Efficacy of mesenchymal stem cell-based therapy on the bone repair of hypertensive rats.

OBJECTIVE: Hypertension disrupts the bone integrity and its repair ability. This study explores the efficiency of a therapy based on the application of mesenchymal stem cells (MSCs) to repair bone defects of spontaneously hypertensive rats (SHR). METHODS: First, we evaluated SHR in terms of bone morphometry and differentiation of MSCs into osteoblasts. Then, the effects of the interactions between MSCs from normotensive rats (NTR-MSCs) cocultured with SHR (SHR-MSCs) on the osteoblast differentiation of both cell populations were evaluated. Also, bone formation into calvarial defects of SHR treated with NTR-MSCs was analyzed. RESULTS: Hypertension induced bone loss evidenced by reduced bone morphometric parameters of femurs of SHR compared with NTR as well as decreased osteoblast differentiation of SHR-MSCs compared with NTR-MSCs. NTR-MSCs partially restored the capacity of SHR-MSCs to differentiate into osteoblasts, while SHR-MSCs exhibited a slight negative effect on NTR-MSCs. An enhanced bone repair was observed in defects treated with NTR-MSCs compared with control, stressing this cell therapy efficacy even in bones damaged by hypertension. CONCLUSION: The use of MSCs derived from a heathy environment can be in the near future a smart approach to treat bone loss in the context of regenerative dentistry for oral rehabilitation of hypertensive patients.

Mesenchymal stem cell-based therapy for osteoporotic bones: Effects of the interaction between cells from healthy and osteoporotic rats on osteoblast differentiation and bone repair.

AIMS: Cell therapy utilizing mesenchymal stem cells (MSCs) from healthy donors (HE-MSCs) is a promising strategy for treating osteoporotic bone defects. This study investigated the effects of interaction between HE-MSCs and MSCs from osteoporotic donors (ORX-MSCs) on osteoblast differentiation of MSCs and of HE-MSCs on bone formation in calvarial defects of osteoporotic rats. MATERIALS AND METHODS: Osteoporosis was induced by orchiectomy (ORX) and its effects on the bone were evaluated by femur microtomography (µCT) and osteoblast differentiation of bone marrow MSCs. HE- and ORX-MSCs were cocultured, and osteoblast differentiation was evaluated using genotypic and phenotypic parameters. HE-MSCs were injected into the calvarial defects of osteoporotic rats, and bone formation was evaluated by µCT, histology, and gene expression of osteoblast markers. KEY FINDINGS: ORX-induced osteoporosis was revealed by reduced bone morphometric parameters and osteoblast differentiation in ORX-MSCs. HE-MSCs partially recovered the osteogenic potential of ORX-MSCs, whereas HE-MSCs were mildly affected by ORX-MSCs. Additionally, the bone morphogenetic protein and wingless-related integration site signaling pathway components were similarly modulated in cocultures involving ORX-MSCs. HE-MSCs induced meaningful bone formation, highlighting the effectiveness of cell therapy even in osteoporotic bones. SIGNIFICANCE: These results provide new perspectives on the development of cell-based therapies to regenerate bone defects in patients with disorders that affect bone tissue.

Mesenchymal Stem Cells Combined with a P(VDF-TrFE)/BaTiO3 Scaffold and Photobiomodulation Therapy Enhance Bone Repair in Rat Calvarial Defects.

BACKGROUND: Tissue engineering and cell therapy have been the focus of investigations on how to treat challenging bone defects. This study aimed to produce and characterize a P(VDF-TrFE)/BaTiO3 scaffold and evaluate the effect of mesenchymal stem cells (MSCs) combined with this scaffold and photobiomodulation (PBM) on bone repair. METHODS AND RESULTS: P(VDF-TrFE)/BaTiO3 was synthesized using an electrospinning technique and presented physical and chemical properties suitable for bone tissue engineering. This scaffold was implanted in rat calvarial defects (unilateral, 5 mm in diameter) and, 2 weeks post-implantation, MSCs were locally injected into these defects (n = 12/group). Photobiomodulation was then applied immediately, and again 48 and 96 h post-injection. The µCT and histological analyses showed an increment in bone formation, which exhibited a positive correlation with the treatments combined with the scaffold, with MSCs and PBM inducing more bone repair, followed by the scaffold combined with PBM, the scaffold combined with MSCs, and finally the scaffold alone (ANOVA, p ≤ 0.05). CONCLUSIONS: The P(VDF-TrFE)/BaTiO3 scaffold acted synergistically with MSCs and PBM to induce bone repair in rat calvarial defects. These findings emphasize the need to combine a range of techniques to regenerate large bone defects and provide avenues for further investigations on innovative tissue engineering approaches.

