CRYAB suppresses ferroptosis and promotes osteogenic differentiation of human bone marrow stem cells via binding and stabilizing FTH1.

BACKGROUND: Bone formation and homeostasis are greatly dependent on the osteogenic differentiation of human bone marrow stem cells (BMSCs). Therefore, revealing the mechanisms underlying osteogenic differentiation of BMSCs will provide new candidate therapeutic targets for osteoporosis. METHODS: The osteogenic differentiation of BMSCs was measured by analyzing ALP activity and expression levels of osteogenic markers. Cellular Fe and ROS levels and cell viability were applied to evaluate the ferroptosis of BMSCs. qRT-PCR, Western blotting, and co-immunoprecipitation assays were harnessed to study the molecular mechanism. RESULTS: The mRNA level of CRYAB was decreased in the plasma of osteoporosis patients. Overexpression of CRYAB increased the expression of osteogenic markers including OCN, OPN, RUNX2, and COLI, and also augmented the ALP activity in BMSCs, on the contrary, knockdown of CRYAB had opposite effects. IP-MS technology identified CRYAB-interacted proteins and further found that CRYAB interacted with ferritin heavy chain 1 (FTH1) and maintained the stability of FTH1 via the proteasome mechanism. Mechanically, we unraveled that CRYAB regulated FTH1 protein stability in a lactylation-dependent manner. Knockdown of FTH1 suppressed the osteogenic differentiation of BMSCs, and increased the cellular Fe and ROS levels, and eventually promoted ferroptosis. Rescue experiments revealed that CRYAB suppressed ferroptosis and promoted osteogenic differentiation of BMSCs via regulating FTH1. The mRNA level of FTH1 was decreased in the plasma of osteoporosis patients. CONCLUSIONS: Downregulation of CRYAB boosted FTH1 degradation and increased cellular Fe and ROS levels, and finally improved the ferroptosis and lessened the osteogenic differentiation of BMSCs.

Recent Advances in Photodynamic Therapy for Vascular Abnormalities.

Abstract Background: Photodynamic therapy (PDT) is a minimally invasive therapy that was gradually established as a first-line treatment for vascular abnormalities. Its action depends on the appropriate wavelength of light and photosensitizer to produce toxic oxygen species and cause cell death. Objective: Several new clinical improvements and trends in PDT have been described in recent years. The aim of this review is to provide an overview of the current data from clinical trials. Methods: In this review, we introduce and generalize the wavelength, duration, dose, strength, and photosensitizer of PDT for the treatment of vascular abnormalities, such as circumscribed choroidal hemangiomas (CCH), choroidal neovascularization (CNV) and capillary malformation (CM). Results: The systematic review findings indicate that the application of PDT is a safe effective method to treat CCH, CNV and CM. However, PDT also has early onset side effects and late onset side effects. Conclusions: Based on the discussion of the effectiveness of PDT, we conclude that PDT has great potential for clinical use, although PDT has possible side effects.

LncRNA MEG3 suppresses erastin-induced ferroptosis of chondrocytes via regulating miR-885-5p/SLC7A11 axis.

BACKGROUND: Ferroptosis is involved in osteoarthritis development; however, the roles of long noncoding RNAs (lncRNAs), including lncRNA MEG3, in the regulation of ferroptosis in osteoarthritis are still unclear. METHODS: In this study, qRT‒PCR and Western blotting assays were used to detect the expression of lncRNA MEG3, miR-885-5p, SLC7A11 and GPX4; MDA and CCK-8 assays were applied to analyse cellular MDA levels and cell viability, respectively. RESULT: Erastin elevated cellular MDA levels and decreased the viability of chondrocytes and the erastin-induced decline in cell viability was reversed by a ferroptosis inhibitor (ferrostatin-1). Erastin downregulated lncRNA MEG3, SLC7A11 and GPX4 and upregulated miR-885-5p. Silencing of lncRNA MEG3 increased miR-885-5p and downregulated SLC7A11 and GPX4 and further sensitized chondrocytes to erastin-induced ferroptosis. In contrast, overexpression of lncRNA MEG3 had opposite effects. Dual luciferase assays confirmed binding between lncRNA MEG3 and miR-885-5p and between miR-885-5p and the 3'UTR of SLC7A11. In the synovial fluids from patients with osteoarthritis compared with synovial fluids from normal controls, the RNA levels of lncRNA MEG3 and SLC7A11 were decreased and the miR-885-5p expression level was increased. CONCLUSION: Our findings indicated that lncRNA MEG3 overexpression alleviated ferroptosis in chondrocytes by affecting the miR-885-5p/SLC7A11 signalling pathway.

