Microfluidics and organ-on-a-chip technologies: A systematic review of the methods used to mimic bone marrow.

Bone marrow (BM) is an organ responsible for crucial processes in living organs, e. g., hematopoiesis. In recent years, Organ-on-a-Chip (OoC) devices have been used to satisfy the need for in vitro systems that better mimic the phenomena occurring in the BM microenvironment. Given the growing interest in these systems and the diversity of developed devices, an integrative systematic literature review is required. We have performed this review, following the PRISMA method aiming to identify the main characteristics and assess the effectiveness of the devices that were developed to represent the BM. A search was performed in the Scopus, PubMed, Web of Science and Science Direct databases using the keywords (("bone marrow" OR "hematopoietic stem cells" OR "haematopoietic stem cells") AND ("organ in a" OR "lab on a chip" OR "microfluidic" OR "microfluidic*" OR ("bioreactor" AND "microfluidic*"))). Original research articles published between 2009 and 2020 were included in the review, giving a total of 21 papers. The analysis of these papers showed that their main purpose was to study BM cells biology, mimic BM niches, model pathological BM, and run drug assays. Regarding the fabrication protocols, we have observed that polydimethylsiloxane (PDMS) material and soft lithography method were the most commonly used. To reproduce the microenvironment of BM, most devices used the type I collagen and alginate. Peristaltic and syringe pumps were mostly used for device perfusion. Regarding the advantages compared to conventional methods, there were identified three groups of OoC devices: perfused 3D BM; co-cultured 3D BM; and perfused co-cultured 3D BM. Cellular behavior and mimicking their processes and responses were the mostly commonly studied parameters. The results have demonstrated the effectiveness of OoC devices for research purposes compared to conventional cell cultures. Furthermore, the devices have a wide range of applicability and the potential to be explored.

Bovine fetal mesenchymal stem cells exert antiproliferative effect against mastitis causing pathogen Staphylococcus aureus.

Staphylococcus aureus is the most commonly isolated pathogen from clinical bovine mastitis samples and a difficult pathogen to combat. Mesenchymal stem cells (MSC) are multipotent progenitor cells equipped with a variety of factors that inhibit bacterial growth. The aim of the present study was to evaluate the in vitro antibacterial potential against S. aureus of conditioned medium (CM) from MSC derived from fetal bovine bone marrow (BM-MSC) and adipose tissue (AT-MSC). BM-MSC, AT-MSC and fetal fibroblasts (FB) cultures were activated by infection with S. aureus. Bacterial growth was evaluated in presence of CM, concentrated CM (CCM), activated CM (ACM) and concentrated ACM (CACM) from BM-MSC, AT-MSC and FB. Gene expression of ß-defensin 4A (bBD-4A), NK-lysine 1 (NK1), cathelicidin 2 (CATHL2), hepcidin (HEP) and indoleamine 2,3 dioxygenase (IDO) and protein expression of bBD-4A were determined in activated and non-activated cells. The majority of BM-MSC and AT-MSC expressed CD73, Oct4 and Nanog, and were negative for CD34. Growth of S. aureus decreased when it was exposed to CM from BM-MSC, AT-MSC and FB. Moreover, growth of S. aureus in CCM, ACM and CACM was lower compared to controls of CM from BM-MSC and AT-MSC. Activated AT-MSC increased mRNA levels of bBD4A and NK1, and protein levels of bBD4A in CM. Thus, CM from fetal bovine BM-MSC and AT-MSC has the capacity to reduce in average ~30% of S. aureus relative growth under in vitro conditions. The in vitro antibacterial effect of fetal bovine MSC may be mediated by bBD4A and NK1 activity.

Umbilical Cord-Derived Mesenchymal Stromal Cells (MSCs) for Knee Osteoarthritis: Repeated MSC Dosing Is Superior to a Single MSC Dose and to Hyaluronic Acid in a Controlled Randomized Phase I/II Trial.