Normoglycemia partially recovers the disrupted osteoblast differentiation of mesenchymal stem cells induced by type 1 but not type 2 diabetes mellitus.

Type 1 (T1DM) and type 2 (T2DM) diabetes mellitus are characterized by changes in glucose metabolism and cause bone damage via a variety of mechanisms, including effects on osteoblasts. We aimed to evaluate the osteoblast differentiation of mesenchymal stem cells (MSCs) from rats with T1DM or T2DM and the effects of removing the hyperglycemic stimulus on the osteogenic potential of these cells. MSCs from healthy rats were cultured in normoglycemic medium, whereas MSCs from rats with T1DM or T2DM were cultured in hyperglycemic or normoglycemic medium. T1DM and T2DM reduced osteoblast differentiation of MSCs grown in hyperglycemic media, with T1DM having a more pronounced effect, as evidenced by alkaline phosphatase activity, RUNX2 protein expression, and extracellular matrix mineralization, and modulated the gene expression of several components of the bone morphogenetic protein signaling pathway. The restoration of the normoglycemic environment partially recovers the osteogenic potential of MSCs from rats with T1DM but not with T2DM. Our findings highlight the need for specific therapies to treat T1DM- or T2DM-induced bone loss, as both disrupt osteoblast differentiation at distinct levels and likely through different mechanisms.

Collagen/κ-Carrageenan-Based Scaffolds as Biomimetic Constructs for In Vitro Bone Mineralization Studies.

Tissue engineering offers attractive strategies to develop three-dimensional scaffolds mimicking the complex hierarchical structure of the native bone. The bone is formed by cells incorporated in a molecularly organized extracellular matrix made of an inorganic phase, called biological apatite, and an organic phase mainly made of collagen and noncollagenous macromolecules. Although many strategies have been developed to replicate the complexity of bone at the nanoscale in vitro, a critical challenge has been to control the orchestrated process of mineralization promoted by bone cells in vivo and replicate the anatomical and biological properties of native bone. In this study, we used type I collagen to fabricate mineralized scaffolds mimicking the microenvironment of the native bone. The sulfated polysaccharide κ-carrageenan was added to the scaffolds to fulfill the role of noncollagenous macromolecules in the organization and mineralization of the bone matrix and cell adhesion. Scanning electron microscopy images of the surface of the collagen/κ-carrageenan scaffolds showed the presence of a dense and uniform network of intertwined fibrils, while images of the scaffolds' lateral sides showed the presence of collagen fibrils with a parallel alignment, which is characteristic of dense connective tissues. MC3T3-E1 osteoblasts were cultured in the collagen scaffolds and were viable after up to 7 days of culture, both in the absence and in the presence of κ-carrageenan. The presence of κ-carrageenan in the collagen scaffolds stimulated the maturation of the cells to a mineralizing phenotype, as suggested by the increased expression of key genes related to bone mineralization, including alkaline phosphatase (Alp), bone sialoprotein (Bsp), osteocalcin (Oc), and osteopontin (Opn), as well as the ability to mineralize the extracellular matrix after 14 and 21 days of culture. Taken together, the results described in this study shed light on the potential use of collagen/κ-carrageenan scaffolds to study the role of the structural organization of bone-mimetic synthetic matrices in cell function.

Effects of Modulation of the Hedgehog and Notch Signaling Pathways on Osteoblast Differentiation Induced by Titanium with Nanotopography.

