Behavior of graft and host cells in underlying subchondral bone after transplantation of osteochondral autograft

Transplantation of osteochondral autograft is widely used as a therapeutic strategy for the defect of articular cartilage. In the repair process, although underlying subchondral bone becomes necrotic and then is followed by bone reconstruction, the fate of graft and host cells during remodeling of underlying subchondral bone has not been elucidated. The objectives of this study were to establish a method to follow graft and host cells after transplantation of osteochondral autograft, and to elucidate the fate of both graft and host cells during remodeling of underlying subchondral bone. For these purposes, autologous transplantation models employing transgenic rats and wild-type rats, which were genetically identical to each other except for transgenes, were used. Two transplantation models were designed so that either the graft or the host cells had transgenes. Model I: transgenic rats were the donor, and wild-type rats were the recipient; model II: conversely, wild-type rats were the donor, and transgenic rats were the recipient. The grafted bone marrow cells and osteocytes in the trabeculae survived in the graft at 3 weeks after transplantation. Invasion of the host bone marrow cells into the graft was also found. Thus, bone marrow cells in the host as well as both bone marrow cells and osteocytes in the graft could potentially participate in the remodeling of underlying subchondral bone. Furthermore, the interface between graft and host was consisted with both graft and host derived cells. Since new bone formation was found in this space, both graft and host cells could have the potential to contribute to remodeling of underlying subchondral bone. The two models of the transplantations using the transgenic rats were found to be beneficial in following graft cells as well as host cells and in understanding their function on healing after autologous transplantation.     

Numerical simulation and experimental validation for a novel dielectrophoresis activated cell sorter to achieve high throughput and efficiency

Although previously developed Dielectrophoresis activated cell sorters (DACSes) have achieved high separation efficiency, limitations still exist with respect to the low throughput. In this paper, therefore, a numerical analysis is presented for a novel DACS to achieve not only high efficiency but also high throughput. Additionally, experimental validation is performed based on the results of a numerical simulation. First, streamlines in the channel are investigated according to various flow rate ratios in each outlet. After cells are deflected by a sufficient amount of dielectrophoretic force, they arrive at the tip of the last electrode pair. Their movement is then determined by hydrodynamic force. Therefore, it is important to confirm whether or not the streamline at the tip of the last electrode pair connects to the target area. Based on the streamline analysis results, the particle trajectory under various AC electric fields is investigated using a three-dimensional analysis. Accordingly, an input voltage condition (7 Vp-p at 10 kHz) is selected to confirm the influence of dielectrophoretic force. Conclusively, optimal flow rates under the determined input voltage condition are selected as 10 μl/min and 20 μl/min in outlet A (target area) and outlet B (non-target area), respectively. In order to validate the feasibility of the selected flow rates, a separation test is performed employing a K562 cell–bone marrow cancer cell–as a target cell. In the experiment, the proposed DACS achieves a high throughput of 45000 cells/min as well as a separation efficiency of 96.5% and recovery rate of 47.25%.

     

Mesenchymal stem cells,the secretome and biomaterials: Regenerative medicine application

Mesenchymal stem cells (MSCs) are multipotent cells usually isolated from bone marrow, endometrium, adipose tissues, skin, and dental pulp. MSCs played a crucial role in regenerative therapy and have been introduced as an interdisciplinary field between cell biology and material science. Recently, MSCs have been widely explored for their application in regenerative medicine and COVID-19 treatment. Different approaches to evaluate the future of biomaterials and stem cell properties have been developed. However, misconceptions and ethical issues still exist, such as MSCs being non-angiogenic, anti-apoptotic, and immunoregulatory competencies. Embryonic stem cells isolation primarily requires the consent of donors and can include the killing of fertilized eggs. These issues generate questions related to ethical and moral issues. However, MSCs have gained considerable attention for tissue regeneration owing to their differentiation ability with immunomodulatory effects. They are capable of secreting a broad range of biomolecules such as proteins, nucleic acids, exosomes, microRNAs, and membrane vesicles, collectively known as secretomes. Secretomes are released in response to the surrounding microenvironment. In this article, we briefly address topics related to the therapeutic potential of MSCs as an advanced approach in the field of regenerative medicine and various perspectives.     