Knee osteoarthritis (OA) is a leading cause of pain and disability. Although conventional treatments show modest benefits, pilot and phase I/II trials with bone marrow (BM) and adipose-derived (AD) mesenchymal stromal cells (MSCs) point to the feasibility, safety, and occurrence of clinical and structural improvement in focal or diffuse disease. This study aimed to assess the safety and efficacy of the intra-articular injection of single or repeated umbilical cord-derived (UC) MSCs in knee OA. UC-MSCs were cultured in an International Organization for Standardization 9001:2015 certified Good Manufacturing Practice-type Laboratory. Patients with symptomatic knee OA were randomized to receive hyaluronic acid at baseline and 6 months (HA, n = 8), single-dose (20 × 106 ) UC-MSC at baseline (MSC-1, n = 9), or repeated UC-MSC doses at baseline and 6 months (20 × 106 × 2; MSC-2, n = 9). Clinical scores and magnetic resonance images (MRIs) were assessed throughout the 12 months follow-up. No severe adverse events were reported. Only MSC-treated patients experienced significant pain and function improvements from baseline (p = .001). At 12 months, Western Ontario and Mc Master Universities Arthritis Index (WOMAC-A; pain subscale) reached significantly lower levels of pain in the MSC-2-treated group (1.1 ± 1.3) as compared with the HA group (4.3 ± 3.5; p = .04). Pain Visual Analog scale was significantly lower in the MSC-2 group versus the HA group (2.4 ± 2.1 vs. 22.1 ± 9.8, p = .03) at 12 months. For total WOMAC, MSC-2 had lower scores than HA at 12 months (4.2 ± 3.9 vs. 15.2 ± 11, p = .05). No differences in MRI scores were detected. In a phase I/II trial (NCT02580695), repeated UC-MSC treatment is safe and superior to active comparator in knee OA at 1-year follow-up. Stem Cells Translational Medicine 2019;8:215&224.

The effect of vitamin D and zoledronic acid in bone marrow adiposity in kidney transplant patients: A post hoc analysis.

PURPOSE: Osteoblasts and adipocytes are derived from mesenchymal stem cells. An imbalance in the differentiation of these lineages could affect the preservation of bone integrity. Several studies have suggested the importance of this imbalance in the pathogenesis of osteoporosis after kidney transplant (KT), but the role of bone marrow adiposity in this process is not well known, and if the treatment with the anti-absorptive (zoledronic acid-ZA) drugs could attenuate bone loss. Thus, our objective was compare bone marrow adiposity, osteoblasts and osteocytes before and after KT, verify an association between bone remodeling process (Turnover, Volume, and Mineralization-TMV classification), the osteocyte sclerostin expression to evaluate if there is a role of Wnt pathway, as well as the effect of ZA on these cells. METHODS: We studied 29 new living-donor KT patients. One group received ZA at the time of KT plus cholecalciferol for twelve months, and the other group received only cholecalciferol. Bone biopsies were performed at baseline and after 12 months of treatment. Histomorphometric evaluation was performed in bone and bone marrow adipocytes. Sclerostin (Scl) expression in osteocytes was evaluated by immunohistochemistry. RESULTS: Some bone marrow adiposity parameters were increased before KT. After KT, some of them remained increased and they worsened with the use of ZA. In the baseline, lower bone Volume and Turnover, were associated with increased bone marrow adiposity parameters (some of them). After KT, both groups showed the same associations. Osteocyte Scl expression after KT decreased with the use of ZA. We observed also an inverse association between bone adiposity parameters and lower osteocyte sclerostin expression 12 months after KT. CONCLUSION: In conclusion, the present study suggests that KT fails to normalize bone marrow adiposity, and it even gets worse with the use of ZA. Moreover, bone marrow adiposity is inversely associated with bone Volume and Turnover, which seems to be accentuated by the antiresorptive therapy.

Adipocytes role in the bone marrow niche.

Adipocyte infiltration in the bone marrow follows chemotherapy or irradiation. Previous studies indicate that bone marrow fat cells inhibit hematopoietic stem cell function. Recently, Zhou et al. (2017) using state-of-the-art techniques, including sophisticated Cre/loxP technologies, confocal microscopy, in vivo lineage-tracing, flow cytometry, and bone marrow transplantation, reveal that adipocytes promote hematopoietic recovery after irradiation. This study challenges the current view of adipocytes as negative regulators of the hematopoietic stem cells niche, and reopens the discussion about adipocytes' roles in the bone marrow. Strikingly, genetic deletion of stem cell factor specifically from adipocytes leads to deficiency in hematopoietic stem cells, and reduces animal survival after myeloablation, The emerging knowledge from this research will be important for the treatment of multiple hematologic disorders. © 2017 International Society for Advancement of Cytometry.

Sources and Clinical Applications of Mesenchymal Stem Cells: State-of-the-art review.