BACKGROUND: The events of bone formation and osteoblast/titanium (Ti) interactions may be affected by Hedgehog and Notch signalling pathways. Herein, we investigated the effects of modulation of these signalling pathways on osteoblast differentiation caused by the nanostructured Ti (Ti-Nano) generated by H2SO4/H2O2. METHODS: Osteoblasts from newborn rat calvariae were cultured on Ti-Control and Ti-Nano in the presence of the Hedgehog agonist purmorphamine or antagonist cyclopamine and of the Notch antagonist N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) or agonist bexarotene. Osteoblast differentiation was evaluated by alkaline phosphatase activity and mineralization, and the expression of Hedgehog and Notch receptors was also evaluated. RESULTS: In general, purmorphamine and DAPT increased while cyclopamine and bexarotene decreased osteoblast differentiation and regulated the receptor expression on both Ti surfaces, with more prominent effects on Ti-Nano. The purmorphamine and DAPT combination exhibited synergistic effects on osteoblast differentiation that was more intense on Ti-Nano. CONCLUSION: Our results indicated that the Hedgehog and Notch signalling pathways drive osteoblast/Ti interactions more intensely on nanotopography. We also demonstrated that combining Hedgehog activation with Notch inhibition exhibits synergistic effects on osteoblast differentiation, especially on Ti-Nano. The uncovering of these cellular mechanisms contributes to create strategies to control the process of osseointegration based on the development of nanostructured surfaces.

Atypical traumatic bone cyst involving impacted lower third molar: report of case / Cisto ósseo traumático atípico envolvendo terceiro molar inferior impactado: relato de caso

ABSTRACT The traumatic bone cyst is an uncommon nonneoplastic lesion of the jaws that is considered as a "pseudocyst" because of the lack of an epithelial lining. This lesion is particularly asymptomatic and, therefore, is diagnosed by routine dental radiographic examination as a unilocular radiolucency with scalloped borders, mainly in the posterior mandibular region. The exact etiopathogenesis of the lesion remains uncertain, though it is often associated with trauma. The objective of this paper is to report one case of atypical traumatic bone cyst involving impacted lower third molar, addressing its clinical and radiographic characteristics, differential diagnosis, treatment through surgical exploration and case follow-up.

RESUMO O cisto ósseo traumático é uma lesão não neoplásica incomum dos maxilares, considerada um "pseudocisto" devido à ausência de um revestimento epitelial. Esta lesão é particularmente assintomática e, portanto, é diagnosticada pelo exame radiográfico odontológico de rotina como uma radioluscência unilocular com bordas recortadas, principalmente na região mandibular posterior. A etiopatogenia exata da lesão permanece incerta, embora esteja frequentemente associada a trauma. O objetivo deste trabalho é relatar um caso de cisto ósseo traumático atípico envolvendo terceiro molar inferior impactado abordando suas características clínicas, radiográficas, diagnóstico diferencial, tratamento por meio de exploração cirúrgica e proservação do caso.

Preparação para o parto: as capacidades como superpoder que a mulher leva para o seu trabalho de parto / Preparation for childbirth: The capacities as a superpower that a woman brings to her labor

O presente documento trata-se de um relatório de estágio de natureza profissional desenvolvido no âmbito do Mestrado em Enfermagem de Saúde Materna e Obstétrica, no período compreendido entre 17 de novembro de 2020 e 30 de junho de 2021, num hospital do grande Porto. Este reflete a aquisição e o desenvolvimento de competências enquanto Enfermeira Especialista em Enfermagem de Saúde Materna e Obstétrica e consiste na minha reflexão relativamente às experiências vividas nos diferentes âmbitos de atuação: gravidez, trabalho de parto, parto e puerpério. Este processo de desenvolvimento de competências tem por base o Regulamento de Competências Específicas do Enfermeiro Especialista em Enfermagem de Saúde Materna e Obstétrica definidos pela Ordem dos Enfermeiros. Um dos objetivos do presente relatório prendeu-se na realização de uma Revisão Integrativa da Literatura, sendo que me suscitou interesse a procura de evidência científica relativamente às possíveis capacidades que a mulher poderia adquirir na preparação para o parto e que seriam facilitadoras, como um superpoder, do processo de trabalho de parto, ou seja, de que forma é que o Enfermeiro Especialista em Enfermagem de Saúde Materna e Obstétrica poderia contribuir, ainda durante a gravidez, para o empoderamento da mulher enquanto parturiente. Através da pesquisa de evidência sobre o tema concluí que o superpoder que a mulher pode levar para o seu trabalho de parto é a perceção da autoeficácia. Existem fatores, internos e externos à grávida, que predizem maiores níveis de perceção da autoeficácia, nesse sentido, e uma vez que maiores níveis de autoeficácia se traduzem numa maior satisfação durante o trabalho de parto e parto, compete ao(à) EEESMO trabalhar com a grávida a sua perceção de autoeficácia durante a gravidez, empoderando-a. Esta pesquisa permitiu-me também perceber que não existe ainda muita evidência sobre o tema, principalmente em Portugal, e seria interessante investir-se na realização de mais estudos.