Photobiomodulation (800 nm Light-Emitting Diode) Treatment Promotes Bone Mesenchymal Stem Cell Proliferation Via Long Noncoding RNA MEG3-MicroRNA-217-5P Pathway.

Background: Patients with osteoporosis (OP) have a high risk of bone fracture. Abnormal bone mesenchymal stem cell (BMSC) differentiation is an essential process of OP development. In recent years, photobiomodulation has been shown to effectively promote BMSC proliferation. However, the mechanism by which photobiomodulation promotes BMSC proliferation is unclear. Long noncoding RNAs (lncRNAs) are essential mediators in multiple biological processes. The lncRNA maternally expressed gene 3 (MEG3) is a novel lncRNA gene and is related to cell proliferation. Studies have indicated that MEG3 serves as a promotor in BMSC proliferation. Objective: To investigate the effects and mechanisms of 800 nm light-emitting diode (LED) photobiomodulation in BMSC proliferation. Materials and methods: The BMSCs collected from mouse tibias and femurs were irradiated by 800 nm LED for 180 sec. CCK-8 assay was used to detect the cell viability. A dual-luciferase reporter assay was used to determine IncRNA MEG3 acted as a miR-217-5p sponge. We used reverse transcription-polymerase chain reaction (RT-PCR) and western blot to detect the mRNA and protein levels of MEG3, miR-217-5p, Notch2, Hes1, Hey2. Results: In the present study, we revealed that photobiomodulation (800 nm LED) could increase the mRNA level of MEG3, and protein levels of Notch2, Hes1, and Hey2. Moreover, we also identified that upregulated MEG3 could act as a miR-217-5p sponge to activate the Notch signaling pathway. Conclusions: The current study revealed the MEG3-related mechanism of photobiomodulation treatment in OP and identified potential gene therapies for OP.

LLLI promotes BMSC proliferation through circRNA_0001052/miR-124-3p.

Osteoporosis (OP) is a multifactorial bone disease that occurs worldwide. The treatment of OP is still unsatisfactory. Bone mesenchymal stem cell (BMSC) differentiation is a key process in OP pathogenesis. Low-level laser irradiation (LLLI) has been reported to regulate BMSC proliferation, but the role of circRNAs in the LLLI-based promotion of BMSC proliferation remains unclear. CircRNAs are essential molecular regulators that participate in numerous biological processes and have therapeutic potential. miR-124-3p is an essential microRNA (miRNA), and its expression changes are related to BMSC proliferation ability. In the present study, gain-loss function of experiments demonstrated that circRNA_0001052 could regulate the proliferation of BMSCs by acting as a miR-124-3p sponge through the Wnt4/ß-catenin pathway. The results of this study strongly suggest that circRNA_0001052 plays an essential role in BMSC proliferation in response to LLLI treatment, which is a potential therapeutic manipulation with clinical applications.

The role of TRPV channels in osteoporosis.

As the world's population ages, the treatment of osteoporosis is a major problem to be addressed. The cause of osteoporosis remains unclear. Ca2+ is not only an important component of bones but also plays a key role in osteoporosis treatment. Transient receptor potential vanilloid (TRPV) channels are one of the TRP channel families that is widely distributed in various organs, playing an important role in the physiological regulation of the human body. Bone formation and bone absorption may require Ca2+ transport via TRPV channels. It has been proven that the TRPV subtypes 1, 2, 4, 5, 6 (TRPV1, TRPV2, TRPV4, TRPV5, TRPV6) may affect bone metabolism balance through selective regulation of Ca2+. They significantly regulate osteoblast/osteoclast proliferation, differentiation and function. The purpose of this review is to explore the mechanisms of TRPV channels involved in regulation of the differentiation of osteoblasts and osteoclasts, as well as to discuss the latest developments in current researches, which may provide new clues and directions for an in-depth study of osteoporosis and other related bone metabolic diseases.