Cell therapy for fibrotic interstitial pulmonary disease: experimental study

Parte superior do formulário Digite um texto ou endereço de um site ou traduza um documento. The aim of this study is to evaluate the histological changes in lung parenchyma of pigs affected by interstitial lung disease induced after the infusion of bone marrow mononuclear cells (BMMCs). Ten female swines were submitted to pulmonary fibrosis induced by a single dose of intratracheal bleomicine sulfate. Animals were arranged into two groups: Group 1: induced‐disease control and Group 2: cell therapy using BMMCs. Both groups were clinically evaluated for 180 days. High‐resolution computed tomography (HRCT) was performed at 90 and 180 days. BMMC sampling was performed in cell therapy group at 90 days. Euthanasia was performed, and samples were collected for histology and immunohistochemistry. The 90‐days HRCT demonstrated typical interstitial lesions in pulmonary parenchyma similarly to human disease. The 180‐days HRCT in Group 1 demonstrated advanced stages of the disease when compared with Group 2. Immunohistochemistry analysis suggests the presence of pre‐existent vessels and neoformed vessels as well as predominant young cells in the injured parenchyma of Group 2. Immunohistochemistry analysis suggests that cell therapy would promote a reconstructive response. Histology and HRCT analysis suggest a positive application of swine as a model for a bleomicine inducing of fibrotic interstitial pulmonary disease. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Microscopy analysis of bone marrow-derived osteoprogenitor cells cultured on hydrogel 3-D scaffold

Bone marrow contains progenitor cells that are able to differentiate into several mesenchymal lineages, including bone. These cells may also provide a potential therapy for bone repair. The purpose of this study was to select the osteoprogenitor cell subpopulation from bone marrow-derived mesenchymal stem cells (MSCs) and to test the ability of a hydrogel scaffold to support growth and osteogenic differentiation. MSCs isolated from rat femur bone marrow were cultured in DMEM medium supplemented with antibiotics, FCS, and L-glutamine. Osteogenic supplements (dexamethasone, sodium beta-glycerophosphate, and ascorbic acid) were added for one, two or three weeks. A selective subpopulation of osteoprogenitor cells was identified by immunohistochemistry, general morphology, scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Committed osteogenic cells were transferred to a 3-D hydrogel scaffold and cultured for an additional week. In standard culture, the osteoprogenitor cells formed cell clusters identified by Alizarin red S staining and by positive osteocalcin immunostaining. The number of osteoprogenitor cells, matrix synthesis, and mineralization increased gradually up to three weeks in culture. Mineral deposition in the matrix analyzed by EDS revealed the presence of calcium and phosphate ions at a Ca/P molar ratio of 1.73 in both the osteogenic cultures and the scaffold osteoprogenitor culture. Histological preparations revealed cell clusters within the hydrogel scaffold and SEM analysis revealed cell clusters attached to the scaffold surface. It is concluded that the hydrogel scaffold can support growth and differentiation of osteogenic cultures including mineralization and can potentially serve as a bone graft substitute containing committed osteoprogenitor cells.     

Hematopoietic progenitor constituents and adherent cell progenitor morphology isolated from black-rumped agouti (Dasyprocta prymnolopha, Wagler 1831) bone marrow

Stem cells are present in the adult tissues of most diverse species. Bone marrow is recognized to be the most exploited site to obtain stem cells and cell progenitors. The objective of the present study was to characterize hematopoietic progenitor (HP) morphology and analyze the performance of adherent cell progenitors (ACPs) cultivated in vitro from black‐rumped agouti bone marrow (Dasyprocta prymnolopha). Bone marrow aspirates were obtained from tibia crest and used to prepare histological slides and identify cell morphology. Cells were also scattered on culture plates for later isolation, expansion, and quantification. Smears obtained from bone marrow demonstrated HPs at different stages of maturity. In culture, these cells showed fibroblastoid morphology and a strong tendency to form colonies, demonstrated by the presence of cell aggregates, cytoplasmic elongations lying side by side. An 80% cell confluence was observed at 18 days in culture and progressive reduction in the percentage of nonadherent mononuclear cells. After eight passes, a mean cell viability of 96.07% was observed, from a pool of 1.6 × 10⁷ cells (ACP). Thirteen 25‐cm² culture bottles were trypsinized, resuspended in freezing medium, stored in 14 criotubes at a concentration of 1 × 10⁶ cells per milliliter, and placed in liquid nitrogen at ?196°C. Agouti bone marrow demonstrated high plasticity, moreover different HP lines, and a population of adherent cells demonstrated morphology similar to mesenchymal stem cells in culture. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Influence of the microenvironment on gene and protein expression of odontogenic-like and osteogenic-like cells