This document is a professional internship report developed within the scope of the Master's Degree in Maternal and Obstetric Health Nursing, between November 17, 2020 and June 30, 2021, at an hospital in Porto. It reflects the acquisition and development of skills as a Midwife and consists on my reflection on the experiences lived in the different areas of action: pregnancy, labor, childbirth and puerperium. This process of skills development is based on the Regulation of Specific Skills for Specialist Nurses in Maternal and Obstetric Health Nursing defined by the Ordem dos Enfermeiros. One of the objectives of this report was to carry out an Integrative Literature Review, and I was interested in the search for scientific evidence regarding the possible skills that women could acquire in preparation for childbirth and that would be facilitators, like a superpower, of the labor process, that is, how Midwives could contribute, even during pregnancy, to the empowerment of women as parturients. Through researching evidence on the subject, I concluded that the superpower that a woman can bring to her labor is the perception of self-efficacy. There are factors, internal and external to women, that predict higher levels of perception of self-efficacy, in that regard, and since higher levels of self-efficacy translate into greater satisfaction during labor and delivery, it is up to the Midwives to work with women their perception of self-efficacy during pregnancy, empowering them. This research also allowed me to realize that there is still not much evidence on the subject, especially in Portugal, and it would be interesting to invest in further studies.

The integrated work between medicine and psychology with infertile couples at a public hospital in Rio de Janeiro/Brazil.

This study examines the coping process of infertile couple during diagnostic investigation and treatment, which is held in a public hospital in the Assisted Reproduction Treatment department. This analysis discusses the results of a qualitative research whose goal is to emphasize the importance of integrating the medical and psychological care to infertile couples. To collect the data, it uses a semistructured psychological interview. This feature promotes a differentiated listening emotional aspects experienced by patients, allowing couples to greater reflection about their difficulties before the process of investigation and treatment of infertility. From the data, it appears that in addition to medical treatment, patients require in a psychological intervention centered emotional effects experienced by them throughout the process. Given the complexity of the process of Assisted Reproduction, it is essential to consider both the medical factors, essential for achieving the concept of biological child, as psychological factors, at the individual, marital and social. We conclude that the psychological dimension integrated medical help these patients to maximize their resources and seek more adaptive ways to minimize the possible dilemmas and anxiety experienced.

Indigofera tinctoria L. leaf powder promotes initiation of indigo reduction by inducing of rapid transition of the microbial community.