Silencing long non-coding RNA MEG8 inhibits the proliferation and induces the ferroptosis of hemangioma endothelial cells by regulating miR-497-5p/NOTCH2 axis.

Even though long non-coding RNA (lncRNA) MEG8 plays vital roles in carcinogenesis of malignances, its roles and mechanisms in hemangioma remain unknown. Therefore, we evaluate the oncogenic roles of MEG8 in hemangioma. Small interfering RNA (siRNA)-mediated depletion of MEG8 inhibited the proliferation and increased MDA level in human hemangioma endothelial cells (HemECs). The inhibitors of ferroptosis (ferrostatin-1 and liproxstatin-1) abolished the MEG8 silence induced cell viability loss. Knockdown of MEG8 increased the miR-497-5p expression and reduced the mRNA and protein levels of NOTCH2. Using a dual-luciferase assay, we confirmed the binding between MEG8 and miR-497-5p, and between the miR-497-5p and 3'UTR of NOTCH2. We further found that silencing MEG8 significantly decreased the expressions of SLC7A11 and GPX4 both in mRNA and protein level and had no effect on the level of AIFM2. Importantly, blocking miR-497-5p abrogated the effects of MEG8 loss on cell viability, MDA level and expression levels of NOTCH2, SLC7A11 and GPX4 in HemECs. Taken together, our results suggested that knockdown of long non-coding RNA MEG8 inhibited the proliferation and induced the ferroptosis of hemangioma endothelial cells by regulating miR-497-5p/NOTCH2 axis.

Mesenchymal stem cells in osteoarthritis therapy: a review.

Osteoarthritis (OA) is a chronic joint disease that generally occurs worldwide with pain and disability. The progression is slow, and it is mostly diagnosed midlife and often disturbs the knees, hips, feet, hands, and spine. Sex, age, obesity, occupation, and hereditary factors are risk factors that increase the opportunity for OA. Physical examinations involving X-rays and MRI, joint fluid analysis and blood tests are common tools for the diagnosis of OA. Interventions including exercise, manual therapy, lifestyle modification, and medication can help relieve pain and maintain mobility in the affected joints, yet none of the therapies enables the promotion of regeneration of degenerated tissues. Mesenchymal stem cells (MSCs) are a promising source for the treatment of OA due to their multipotency for differentiation into chondrocytes and their ability to modulate the immune system. Herein, we review the pathogenesis and treatment of OA and address the current status of MSCs as a novel potential therapeutic agent in OA treatment.

The role of human immortal skin keratinocytes-acellular dermal matrix scaffold in skin repair and regeneration.

In this study, we aimed to investigate the phenotypic characteristics of human immortal skin keratinocytes (HaCaT) cells and the role of acellular dermal matrix (ADM) in coculture system of HaCaT cells and ADM. Flow cytometry was used to examine the cluster of differentiation (CD) makers of HaCaT cells. Apoptosis analysis was applied to detect the apoptosis rate of HaCaT cells. Morphological observation of ADM isolated from the reticular layer of Sprague-Dawley rat dermis was utilized to evaluate the morphological structure of ADM. Methylthiazolyl tetrazolium (MTT) assay and morphological experiments were further used to confirm the scaffold role of ADM in HaCaT cells. A wound-healing mice model accompanied by HaCaT-ADM scaffold transplantation was performed to further verify the function of HaCaT-ADM scaffold. Our results showed that CD71, CD49f, K19, and CD29 were highly expressed in HaCaT cells, and the percentage of apoptosis cells was significantly increased, which represented that HaCaT cells had much stronger capacities of adhesion and proliferation than normal human keratinocytes. Additionally, the morphological structure of ADM presented many natural microbores, which made cells rapidly grow on ADM. The results exhibited that the HaCaT cells indeed promptly proliferate on ADM and easily grow into the microbores of ADM. Finally, an in vivo experiment further confirmed that the transplantation of the HaCaT-ADM scaffold into the dorsal skin of a wound-healing mice model could gradually repair the injured wound. Thus, these findings indicated that HaCaT cells might be as seed cells to develop skin tissue engineering and the HaCaT-ADM scaffold might be a better candidate to promote skin repair and regeneration.