First discovered by Friedenstein in 1976, mesenchymal stem cells (MSCs) are adult stem cells found throughout the body that share a fixed set of characteristics. Discovered initially in the bone marrow, this cell source is considered the gold standard for clinical research, although various other sources-including adipose tissue, dental pulp, mobilised peripheral blood and birth-derived tissues-have since been identified. Although similar, MSCs derived from different sources possess distinct characteristics, advantages and disadvantages, including their differentiation potential and proliferation capacity, which influence their applicability. Hence, they may be used for specific clinical applications in the fields of regenerative medicine and tissue engineering. This review article summarises current knowledge regarding the various sources, characteristics and therapeutic applications of MSCs.

Morphometric evaluation of the early stages of healing at cortical and marrow compartments at titanium implants: an experimental study in the dog.

OBJECTIVE: To study the early sequential stages of tissue composition in the cortical and marrow compartments of the alveolar bone crest at implants with a moderately rough surface. MATERIALS AND METHODS: Three month after tooth extraction in 12 Labrador dogs, full-thickness flaps were elevated in the edentulous region of the right side of the mandible and one implant was installed. The flaps were sutured to allow a fully submerged healing. The timing of the installations in the left side of the mandible and of sacrifices were scheduled in such a way to obtained biopsies representing the healing after 5, 10, 20, and 30 days. Ground sections (n = 6 per each healing period) were prepared, and the percentages of osteoid/new bone, old bone, new soft tissues (provisional matrix and primitive marrow), mature bone marrow, vessels, and other tissues (bone debris/particles and clot) were evaluated laterally to the implant surface up to a distance of about 0.4 mm from it. RESULTS: Osteoid/new bone was found after 5 days at percentages of 10.8 ± 4.3% at the marrow and 0.6 ± 0.6% at the cortical compartments. After 30 days, these percentages increased up to 56.4 ± 4.0% and 23.3 ± 6.1%, respectively. Old parent bone was resorbed between 5 and 30 days from 28.7 ± 10.9% to 14.9 ± 3.4% at the marrow (~48% of resorption) and from 81.2 ± 9.4% to 67.6 ± 5.6% at the cortical (~17% of resorption) compartments. All differences were statistically significant. CONCLUSION: Bone apposition to an implant surface followed a significantly different pattern in the compact and the marrow compartments around the implants. While in the compact compartments, bone apposition had to develop through the BMUs following resorption, it developed in very dense layers through an early apposition in the marrow compartments.

Role of SDF-1 (CXCL12) in regulating hematopoietic stem and progenitor cells traffic into the liver during extramedullary hematopoiesis induced by G-CSF, AMD3100 and PHZ.

The stromal cell derived factor 1 (SDF-1/CXCL12) plays an essential role in the homing of hematopoietic stem and progenitor cells (HSPCs) to bone marrow (BM). It is not known whether SDF-1 may also regulate the homing of HSPCs to the liver during extramedullary hematopoiesis (EMH). Here, we investigated the possible role of SDF-1 in attracting HSPCs to the liver during experimental EMH induced by the hematopoietic mobilizers G-CSF, AMD3100 and phenylhydrazine (PHZ). Mice treated with G-CSF, AMD3100 and PHZ showed a significant increase in the expression of SDF-1 in the liver sinusoidal endothelial cells (LSECs) microenvironments. Liver from mice treated with the hematopoietic mobilizers showed HSPCs located adjacent to the LSEC microenvironments, expressing high levels of SDF-1. An inverse relationship was found between the hepatic SDF-1 levels and those in the BM. In vitro, LSEC monolayers induced the migration of HSPCs, and this effect was significantly reduced by AMD3100. In conclusion, our results provide the first evidence showing that SDF-1 expressed by LSEC can be a major player in the recruitment of HSPCs to the liver during EMH induced by hematopoietic mobilizers.

Pore cross-section area on predicting elastic properties of trabecular bovine bone for human implants.

A clear understanding of the dependence of mechanical properties of bone remains a task not fully achieved. In order to estimate the mechanical properties in bones for implants, pore cross-section area, calcium content, and apparent density were measured in trabecular bone samples for human implants. Samples of fresh and defatted bone tissue, extracted from one year old bovines, were cut in longitudinal and transversal orientation of the trabeculae. Pore cross-section area was measured with an image analyzer. Compression tests were conducted into rectangular prisms. Elastic modulus presents a linear tendency as a function of pore cross-section area, calcium content and apparent density regardless of the trabecular orientation. The best variable to estimate elastic modulus of trabecular bone for implants was pore cross-section area, and affirmations to consider Nukbone process appropriated for marrow extraction in trabecular bone for implantation purposes are proposed, according to bone mechanical properties. Considering stress-strain curves, defatted bone is stiffer than fresh bone. Number of pores against pore cross-section area present an exponential decay, consistent for all the samples. These graphs also are useful to predict elastic properties of trabecular samples of young bovines for implants.