Water-insoluble indigo is solubilized by the reducing action of microorganisms which occurs during fermentation. In natural indigo fermentation, composted leaves of Polygonum tinctorium L. (sukumo) are the raw material that has been used as both the indigo source and the bacterial inoculum. Ideally, indigo reduction occurs shortly after preparation of the fermentation vat. The time-to-reduction depends on the quality of the sukumo and the methods for preparation and management of the fermentation batch. We estimated the effect of adding Indigofera tinctoria L. leaf powder (LP) to indigo fermentation in two fermentations originally exhibiting either rapid or slow time-to-reduction (T-sukumo and D-sukumo, respectively). Alkalihalobacillus spp. (97.7%-98.4% similarities with Alkalihalobacillus macyae) were observed only in the LP-added T-sukumo fermentation liquor. They appeared from day 1 (0.7%) and increased to 24.4% on day 6, and their presence was related to indigo reduction. Differences in functional ratio between LP-added and its control batches revealed enhancement of pathways related to reconstitution of cellular functions and substrate metabolisms, to all of which Alkalihalobacillus spp. contributed intensively. In D-sukumo batch, appearance of bacteria necessary to initiate indigo reduction (principally Anaerobacillus/Polygonibacillus) was comparatively slower. LP promotes earlier indigo reduction in both T- and D-sukumo-based batches, owing to its promotion of microbiota transition. The effect of the LP was intensified from day 1 to day 2 in both sukumo using batches according to the assumed function of the microbiota. The initial effect of LP on the T-sukumo batches was more intense than that in the D-sukumo batches and was continued until day 3, while the duration in the T-sukumo batches was continued until day 5. Based on these observations, we propose that the LP functions through its phytochemicals that eliminate oxygen, stimulate the microbiota, and accelerate its transitional changes toward a suitable function that opens the pathway for the extracellular electron transfer using carbohydrates as a substrate.

Curcumin-loaded carrageenan nanoparticles: Fabrication, characterization, and assessment of the effects on osteoblasts mineralization.

The use of Curcumin (CR) as a bioactive molecule to prevent and treat inflammation- related diseases is widespread. However, the high hydrophobicity hinders the in vivo bioavailability of CR, reducing its therapeutic index. In the present study, we described the use of nanoparticles (NPs) made of kappa-carrageenan (κ-Carr), a sulphated polysaccharide, as cost-effective, biodegradable and biocompatible CR carriers. CR-loaded κ-Carr nanoparticles (CR@Carr NPs) were prepared by mixing a κ-Carr aqueous solution with a CR ethanolic solution. The final suspension was centrifuged and re-suspended in phosphate buffer solution. The NPs' size was tuned by changing the concentration of the polysaccharide. CR@CarrNPs displayed high CR incorporation efficiency (~80 wt%) and a double-exponential curve of CR release at physiological conditions (37 °C and pH 7.4) with a cumulative drug release of 32 wt% after 24 h for the smaller NP. Our results also showed that CR@CarrNPs were not cytotoxic to osteoblasts at concentrations up to 1 µM. Confocal microscopy images revealed the internalization of CR by the cells guided by the NPs. Cells treated with CR@CarrNPs exhibited higher activity of alkaline phosphatase and higher expression of the main osteogenic genes (Sp7, Col1 and Runx2), and mineralized the extracellular matrix in a higher extent compared to the cells cultivated in absence of the NPs. We posited that these effects were related to the NP-driven internalization of CR by osteoblasts. Our study sheds light on the possible use of CR@CarrNPs as efficient and safe therapeutic tools for the treatment of bone-related diseases.

A new modelling approach for predicting process evolution of cork-rubber composites slabs vulcanization.

In order to predict the evolution of the vulcanization process of cork-rubber slabs, a numerical approach was developed combining heat transfer by conduction and kinetics models. A one-dimensional model was applied to predict the evolution of temperature and degree of cure at different stages of the vulcanization of a cork-rubber composite. Also, due to the degradation verified by the compound, an existent reversion model was added to the problem. Based on rheometer data, cure and reversion parameters were determined. Experimental data were used to determine the thermal properties of the compound, assuming a constant value or according to its degree of cure and temperature. The results obtained by simulation showed a good correspondence with experimental results, even when assuming constant thermal properties. The application of the proposed methodology provides information about the optimum process parameters for each thickness slab, without compromising the homogeneity and characteristics of the final product, which can be a valuable tool during the development and product stages of cork-rubber composites.

Human periodontal ligament stem cells with distinct osteogenic potential induce bone formation in rat calvaria defects.