Efficacy of combined vancomycin and fosfomycin against methicillin-resistant Staphylococcus aureus in biofilms in vivo.

Infection by methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. The aim of this study was to demonstrate the in vivo bactericidal effects of a combination of vancomycin (VAN) and fosfomycin (FOS) against MRSA in a rat carboxymethyl cellulose-pouch biofilm model. The results of the time-kill assay showed that the combination therapy was capable of killing at low minimal inhibitory concentrations (MIC) (½ × MIC VAN +1 × MIC FOS and 1 × MIC VAN + 1 × MIC FOS). In the in vivo study, a synergistically bactericidal effect was observed when using the combination therapy on MRSA embedded in the mature biofilm model. In comparison with the untreated control group and the groups receiving either VAN or FOS alone, the rats treated with combination therapy had lower MRSA colony counts in exudates from the pouch, lower white blood cell and neutrophil counts, and C-reactive protein (CRP) in peripheral blood. Furthermore, histological analysis of the pouch wall indicated combination therapy resulted in disappearance of biofilm-like structures, marked decrease in necrosis, and formation of granular tissue. In conclusion, the combination of VAN with FOS had a synergistic bactericidal effect on chronic MRSA infection embedded in biofilm, providing an alternative approach to treating this condition.

Percutaneous kyphoplasty combined with the posterior screw-rod system in treatment of osteoporotic thoracolumbar fractures.

BACKGROUND: The osteoporotic vertebral compression fractures (OVCF) have attracted more and more attention due to increase in life span globally and aging population. Percutaneous vertebroplasty (PVP) and percutaneous kyphoplasty (PKP) have been popularized rapidly by virtue of their unique advantage in minimal invasiveness. We analysed our results in osteoporotic thoracolumbar fractures using percutaneous kyphoplasty and posterior screw rod system. To investigate the possibility of treatment of rupture of the posterior vertebral osteoporotic fractures by means of kyphoplasty combined with the posterior screw-rod system. MATERIALS AND METHODS: Twenty six patients (65 years of age or older) with the single spine fractures included in study. The preoperative bone mineral density was measured by dual-energy X-ray. The PKP was done in all the cases. Decompression was done if neurological symptoms were present. RESULTS: The results demonstrated osteoporosis with BMD T value ≤ -2.5; injured posterior vertebral body (3 cases) had shown the whole damage accompanied by neurological symptoms through X-ray or CT. After 2 days, the remaining patients of back pain symptoms were relieved or disappeared except for three cases of patients with decompression incision. VAS score and Cobb angle changed from preoperative 8.23 ± 0.17 and 28.7 ± 0.33° respectively to postoperative 3.77 ± 0.44 and 3.8 ± 0.2° respectively. CONCLUSION: Treatment of rupture of the posterior vertebral osteoporotic thoracolumbar fractures by means of kyphoplasty combined with posterior screw-rod system is a safe, effective procedure.

Correlation between Innervation of Skeletal Muscles and Myoblast Stem Cells / Correlación entre la Inervación de los Músculos Esqueléticos y las Células Precursoras Mioblásticas

In order to explore the change rule of myoblast stem cells (satellite cells, SCs) in the denervated and re-innervated muscle and to investigate the cellular mechanism of the morphological and functional changes of the muscle, denervated muscle atrophy and nerve regeneration models were established in one-month-old rats. Postoperative indexes such as muscle wet weight, cell section areas, content of collagen fibers and DNA, electrophysiology, numbers of SCs in the triceps muscle of calf were dynamically tested. After denervation, the muscle wet weight and cell area reduced rapidly, and the collagen fiber content increased slowly. The number of SCs increased at first, and then declined suddenly two months later. From 4 to 5 weeks after re-neuralization, muscle action potentials could be evoked, but the best innervation effect was found in the groups, which received re-neuralization at 2 months and 3 months after denervation. Denervation causes a progressive progress of muscle atrophy. SCs proliferate within 3 months after denervation, and then atrophy becomes irreversible from 4 months. At 4 or 5 weeks after re-neuralization, muscle action potentials can be evoked. Re-neuralization at 2 months and 3 months after denervation can achieve a good effect on the functional recovery of the atrophic muscle.