Influence of bone marrow on osseointegration in long bones: an experimental study in sheep.

AIM: To evaluate the influence of yellow bone marrow on osseointegration of titanium oral implants using a long bone model. MATERIAL AND METHODS: The two tibiae of eight sheep were used as experimental sites. Two osteotomies for implant installation were prepared in each tibia. At the control sites, no further treatments were performed while, at the test sites, bone marrow was removed from the osteotomy site with a curette to an extent that exceeded the implant dimensions. As a result, the apical portion of the implants at the control sites was in contact with bone marrow while, at the test sites, it was in contact with the blood clot. After 2 months, the same procedures were performed in the contralateral side. After another month, the animal was sacrificed. Ground sections were obtained for histological analysis. RESULTS: After 1 month of healing, no differences between test and control sites were found in the apical extension of osseointegration and the percentage of new bone-to-implant contact. However, after 3 months of healing, a higher percentage of new bone-to-implant contact was found at the test compared to the control sites in the marrow compartment. The apical extension of osseointegration, however, was similar to that found at the 1-month healing period both for test and control sites. CONCLUSIONS: Osseointegration appeared to be favored by the presence of a blood clot when compared to the presence of yellow fatty bone marrow. Moreover, the contact with cortical bone appeared to be a prerequisite for the osseointegration process in the long bone model.

Morphology and morphometry of feline bone marrow-derived mesenchymal stem cells in culture / Morfologia e morfometria das células-tronco mesenquimais isoladas de medula óssea de gato

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for treatment of a variety of different diseases in human and veterinary medicine. Stem cells have been isolated from feline bone marrow, however, very few data exist about the morphology of these cells and no data were found about the morphometry of feline bone marrow-derived MSCs (BM-MSCs). The objectives of this study were the isolation, growth evaluation, differentiation potential and characterization of feline BM-MSCs by their morphological and morphometric characteristics. in vitro differentiation assays were conducted to confirm the multipotency of feline MSC, as assessed by their ability to differentiate into three cell lineages (osteoblasts, chondrocytes, and adipocytes). To evaluate morphological and morphometric characteristics the cells are maintained in culture. Cells were observed with light microscope, with association of dyes, and they were measured at 24, 48, 72 and 120h of culture (P1 and P3). The non-parametric ANOVA test for independent samples was performed and the means were compared by Tukey's test. On average, the number of mononuclear cells obtained was 12.29 (±6.05x10(6)) cells/mL of bone marrow. Morphologically, BM-MSCs were long and fusiforms, and squamous with abundant cytoplasm. In the morphometric study of the cells, it was observed a significant increase in average length of cells during the first passage. The cell lengths were 106.97±38.16µm and 177.91±71.61µm, respectively, at first and third passages (24 h). The cell widths were 30.79±16.75 µm and 40.18±20.46µm, respectively, at first and third passages (24 h).The nucleus length of the feline BM-MSCs at P1 increased from 16.28µm (24h) to 21.29µm (120h). However, at P3, the nucleus length was 26.35µm (24h) and 25.22µm (120h). This information could be important for future application and use of feline BM-MSCs.(AU)

As células tronco mesenquimais são utilizadas na terapia de várias doenças na medicina humana e veterinária. As células tronco foram isoladas da medula óssea de gato, entretanto, existem poucos dados referentes a morfologia e não existem informações sobre a morfometria das células tronco isoladas da medula óssea. Os objetivos do presente estudo foram o isolamento, avaliação do crescimento, potencial de diferenciação e caracterização morfológica e morfométrica das células mesenquimais de gato isoladas de medula óssea. A diferenciação in vitro foi realizada para confirmar a multipotencialidade das células mesenquimais de gato (diferenciação em osteoblastos, condrócitos, adipócitos). As células mesenquimais foram mantidas em cultivo para avaliações morfológica e morfométrica. As células foram coradas e observadas em microscopia ótica. As mensurações foram realizadas com 24, 48, 72 e 120h de cultura (primeira e terceira passagens). O teste não paramétrico ANOVA foi utilizado e as médias foram comparadas pelo teste de Tukey. O número médio de células mononucleares obtido foi de 12,29 (±6,05x10(6)) células/mL de medula óssea. As células mesenquimais são longas e fusiformes, e escamosas com citoplasma abundante. No estudo morfométrico, observou-se aumento no comprimento médio das células durante a primeira passagem. As medidas de comprimento das células foram: 106,97±38,16µm e 177,91±71,61µm, respectivamente, na primeira e terceira passagens (24 horas). As medidas de largura das células foram: 30,79±16,75 µm e 40,18±20,46 µm, respectivamente, na primeira e terceira passagens (24 horas). O comprimento do núcleo na primeira passagem aumentou de 16,28µm (24h) para 21,29µm (120h) e na terceira passagem foi de 26,35µm (24h) para 25,22µm (120h). As informações são importantes para futuras aplicações e uso da célula mesenquimal de gato.(AU)

Expression of non-neuronal cholinergic system in maxilla of rat in vivo.