Aim: This study aimed to evaluate the ability of human periodontal ligament stem cells (PDLSCs) with high (HP-PDLSCs) and low (LP-PDLSCs) osteogenic potential, in addition to mixed cells, to repair bone tissue. Methods: Cell phenotype, proliferation and differentiation were evaluated. Undifferentiated PDLSCs were injected into rat calvarial defects and the new bone was evaluated by µCT, histology and real-time PCR. Results: PDLSCs exhibited a typical mesenchymal stem cell phenotype and HP-PDLSCs showed lower proliferative and higher osteogenic potential than LP-PDLSCs. PDLSCs induced similar bone formation and histological analysis suggests a remodeling process, confirmed by osteogenic and osteoclastogenic markers, especially in tissues derived from defects treated with HP-PDLSCs. Conclusion: PDLSCs induced similar bone formation irrespective of their in vitro osteogenic potential.

Bone is one of the most transplanted tissues worldwide and cell-based therapies has been investigated as an alternative for the treatment of bone defects. Dental tissues have been investigated as sources of stem cells and the periodontal ligament has been shown to be a viable source of these cells. Stem cells from periodontal ligament induce significant bone formation in rat calvaria defects and are safe for cell-based therapies, as the cells remain at the bone defect site for up to 4 weeks and do not migrate to vital organs, such as brain, heart, lungs, spleen, kidneys, and liver in the same period. In addition, immune responses were not detected. Considering that, stem cells from periodontal ligament can be useful in cell therapy strategies to induce bone regeneration.

Fundicoccus fermenti sp. nov., an indigo-reducing facultative anaerobic alkaliphile isolated from indigo fermentation liquor used for dyeing.

Two facultative anaerobic and facultative alkaliphilic indigo-reducing strains, designated F-1T and F-2, were isolated from indigo fermentation liquor produced from couched woad fermentation-based Indian indigo fermentation fluid. The 16S rRNA gene phylogeny showed that Fundicoccus ignavus WS4937T (99.5%) was the closest neighbour of F-1T. The isolated bacterial cells were Gram-stain-positive and facultative anaerobic coccoids. Strain F-1T grew at between 5 and 37 °C with optimum growth between 28‒32 °C. The isolate grew in a pH range of 7.0‒10.5, with optimum growth between pH 9.0‒10.5. The DNA G+C content was 37.6 mol% (HPLC). The whole-cell fatty acid profile mainly consisted (>10 %) of C16 : 0, C16 : 1 ω9c, C18 : 0 and C18 : 1 ω9c. The digital DNA-DNA hybridization value between strain F-1T and F. ignavus WS4937T was 52.9 %. Based on their physiological and biochemical characteristics, and phylogenetic and genomic data, the isolates can be discriminated from F. ignavus WS4937T. The name Fundicoccus fermenti sp. nov. is proposed. The type strain of this species is F-1T (JCM 34140T=NCIMB 15255T).

Indigofera tinctoria leaf powder as a promising additive to improve indigo fermentation prepared with sukumo (composted Polygonum tinctorium leaves).

Being insoluble in the oxidize form, indigo dye must be solubilized by reduction for it to penetrate textile. One of the procedures is the reduction by natural bacterial fermentation. Sukumo, composted leaves of Polygonum tinctorium, is a natural source of indigo in Japan. Although sukumo has an intrinsic bacterial seed, the onset of indigo reduction with this material may vary greatly. Certain additives improve indigo fermentation. Here, we studied the effects of Indigofera tinctoria leaf powder (LP) on the initiation of indigo reduction, bacterial community, redox potential (ORP), and dyeing intensity in the initial stages and in aged fermentation fluids prepared with sukumo. I. tinctoria LP markedly decreased ORP at day 1 and stabilised it during early fermentation. These effects could be explained by the phytochemicals present in I. tinctoria LP that act as oxygen scavengers and electron mediators. Using next generation sequencing results, we observed differences in the bacterial community in sukumo fermentation treated with I. tinctoria LP, which was not influenced by the bacterial community in I. tinctoria LP per se. The concomitant decrease in Bacillaceae and increase in Proteinivoraceae at the onset of fermentation, increase in the ratio of facultative to obligate anaerobes (F/O ratio), or the total abundance of facultative anaerobes (F) or obligate anaerobes (O) (designated F + O) are vital for the initiation and maintenance of indigo reduction. Hence, I. tinctoria LP improved early indigo reduction by decreasing the ORP and hasten the appropriate transitions in the bacterial community in sukumo fermentation.