Progenitor cells play an important biological role in tooth and bone formation, and previous analyses during bone and dentine induction have indicated that they may be a good alternative for tissue engineering. Thus, to clarify the influence of the microenvironment on protein and gene expression, MDPC23 cells (mouse dental papilla cell line) and KUSA/A1 cells (bone marrow stromal cell line) were used, both in vitro cell culture and in intra-abdominal diffusion chambers implanted in 4-week-old male immunodefficient mice (SCID mice). Our results indicate that KUSA/A1 cells differentiated into osteoblast-like cells and induced bone tissue inside the chamber, whereas, MDPC-23 showed odontoblast-like characteristics but with a low ability to induce dentin formation. This study shows that MDPC-23 cells are especial cells, which possess morphological and functional characteristics of odontoblast-like cells expressing dentin sialophosphoprotein in vivo. In contrast, dentin sialophosphoprotein gene and protein expression was not detected in both cell lines in vitro. The intra-abdominal diffusion chamber appears as an interesting experimental model for studying phenotypic expression of dental pulp cells in vivo.     

Effects of filgrastim on granulopoietic cells of mice pretreated with methotrexate.

We have evaluated the effect of filgrastim on proliferation and differentiation activity of granulopoietic cells in mice pretreated with methotrexate. Filgrastim was injected daily, from day 8 to 28 after cytotoxic agent administration. The granulopoiesis changes were measured by assessment of GM-CFU cells content, marrow and spleen granuloid cells pool as well as circulating neutrophils. In MTX pretreated mice, bone marrow GM-CFU oscillating values were higher than normal values, but these changes were not followed by high proliferative activity in granuloid precursor cell compartment. After MTX treatment, filgrastim administration was unable to stimulate marrow granulopoiesis as observed in normal mice. In the spleen, MTX led to dramatic changes in the proliferative activity of GM-CFU cells, but did not result in spleen granuloid cell changes. However, filgrastim treatment induced a spleen granuloid amplification, similar to the changes observed in circulating neutrophils values. We suggest that these findings can be explained by inhibition of differentiation of marrow GM-CFU cells into the more mature granulopoietic cells and/or by an inhibited proliferative activity of marrow granuloid cells. They can be also explained in terms of an unfavorable marrow microenvironment for granulopoiesis, contrary to a supportive spleen microenvironment.     

Ultrastructural characteristics of ovine bone marrow‐derived mesenchymal stromal cells cultured with a silicon stabilized tricalcium phosphate bioceramic

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM‐MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM‐MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM‐MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM‐MSCs were isolated from the iliac crest, cultured until they reached near‐confluence and incubated with SiTCP. After 48 hr the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT‐PCR analysis. RT‐PCR displayed that oBM‐MSCs express typical surface marker for MSCs. TEM revealed the presence of electron‐lucent cells and electron‐dense cells, both expressing the CD90 surface antigen. The prominent feature of electron‐lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM‐MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM‐MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic. Skelite cultured ovine BM‐MSCs display electron‐dense and electron‐lucent cells which are differently affected by this bioceramic. This suggests that they could play a different role in bioceramic based therapy.     

Computer aided detection and classification of acute lymphoblastic leukemia cell subtypes based on microscopic image analysis

Acute lymphoblastic leukemia (ALL) is a cancer that starts from the early version of white blood cells called lymphocytes in the bone marrow. It can spread to different parts of the body rapidly and if not treated, would probably be deadly within a couple of months. Leukemia cells are categorized into three types of L1, L2, and L3. The cancer is detected through screening of blood and bone marrow smears by pathologists. But manual examination of blood samples is a time‐consuming and boring procedure as well as limited by human error risks. So to overcome these limitations a computer‐aided detection system, capable of discriminating cancer from noncancer cases and identifying the cancerous cell subtypes, seems to be necessary. In this article an automatic detection method is proposed; first cell nucleus is segmented by fuzzy c‐means clustering algorithm. Then a rich set of features including geometric, first‐ and second‐order statistical features are obtained from the nucleus. A principal component analysis is used to reduce feature matrix dimensionality. Finally, an ensemble of SVM classifiers with different kernels and parameters is applied to classify cells into four groups, that is noncancerous, L1, L2, and L3. Results show that the proposed method can be used as an assistive diagnostic tool in laboratories.     

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells.     