BACKGROUND: Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, which is also produced in a variety of non-neuronal tissues and cell. The existence of ACh in maxilla in vivo and potential regulation role for osteogenesis need further study. RESULTS: Components of the cholinergic system (ACh, esterase, choline acetyltransferase, high-affinity choline uptake, n- and mAChRs) were determined in maxilla of rat in vivo, by means of Real-Time PCR and immunohistochemistry. Results showed RNA for CarAT, carnitine/acylcarnitine translocase member 20 (Slc25a20), VAChT, OCTN2, OCT1, OCT3, organic cation transporter member 4 (Slc22a4), AChE, BChE, nAChR subunits α1, α2, α3, α5, α7, α10, ß1, ß2, ß4, γ and mAChR subunits M1, M2, M3, M4, M5 were detected in rat's maxilla. RNA of VAChT, AChE, nAChR subunits α2, ß1, ß4 and mAChR subunits M4 had abundant expression (2(-ΔCt) > 0.03). Immunohistochemical staining was conducted for ACh, VAChT, nAChRα7 and AChE. ACh was expressed in mesenchymal cells, chondroblast, bone and cartilage matrix and bone marrow cells, The VAChT expression was very extensively while ACh receptor α7 was strongly expressed in newly formed bone matrix of endochondral and bone marrow ossification, AchE was found only in mesenchymal stem cells, cartilage and bone marrow cells. CONCLUSIONS: ACh might exert its effect on the endochondral and bone marrow ossification, and bone matrix mineralization in maxilla.

Expression of non-neuronal cholinergic system in maxilla of rat in vivo

BACKGROUND: Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, which is also produced in a variety of non-neuronal tissues and cell. The existence of ACh in maxilla in vivo and potential regulation role for osteogenesis need further study. RESULTS: Components of the cholinergic system (ACh, esterase, choline acetyltransferase, high-affinity choline uptake, n- and mAChRs) were determined in maxilla of rat in vivo, by means of Real-Time PCR and immunohistochemistry. Results showed RNA for CarAT, carnitine/acylcarnitine translocase member 20 (Slc25a20), VAChT, OCTN2, OCT1, OCT3, organic cation transporter member 4 (Slc22a4), AChE, BChE, nAChR subunits α1, α2, α3, α5, α7, α10, ß1, ß2, ß4, γ and mAChR subunits M1, M2, M3, M4, M5 were detected in rat's maxilla. RNA of VAChT, AChE, nAChR subunits α2, ß1, ß4 and mAChR subunits M4 had abundant expression (2(-ΔCt) > 0.03). Immunohistochemical staining was conducted for ACh, VAChT, nAChRα7 and AChE. ACh was expressed in mesenchymal cells, chondroblast, bone and cartilage matrix and bone marrow cells, The VAChT expression was very extensively while ACh receptor α7 was strongly expressed in newly formed bone matrix of endochondral and bone marrow ossification, AchE was found only in mesenchymal stem cells, cartilage and bone marrow cells. CONCLUSIONS: ACh might exert its effect on the endochondral and bone marrow ossification, and bone matrix mineralization in maxilla.

Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis.

In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the "quiescent" and "proliferative" niches in which hematopoietic stem cells and progenitors reside.