The Mechanism Underlying of Long-Term Stable Indigo Reduction State in Indigo Fermentation Using Sukumo (Composted Polygonum tinctorium Leaves).

Indigo fermentation fluid maintains its indigo-reducing state for more than 6 months under open-air. To elucidate the mechanism underlying the sustainability of this indigo reduction state, three indigo fermentation batches with different durations for the indigo reduction state were compared. The three examined batches exhibited different microbiota and consisted of two phases. In the initial phase, oxygen-metabolizing-bacteria derived from sukumo established an initial network. With decreasing redox potential (ORP), the initial bacterial community was replaced by obligate anaerobes (mainly Proteinivoraceae; phase 1). Approximately 1 month after the beginning of fermentation, the predominating obligate anaerobes were decreased, and Amphibacillus and Polygonibacillus, which can decompose macromolecules derived from wheat bran, were predominantly observed, and the transition of microbiota became slow (phase 2). Considering the substrate utilization ability of the dominated bacterial taxa, the transitional change from phase 1 to phase 2 suggests that this changed from the bacterial flora that utilizes substrates derived from sukumo, including intrinsic substrates in sukumo and weakened or dead bacterial cells derived from early events (heat and alkaline treatment and reduction of ORP) to that of wheat bran-utilizers. This succession was directly related to the change in the major substrate sustaining the corresponding community and the turning point was approximately 1 month after the start of fermentation. As a result, we understand that the role of sukumo includes changes in the microbial flora immediately after the start of fermentation, which has an important function in the start-up phase of fermentation, whereas the ecosystem comprised of the microbiota utilizing wheat bran underpins the subsequent long-term indigo reduction.

The extracellular matrix protein Agrin is expressed by osteoblasts and contributes to their differentiation.

The extracellular matrix protein Agrin has been detected in chondrocytes and endosteal osteoblasts but its function in osteoblast differentiation has not been investigated yet. Thus, it is possible that Agrin contributes to osteoblast differentiation and, due to Agrin and wingless-related integration site (Wnt) sharing the same receptor, transmembrane low-density lipoprotein receptor-related protein 4 (Lrp4), and the crosstalk between Wnt and bone morphogenetic protein (BMP) signalling, both pathways could be involved in this Agrin-mediated osteoblast differentiation. Confirming this, Agrin and its receptors Lrp4 and α-dystroglycan (Dag1) were expressed during differentiation of osteoblasts from three different sources. Moreover, the disruption of Agrin impaired the expression of its receptors and osteoblast differentiation, and the treatment with recombinant Agrin slightly increase this process. In addition, whilst Agrin knockdown downregulated the expression of genes related to Wnt and BMP signalling pathways, the addition of Agrin had no effect on these genes. Altogether, these data uncover the contribution of Agrin to osteoblast differentiation and suggest that, at least in part, an Agrin-Wnt-BMP circuit is involved in this process. This makes Agrin a candidate as target for developing new therapeutic strategies to treat bone-related diseases and injuries.

Analysis of bacterial flora of indigo fermentation fluids utilizing composted indigo leaves (sukumo) and indigo extracted from plants (Ryukyu-ai and Indian indigo).

Indigo is a fabric dye that requires reduction by microbial activity or chemical reagents to render it soluble in water. Sources of indigo for fermentation are primarily divided into composted indigo-containing plants and indigo extracted from plants. To elucidate the factors responsible for bacterial diversity, and for sustaining reduced state of indigo in different preparations, this study assessed fermentation-derived fluids using composted plant leaves, sukumo, and extracted indigo (Ryukyu-ai paste, and Indian indigo cake) prepared using different procedures. Regardless of the indigo source, obligate anaerobic bacteria, including the families Proteinivoraceae and Tissierellaceae, predominate (16.9-46.1%), suggesting their high affinity for this fermentation ecosystem (hyperalkaline and low redox potential). Moreover, bacterial communities in sukumo fermentations are more diverse than those from indigo extracts with the diversity tending to increase based on the fermentation period. Our results further suggest that the microbiota composition in sukumo fermentation is associated with the various bacterial nutrients derived from sukumo, including seed microorganisms. In addition, the debris derived from sukumo can reduce the pH stress experienced by the microorganisms. Further, regardless of 5.4 years difference in the fermentation age, the bacterial flora in two Ryukyu-ai batches exhibit similar features with low microbial diversities. The uniformity of the nutrient, along with the simple, yet strong, bacterial network in Ryukyu-ai fluids may be responsible for the stable bacterial flora composition. Taken together, these results indicate that the microbiota in indigo fermentation is highly influenced by the seed culture, the nutrient derived from raw materials, and the fermentation conditions.