Automatic recognition of myeloma cells in microscopic images using bottleneck algorithm,modified watershed and SVM classifier

Plasma cells are developed from B lymphocytes, a type of white blood cells that is generated in the bone marrow. The plasma cells produce antibodies to fight with bacteria and viruses and stop infection and disease. Multiple myeloma is a cancer of plasma cells that collections of abnormal plasma cells (myeloma cells) accumulate in the bone marrow. The definitive diagnosis of multiple myeloma is done by searching for myeloma cells in the bone marrow slides through a microscope. Diagnosis of myeloma cells from bone marrow smears is a subjective and time‐consuming task for pathologists. Also, because of depending on final decision on human eye and opinion, error risk in decision may occur. Sometimes, existence of infection in body causes plasma cell's increment which could be diagnosed wrongly as multiple myeloma. The computer diagnostic process will reduce the diagnostic time and also can be worked as a second opinion for pathologists. This study presents a computer‐aided diagnostic method for myeloma cells diagnosis from bone marrow smears. At first, white blood cells consist of plasma cells and other marrow cells are separated from the red blood cells and background. Then, plasma cells are detected from other marrow cells by feature extraction and series of decision rules. Finally, normal plasma cells and myeloma cells could be classified easily by a classifier. This algorithm is applied on 50 digital images that are provided from bone marrow aspiration smears. These images contain 678 cells: 132 normal plasma cells, 256 myeloma cells and 290 other types of marrow cells. Applying the computer‐aided diagnostic method for identifying myeloma cells on provided database showed a sensitivity of 96.52%; specificity of 93.04% and precision of 95.28%.     

Using optical tweezers for measuring the interaction forces between human bone cells and implant surfaces: System design and force calibration

Optical tweezers were used to study the interaction and attachment of human bone cells to various types of medical implant materials. Ideally, the implant should facilitate cell attachment and promote migration of the progenitor cells in order to decrease the healing time. It is therefore of interest, in a controlled manner, to be able to monitor the cell adhesion process. Results from such studies would help foresee the clinical outcome of integrating medical implants. The interactions between two primary cell culture models, human gingival fibroblasts and bone forming human osteoblast cells, and three different implant materials, glass, titanium, and hydroxyapatite, were studied. A novel type of optical tweezers, which has a newly designed quadrant detector and a powerful 3 W laser was constructed and force calibrated using two different methods: one method in which the stiffness of the optical trap was obtained by monitoring the phase lag between the trap and the moved object when imposing a forced oscillation on the trapped object and another method in which the maximum trapping force was derived from the critical velocity at which the object escapes the trap. Polystyrene beads as well as cells were utilized for the calibrations. This is the first time that cells have been used directly for these types of force calibrations and, hence, direct measurements of forces exerted on cells can be performed, thus avoiding the difficulties often encountered when translating the results obtained from cell measurements to the calibrations obtained with reference materials. This more straightforward approach represents an advantage in comparison to established methods.     

Response of the donor and recipient cells in mesenchymal cell transplantation to cartilage defect

To facilitate the repair of articular cartilage defects, autologous mesenchymal cells from bone marrow or periosteum were transplanted in a rabbit model. Two weeks after the transplantation of the mesenchymal cells, the whole area of the original defect was occupied by cartilage. From the deep area of the reparative cartilage, which contacted with host bone, chondrocytes became hypertrophic and the invasion of bone with vasculature started, until the replacement reached the natural junction of the host cartilage and the subchondral bone about 4 weeks after transplantation. Twelve weeks after the transplantation, the repair cartilage in the defect became a little thinner than the adjacent normal cartilage, which became a little thinner 24 weeks after the transplantation (the longest observation period in the study). Large, full-thickness defects of the weight-bearing region of the articular cartilage were repaired with hyaline-like cartilage after implantation of autologous mesenchymal cells. The repair process by mesenchymal cell transplantation was explained as follows: The donor transplanted cell differentiated into cartilage and the defects were completely filled with cartilage. Then, mesenchymal cells that entered the chondrogenic lineage rapidly progressed through this lineage to the hypertrophic state, which was then the target for erosion and vascular invasion. Although this vasculature and the newly formed bone were considered to be host-derived, there was no evidence to that effect. To prove this, suitable experimental marking of these donor cells is needed. In the case of chondrocyte transplantation, the repair cartilage maintained its thickness to the full depth of the original defect; the tissue derived from the implanted chondrocytes was not invaded by vessels or replaced by subchondral bone.     