Diferenciação in vitro de células-tronco mesenquimais da medula óssea de cães em precursores osteogênicos / In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

O objetivo principal da nossa pesquisa foi avaliar o potencial de diferenciação osteogênica de células-tronco mesenquimais (MSC) obtidas da medula óssea do cão. As MSC foram separadas pelo método Ficoll e cultivadas sob duas condições distintas: DMEM baixa glicose ou DMEM/F12, ambos contendo L-glutamina, 20% de SFB e antibióticos. Marcadores de MSC foram testados, confirmando células CD44+ e CD34- através da citometria de fluxo. Para a diferenciação osteogênica, as células foram submetidas a quatro diferentes condições: Grupo 1, as mesmas condições utilizadas para a cultura de células primárias com os meios DMEM baixa glicose suplementado; Grupo 2, as mesmas condições do Grupo 1, mais os indutores de diferenciação dexametasona, ácido ascórbico e b-glicerolfosfato; Grupo 3, células cultivadas com meios DMEM/F12 suplementado; e Grupo 4, nas mesmas condições que no Grupo 3, mais indutores de diferenciação de dexametasona, ácido ascórbico e b-glicerolfosfato. A diferenciação celular foi confirmada através da coloração com alizarin red e da imunomarcação com o anticorpo SP7/Osterix. Nós observamos através da coloração com alizarin red que o depósito de cálcio foi mais evidente nas células cultivadas em DMEM/F12. Além disso, usando a imunomarcação com o anticorpo SP/7Osterix obtivemos positividade em 1:6 células para o Meio DMEM/F12 comparada com 1:12 para o meio DMEM-baixa glicose. Com base nos nossos resultados concluímos que o meio DMEM/F12 é mais eficiente para a indução da diferenciação de células-tronco mesenquimais caninas em promotores osteogênicos. Este efeito provavelmente ocorre em decorrência da maior quantidade de glicose neste meio, bem como da presença de diversos aminoácidos.(AU)

The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12.Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.(AU)

Potencial de transdiferenciação neural das células-tronco mesenquimais da medula óssea de equino / Potential of neural transdifferentiation of mesenchymal stem cells from equine bone marrow

Os primeiros estudos demonstrando o potencial de trandiferenciação neural das células-tronco mesenquimais (CTMs) provenientes da medula óssea (MO) foram conduzidos em camundogos e humanos no início da década de 2000. Após esse período, o número de pesquisas e publicações com o mesmo propósito tem aumentado, mas com raros ou escassos estudos na espécie equina. Nesse sentindo, o objetivo desse trabalho foi avaliar o potencial in vitro da transdiferenciação neural das CTMs provenientes da MO de equinos utilizando-se dois protocolos: P1 (forksolin e ácido retinóico) e P2 (2-βmecarptoetanol). Após a confirmação das linhagens mesenquimais, pela positividade para o marcador CD90 (X=97,94%), negatividade para o marcador CD34 e resposta positiva a diferenciação osteogênica, as CTMs foram submetidas a transdiferenciação neural (P1 e P2) para avaliação morfológica e expressão dos marcadores neurais GFAP e β3 tubulina por citometria de fluxo. Os resultados revelaram mudanças morfológicas em graus variados entre os protocolos testados. No protocolo 1, vinte quatro horas após a incubação com o meio de diferenciação neural, grande proporção de células (>80%) apresentaram morfologia semelhante a células neurais, caracterizadas por retração do corpo celular e grande número de projeções protoplasmáticas (filopodia). Por outro lado, de forma comparativa, já nos primeiros 30 minutos após a exposição ao antioxidante β-mercaptoetanol (P2) as CTMs apresentaram rápida mudança morfológica caracterizada principalmente por retração do corpo celular e menor número de projeções protoplasmáticas. Também ficou evidenciado com o uso deste protocolo, menor aderência das células após tempo de exposição ao meio de diferenciação, quando comparado ao P1.(AU)

The first studies showing the potential of neural transdifferentiation of mesenchymal stem cells (MSCs) from bone marrow (BM) were conducted in camundogos and humans in the early 2000s. After this period, the number of research and publications with the same purpose increased, but with rare or scarce studies in horses. The aim of this study was to evaluate in vitro neuronal transdifferentiation potential of MSCs from equine BM using two protocols: P1 (forksolin and retinoic acid) and P2 (2-βmecarptoetanol). After confirming the mesenchymal lineages, by positivity for the marker CD90 (X=97.94%), negative for the marker CD34 and positive response for osteogenic differentiation, MSCs were subjected to neural transdifferentiation (P1 and P2) for morphological analysis and expression of neural markers GFAP and β3 tubulin by flow cytometry. The results revealed morphological changes in varying degrees between the tested protocols. In protocol 1, twenty four hours after incubation with the media of neural differentiation, a large proportion of cells (>80%) had similar morphology to neural cells, characterized by retraction of cellular body and a large number of cytoplasmic extension (filopodia). However, comparatively, within the first 30 minutes after exposure to the antioxidant β-mercaptoethanol (P2) MSCs showed rapid morphological changes characterized mainly by retraction of cellular body and less cytoplasmic extension.(AU)