Mesenchymal stem cells overexpressing BMP-9 by CRISPR-Cas9 present high in vitro osteogenic potential and enhance in vivo bone formation.

Cell therapy is a valuable strategy for the replacement of bone grafts and repair bone defects, and mesenchymal stem cells (MSCs) are the most frequently used cells. This study was designed to genetically edit MSCs to overexpress bone morphogenetic protein 9 (BMP-9) using Clustered Regularly Interspaced Short Palindromic Repeats/associated nuclease Cas9 (CRISPR-Cas9) technique to generate iMSCs-VPRBMP-9+, followed by in vitro evaluation of osteogenic potential and in vivo enhancement of bone formation in rat calvaria defects. Overexpression of BMP-9 was confirmed by its gene expression and protein expression, as well as its targets Hey-1, Bmpr1a, and Bmpr1b, Dlx-5, and Runx2 and  protein expression of SMAD1/5/8 and pSMAD1/5/8. iMSCs-VPRBMP-9+ displayed significant changes in the expression of a panel of genes involved in TGF-ß/BMP signaling pathway. As expected, overexpression of BMP-9 increased the osteogenic potential of MSCs indicated by increased gene expression of osteoblastic markers Runx2, Sp7, Alp, and Oc, higher ALP activity, and matrix mineralization. Rat calvarial bone defects treated with injection of iMSCs-VPRBMP-9+ exhibited increased bone formation and bone mineral density when compared with iMSCs-VPR- and phosphate buffered saline (PBS)-injected defects. This is the first study to confirm that CRISPR-edited MSCs overexpressing BMP-9 effectively enhance bone formation, providing novel options for exploring the capability of genetically edited cells to repair bone defects.

Miniplates coated by plasma electrolytic oxidation improve bone healing of simulated femoral fractures on low bone mineral density rats.

The treatment of polytrauma patients represents a great challenge in the maxillofacial and orthopedic surgery fields. Therefore, this study tested the hypothesis that the use of a bioactive coating (by plasma electrolytic oxidation, PEO) on titanium microplates could improve the fracture healing of low bone mineral density (BMD) rats. Thirty female rats underwent bilateral ovariectomy surgery (OVX), and 35 rats underwent fake surgery (SHAM). Three months later, animals were subjected to femoral fracture simulation and were fixed with either non-coated (CONV) or coated (PEO) titanium miniplates. Eight weeks postoperatively, microplate/bone complexes were analyzed through computed microtomography, histometric, confocal microscopy, molecular, and biomechanical analysis. Bioactive elements (Ca and P) were incorporated on the PEO microplate and the surface was modified in a volcano-like structure. In the microCT analysis the OVX/PEO group had greater values for Tb.Th (bone trabecular thickness), Tb.Sp (separation of bone trabeculae) and Tb.N (number of trabeculae) parameters compared to the OVX/CONV group. According to histometric analysis, the OVX/PEO group showed significantly higher new bone formation than the OVX/CONV group (P < 0.05). For the fluorochrome area, the OVX groups (PEO and CONV) showed greater values for calcein precipitation (old bone) than alizarin red (new bone). Molecular results showed greater values for proteins related to the final phase of bone formation (P < 0.05) in the OVX/PEO group. The OVX/PEO group showed higher bone/miniplate system resilience compared to the others (P < 0.05). It was concluded that PEO coating optimizes bone healing on simulated femoral fractures in low bone mineral density rats. This sheds new light in the treatment of osteoporotic patients with bone fractures.