Single-cell RNA sequencing reveals classical monocytes are the major precursors of rat osteoclasts

To dissect which subset of bone marrow monocyte is the major precursor of osteoclast, 3-month-old rat bone marrow was obtained for single-cell RNA sequencing. A total of 6091 cells were acquired for detailed analysis, with a median number of 1206 genes detected per cell and 17,959 genes detected in total. A total of 19 cell clusters were recognized, with the main lineages identified as B cells, Granulocytes, Monocytes, T cells, Erythrocytes and Macrophages. Monocytes were further divided into classical monocytes and non-classical monocytes. Compared with non-classical monocytes, classical monocytes highly expressed osteoclast differentiation related genes Mitf, Spi1, Fos and Csf1r. Additionally, biological processes of classical monocytes were related to osteoclast differentiation. qPCR revealed differentially expressed genes of classical monocytes played a role in osteoclast differentiation. In conclusion, classical monocytes were identified as the main precursors of osteoclasts in rats, and may contribute to osteoclast differentiation by regulating S100a4, S100a6, S100a10, Fn1, Vcan and Bcl2a1. The results of this study contribute to the understanding of the origin of osteoclasts and may provide potential biomarkers for early diagnosis of bone metabolic diseases, as well as molecular and cellular targets for clinical intervention in bone metabolic diseases.     

Nanoscale distribution of CD20 on B‐cell lymphoma tumour cells and its potential role in the clinical efficacy of rituximab

Rituximab is an exciting monoclonal antibody drug approved for treating B‐cell lymphomas and its target is the CD20 antigen which is expressed on the surface of B cells. In recent years, the variable efficacies of rituximab among different lymphoma patients have become an important clinical issue and urgently need to be solved for further development of antibodies with enhanced efficacies. In this work, atomic force microscopy (AFM) was used to investigate the nanoscale distribution of CD20 on the surface of tumour B cells from lymphoma patients to examine its potential role in the clinical therapeutic effects of rituximab. By performing ROR1 fluorescence labelling (ROR1 is a specific tumour cell surface marker) on the bone marrow cells prepared from B‐cell lymphoma patients, the tumour B cells were recognized, and then AFM tips carrying rituximabs via polyethylene glycol crosslinkers were moved to the tumour cells to probe the specific CD20‐rituximab interactions. By applying AFM single‐molecule force spectroscopy (SMFS) at the local areas (500×500 nm²) on the surface of tumour B cells, the nanoscale distributions of CD20 on the surface of tumour B cells were mapped, visually showing that CD20 distributed heterogeneously on the cell surface. Bone marrow cell samples from three clinical B‐cell lymphoma cases were collected to analyze the binding affinity and nanoscale distribution of CD20 on tumour cells. The experimental results showed that CD20 distribution on tumour cells were to some extent related to the clinical therapeutic outcomes while the CD20‐rituximab binding forces did not have distinct effects to the clinical outcomes. These results can provide novel insights in understanding the rituximab's clinical efficacies from the nanoscale distribution of CD20 on the tumour cells at single‐cell and single‐molecule levels.     

Immuno- and enzyme-histo/cytochemical analysis of resin sections and cell suspensions: a comprehensive diagnosis of bone marrow on a single aspiration sample

A protocol is described in which a single bone marrow aspiration specimen is used to prepare resin sections and cell suspensions. Using this protocol, a full battery of morphological, enzyme-histochemical, immuno-histochemical and cyto-pathological techniques can be applied. This allows a definitive bone marrow diagnosis to be established without resorting to bone marrow biopsy in the majority of patients.     

Behavior of mesenchymal stem cells stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures

4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is a DNA dye widely used to mark and trace stem cells in therapy. We here studied the effect of DAPI staining on the behavior of mesenchymal stem cells cultured in either a control, non-osteogenic medium or in an osteogenic differentiation medium. In the control medium, the number of stem cells/field, as well as the number of fluorescent cells/field increased up to the sixth day in both control and DAPI-treated cultures. Afterwards, both the number of fluorescent cells and their fluorescence intensity decreased. Control cells were fusiform and with some long extensions that apparently linked them to neighboring cells, while DAPI-treated cells were mostly round cells with fine and short extensions. The trypan-blue exclusion method showed 99% cell viability in both groups, however, both alkaline phosphatase activity and the thiazolyl blue formazan assay (indicative of mitochondrial metabolism) gave significantly lower values in DAPI-marked cells. The mitochondrial mass, as indicated by specific staining and flow cytometry, showed no differences between groups. Mesenchymal stem cells gave origin to mineralized nodules in the osteogenic differentiation medium and there were not DAPI-marked cells on the ninth day of culture. Alkaline phosphatase activity, viability assay and number of cells/field and of mineralized nodules/field were similar in both groups. So, DAPI treatment did not change cell viability and proliferation during osteogenic differentiation of mesenchymal stem cells. However, since these cells loose DAPI marking after 9 days in osteogenic cultures suggests that DAPI may not be an effective marker for mesenchymal stem cells implanted in bone tissue for long periods.