mechanical stiffness, and can be used under physiological conditions is still a challenge. In this study, we developed two tetrameric self-assembling ultrashort peptide bioinks that are chemically well-defined and can spontaneously form nanofibrous hydrogels under physiological conditions. The printability of the two ultrashort peptides was demonstrated; different shape constructs were printed with high shape fidelity and stability. Furthermore, the developed ultrashort peptide bioinks gave rise to constructs with different mechanical properties that could be used to guide stem cell differentiation toward specific lineages. Both ultrashort peptide bioinks demonstrated high biocompatibility and supported the chondrogenic differentiation of human mesenchymal stem cells. Additionally, the gene expression analysis of differentiated stem cells with the ultrashort peptide bioinks revealed articular cartilage extracellular matrix formation preference. Based on the different mechanical stiffness of the two ultrashort peptide bioinks, they can be used to fabricate cartilage tissue with different cartilaginous zones, including the articular and calcified cartilage zones, which are essential for engineered tissue integration.

Review of a series of clinical observations of the use of "Cell culture for substitution therapy"

Introduction. The necessity to develop new substitutional medical technologies is associated with a high demand for rehabilitative measures of traumatological, surgical, and dental patients and persons with burn disease. The existing clinical practice of using allogeneic and autogenous cells, previously used for these purposes, has not yet been standardized. The aim of the study was to summarize scientific literature data on the clinical effectiveness of cell-based medical biotechnologies for regenerative medicine. Materials and methods. The authors reviewed 29 published scientific sources on the results of clinical trials of the preparation "Cell culture for substitution therapy" in 12 medical institutions of the Ural and Siberian regions of the Russian Federation during the period from 1998 to 2012. Results and discussion. Developed and effectively tested on the basis of medical institutions of Sverdlovsk, Chelyabinsk, Tyumen, Kemerovo regions, the Republic of Sakha (Yakutia) and Khanty-Mansi Autonomous Okrug immunobiological preparation "Cell culture for replacement therapy" had high clinical effectiveness in pathological conditions associated with tissue damage in patients with burns, surgery, trauma and dental profile. The studies demonstrated the key activity of allogeneic fibroblasts in the lesion nidus while reducing the time of extracellular matrix formation during active tissue regeneration. The use of the drug prevented the development of generalized infectious-inflammatory complications, reduced the risk of secondary infections, reduced the period of hospitalization and rehabilitation of patients. Conclusion. Analysis of the literature data on the high efficiency of immunobiological agents for substitution therapy on the example of using the preparation on the basis of allogeneic human fibroblasts, based on modern medical standardization and ethical norms, sets the actual task of their replacement by xenogenic and autologous preparations, which will increase the efficiency and safety of the presented method of treatment, improve the quality, life expectancy and demographic indicators in Russia.