Con el fin de explorar la regla de cambio de las células precursoras mioblásticas (células satélite, CSs) en el músculo denervado y re-inervado e investigar el mecanismo celular de los cambios morfológicos y funcionales del músculo, se establecieron, en ratas de un mes de edad, modelos de atrofia del músculo denervado y regeneración del nervio. Fueron examinados de manera dinámica índices postoperatorios tales como, el peso húmedo del músculo, áreas celulares de la sección, contenido de fibras de colágeno y ADN, electrofisiología, número de CSs en el músculo tríceps de las crías. Luego de la denervación, el peso del músculo húmedo y el área celular se redujeron rápidamente, mientras que el contenido de fibras colágenas aumentó lentamente. El número de CSs aumentó al inicio, pero más tarde, a los dos meses, disminuyó repentinamente. Entre las 4 a 5 semanas después de la reneuralización, los potenciales de acción muscular pudieron ser evocados, pero el mejor efecto de inervación se encontró en los grupos que recibieron reneuralización a los 2 y 3 meses después de la denervación. La denervación causó un avance progresivo de la atrofia muscular. Las CSs proliferaron dentro de los primeros 3 meses post-denervación, y luego de los 4 meses la atrofia fue irreversible. A las 4 o 5 semanas después de la reneuralizacón, los potenciales de acción muscular pueden ser evocados. La reneuralización a los 2 y 3 meses después de la denervación puede lograr un buen efecto en la recuperación funcional del músculo atrófico.

Liposome combined porous beta-TCP scaffold: preparation, characterization, and anti-biofilm activity.

The objective of this study was to design a novel artificial bone scaffold for therapy and prevention of refractory bacterial infection. Porous beta-tricalcium phosphate (beta-TCP) scaffold was combined with liposomal gentamicin (GS) to form a novel complex drug carrier. The liposome combined beta-TCP scaffold (LCS) was characterized for its liposome binding rate, drug loading, and micromorphology. The anti-biofilm activity of LCS was evaluated by Staphylococcus aureus biofilm in vitro. The drug release from LCS was recognized as an initial high dose of liposomal GS released from the matrix and a further sustained release of free GS from the liposome, respectively, and it is an ideal release pattern for treatment and prevention of post-operative osteomyelitis. The release kinetics was influenced by variation of particle size of liposome. LCS displayed a potential anti-biofilm activity even in the lowest GS concentration (2.5 microg/mL), and the regrowth time was extended from 5.0 h to 9.5 h. At a higher dosage range, the highest anti-biofilm activity was achieved by LCS with liposomal particle size of 800 nm. In conclusion, the development of LCS showed a new pathway for controlled delivery of liposomal antibiotics for treatment of osteomyelitis caused by persistent bacterial infection.

[Morphological observation of human keratinocytes tridimensionally cocultured with xenogenic acellular dermal matrix].

OBJECTIVE: To establish the tridimensional culture method for tissue-engineered skin to observe the histomorphological change in human immortal KC strain (HacaT)cocultured with xenogenic acellular dermal matrix (ADM). METHODS: The ADM was prepared from SD rats by a modified method. HaCaTs were cultured in defined KC-serum free medium. HaCaTs in log growth phase were inoculated on ADM at the cell density of 2 x 10(5)/cm(2). They were submergedly cultured for 5 days and then changed to air-liquid phase culture for another 5 days. ADM and growth of HaCaTs on day 1 and 5 after cocultured with ADM were observed with scanning electron microscope. The histological change in ADM and HaCaTs on day 1, 5, and 10 after cocultured with ADM were examined by HE staining. RESULTS: The gross appearance of ADM was white with smooth and soft texture, and intact collagen bundles without cellular residue. HaCaTs adhered and stretched out pseudopodia on the surface of the ADM on day 1 after combined culture, and a monolayer of cells was formed on day 5, growing into 3-6 layers of cells on day 10 with a tendency to grow into ADM. CONCLUSIONS: SD rats ADM is benefit for the adhesion of HaCaTs and the permeation of nutrient solution, from which an engineered multiple-layered human skin can be obtained within 10 days.