Potencial de transdiferenciação neural das células-tronco mesenquimais da medula óssea de equino / Potential of neural transdifferentiation of mesenchymal stem cells from equine bone marrow

Os primeiros estudos demonstrando o potencial de trandiferenciação neural das células-tronco mesenquimais (CTMs) provenientes da medula óssea (MO) foram conduzidos em camundogos e humanos no início da década de 2000. Após esse período, o número de pesquisas e publicações com o mesmo propósito tem aumentado, mas com raros ou escassos estudos na espécie equina. Nesse sentindo, o objetivo desse trabalho foi avaliar o potencial in vitro da transdiferenciação neural das CTMs provenientes da MO de equinos utilizando-se dois protocolos: P1 (forksolin e ácido retinóico) e P2 (2-βmecarptoetanol). Após a confirmação das linhagens mesenquimais, pela positividade para o marcador CD90 (X=97,94%), negatividade para o marcador CD34 e resposta positiva a diferenciação osteogênica, as CTMs foram submetidas a transdiferenciação neural (P1 e P2) para avaliação morfológica e expressão dos marcadores neurais GFAP e β3 tubulina por citometria de fluxo. Os resultados revelaram mudanças morfológicas em graus variados entre os protocolos testados. No protocolo 1, vinte quatro horas após a incubação com o meio de diferenciação neural, grande proporção de células (>80%) apresentaram morfologia semelhante a células neurais, caracterizadas por retração do corpo celular e grande número de projeções protoplasmáticas (filopodia). Por outro lado, de forma comparativa, já nos primeiros 30 minutos após a exposição ao antioxidante β-mercaptoetanol (P2) as CTMs apresentaram rápida mudança morfológica caracterizada principalmente por retração do corpo celular e menor número de projeções protoplasmáticas. Também ficou evidenciado com o uso deste protocolo, menor aderência das células após tempo de exposição ao meio de diferenciação, quando comparado ao P1. Com relação a análise imunofenotípica foi observado uma maior (P<0,001) expressão dos marcadores GFAP e β3 tubulina ao término do P2 quando comparado ao P1. A habilidade das CTMs em gerar tipos celulares relacionados a linhagem neural é complexa e multifatorial, dependendo não só dos agentes indutores, mas também do ambiente no qual estas células são cultivadas. Desta forma um maior número de estudos é necessário para o melhor entendimento do processo de transdiferenciação neural a partir de CTMs de equinos.

The first studies showing the potential of neural transdifferentiation of mesenchymal stem cells (MSCs) from bone marrow (BM) were conducted in camundogos and humans in the early 2000s. After this period, the number of research and publications with the same purpose increased, but with rare or scarce studies in horses. The aim of this study was to evaluate in vitro neuronal transdifferentiation potential of MSCs from equine BM using two protocols: P1 (forksolin and retinoic acid) and P2 (2-βmecarptoetanol). After confirming the mesenchymal lineages, by positivity for the marker CD90 (X=97.94%), negative for the marker CD34 and positive response for osteogenic differentiation, MSCs were subjected to neural transdifferentiation (P1 and P2) for morphological analysis and expression of neural markers GFAP and β3 tubulin by flow cytometry. The results revealed morphological changes in varying degrees between the tested protocols. In protocol 1, twenty four hours after incubation with the media of neural differentiation, a large proportion of cells (>80%) had similar morphology to neural cells, characterized by retraction of cellular body and a large number of cytoplasmic extension (filopodia). However, comparatively, within the first 30 minutes after exposure to the antioxidant β-mercaptoethanol (P2) MSCs showed rapid morphological changes characterized mainly by retraction of cellular body and less cytoplasmic extension. It was also evidenced with the use of this protocol, lower cellular adhesion after exposure to media when compared to P1. Regarding the immunophenotyping analysis it was observed a higher (P<0.001) expression of the markers GFAP and β3 tubulin at the end of P2 compared to P1. The ability of MSCs to generate cell types related to neural lineage is complex and multifactorial, depending not only of inducing agents, but also the environment in which these cells will be cultivated. Thus a greater number of studies are necessary to better understand the process of neural transdifferentiation of MSCs from equine.

Diferenciação in vitro de células-tronco mesenquimais da medula óssea de cães em precursores osteogênicos / In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

O objetivo principal da nossa pesquisa foi avaliar o potencial de diferenciação osteogênica de células-tronco mesenquimais (MSC) obtidas da medula óssea do cão. As MSC foram separadas pelo método Ficoll e cultivadas sob duas condições distintas: DMEM baixa glicose ou DMEM/F12, ambos contendo L-glutamina, 20% de SFB e antibióticos. Marcadores de MSC foram testados, confirmando células CD44+ e CD34- através da citometria de fluxo. Para a diferenciação osteogênica, as células foram submetidas a quatro diferentes condições: Grupo 1, as mesmas condições utilizadas para a cultura de células primárias com os meios DMEM baixa glicose suplementado; Grupo 2, as mesmas condições do Grupo 1, mais os indutores de diferenciação dexametasona, ácido ascórbico e b-glicerolfosfato; Grupo 3, células cultivadas com meios DMEM/F12 suplementado; e Grupo 4, nas mesmas condições que no Grupo 3, mais indutores de diferenciação de dexametasona, ácido ascórbico e b-glicerolfosfato. A diferenciação celular foi confirmada através da coloração com alizarin red e da imunomarcação com o anticorpo SP7/Osterix. Nós observamos através da coloração com alizarin red que o depósito de cálcio foi mais evidente nas células cultivadas em DMEM/F12. Além disso, usando a imunomarcação com o anticorpo SP/7Osterix obtivemos positividade em 1:6 células para o Meio DMEM/F12 comparada com 1:12 para o meio DMEM-baixa glicose. Com base nos nossos resultados concluímos que o meio DMEM/F12 é mais eficiente para a indução da diferenciação de células-tronco mesenquimais caninas em promotores osteogênicos. Este efeito provavelmente ocorre em decorrência da maior quantidade de glicose neste meio, bem como da presença de diversos aminoácidos.

The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12.Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

Jaw and long bone marrows have a different osteoclastogenic potential.

Osteoclasts, the multinucleated bone-resorbing cells, arise through fusion of precursors from the myeloid lineage. However, not all osteoclasts are alike; osteoclasts at different bone sites appear to differ in numerous respects. We investigated whether bone marrow cells obtained from jaw and long bone differed in their osteoclastogenic potential. Bone marrow cells from murine mandible and tibiae were isolated and cultured for 4 and 6 days on plastic or 6 and 10 days on dentin. Osteoclastogenesis was assessed by counting the number of TRAP(+) multinucleated cells. Bone marrow cell composition was analyzed by FACS. The expression of osteoclast- and osteoclastogenesis-related genes was studied by qPCR. TRAP activity and resorptive activity of osteoclasts were measured by absorbance and morphometric analyses, respectively. At day 4 more osteoclasts were formed in long bone cultures than in jaw cultures. At day 6 the difference in number was no longer observed. The jaw cultures, however, contained more large osteoclasts on plastic and on dentin. Long bone marrow contained more osteoclast precursors, in particular the myeloid blasts, and qPCR revealed that the RANKL:OPG ratio was higher in long bone cultures. TRAP expression was higher for the long bone cultures on dentin. Although jaw osteoclasts were larger than long bone osteoclasts, no differences were found between their resorptive activities. In conclusion, bone marrow cells from different skeletal locations (jaw and long bone) have different dynamics of osteoclastogenesis. We propose that this is primarily due to differences in the cellular composition of the bone site-specific marrow.

Localization of hematopoietic cells in the bullfrog (Lithobates catesbeianus).

Amphibians represent the first phylogenetic group to possess hematopoietic bone marrow. However, adult amphibian hematopoiesis has only been described in a few species and with conflicting data. Bone marrow, kidney, spleen, liver, gut, stomach, lung, tegument, and heart were therefore collected from adult Lithobates catesbeianus and investigated by light microscopy and immunohistochemical methods under confocal laser microscopy. Our study demonstrated active hematopoiesis in the bone marrow of vertebrae, femur, and fingers and in the kidney, but no hematopoietic activity inside other organs including the spleen and liver. Blood cells were identified as a heterogeneous cell population constituted by heterophils, basophils, eosinophils, monocytes, erythrocytic cells, lymphocytes, and their precursors. Cellular islets of the thrombocytic lineage occurred near sinusoids of the bone marrow. Antibodies against CD34, CD117, stem cell antigen, erythropoietin receptor, and the receptor for granulocyte colony-stimulating factor identified some cell populations, and some circulating immature cells were seen in the bloodstream. Thus, on the basis of these phylogenetic features, we propose that L. catesbeianus can be used as an important model for hematopoietic studies, since this anuran exhibits hematopoiesis characteristics both of lower vertebrates (renal hematopoiesis) and of higher vertebrates (bone marrow hematopoiesis).