scholarly journals Osteogenic preconditioning in perfusion bioreactors improves vascularization and bone formation by human bone marrow aspirates

2020 ◽  
Vol 6 (7) ◽  
pp. eaay2387 ◽  
Author(s):  
J. N. Harvestine ◽  
T. Gonzalez-Fernandez ◽  
A. Sebastian ◽  
N. R. Hum ◽  
D. C. Genetos ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Stromal Cells ◽  
Bone Marrow Aspirate ◽  
De Novo ◽  
Trophic Factor ◽  
Calvarial Defect ◽  
Factor Secretion

Cell-derived extracellular matrix (ECM) provides a niche to promote osteogenic differentiation, cell adhesion, survival, and trophic factor secretion. To determine whether osteogenic preconditioning would improve the bone-forming potential of unfractionated bone marrow aspirate (BMA), we perfused cells on ECM-coated scaffolds to generate naïve and preconditioned constructs, respectively. The composition of cells selected from BMA was distinct on each scaffold. Naïve constructs exhibited robust proangiogenic potential in vitro, while preconditioned scaffolds contained more mesenchymal stem/stromal cells (MSCs) and endothelial cells (ECs) and exhibited an osteogenic phenotype. Upon implantation into an orthotopic calvarial defect, BMA-derived ECs were present in vessels in preconditioned implants, resulting in robust perfusion and greater vessel density over the first 14 days compared to naïve implants. After 10 weeks, human ECs and differentiated MSCs were detected in de novo tissues derived from naïve and preconditioned scaffolds. These results demonstrate that bioreactor-based preconditioning augments the bone-forming potential of BMA.

scholarly journals Stromal cells in myeloid and lymphoid long-term bone marrow cultures can support multiple hemopoietic lineages and modulate their production of hemopoietic growth factors

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
Author(s):  
A Johnson ◽  
K Dorshkind
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Culture Conditions ◽  
B Lymphopoiesis ◽  
Bone Marrow Cultures ◽  
Factor Secretion ◽  
Marrow Cultures

Abstract Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.

scholarly journals Stromal cells in myeloid and lymphoid long-term bone marrow cultures can support multiple hemopoietic lineages and modulate their production of hemopoietic growth factors

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
Author(s):  
A Johnson ◽  
K Dorshkind
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Culture Conditions ◽  
B Lymphopoiesis ◽  
Bone Marrow Cultures ◽  
Factor Secretion ◽  
Marrow Cultures

Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.

Development Of 3D Models For Studying Megakaryopoiesis In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3696-3696
Author(s):  
Lindsay Wray ◽  
Christian A Di Buduo ◽  
David L. Kaplan ◽  
Alessandra Balduini
Keyword(s):  
Mechanical Properties ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Peripheral Blood ◽  
Platelet Production ◽  
Vascular Niche ◽  
Proplatelet Formation ◽  
Bone Marrow Vascular Niche

Abstract Introduction Silk fibroin, derived from Bombyx mori silkworm cocoons, is used extensively in biomaterials and regenerative medicine. The useful characteristics of this protein include self-assembly, robust mechanical properties, biocompatibility and biodegradability. Moreover, silk can be enhanced through a variety of chemical modifications that affect cell attachment, growth and differentiation. Thrombocytopenia occurs when a patient suffers from an abnormally low platelet count in the peripheral blood; usually a result of disease, trauma, or cancer treatment. To treat these patients, it is estimated that two million platelet transfusions are performed in the U.S. each year. This high demand for platelets has created a clinical demand for studying the causes of thrombocytopenia and alternative routes for treatment. Platelets are anuclear cells that are released into the bloodstream in the bone marrow by megakaryocytes via the extension of long filaments called proplatelets. It is hypothesized that platelet production from megakaryocytes is regulated by environmental factors at the site of bone marrow vascular niche. Studies of megakaryopoiesis are typically performed on extracellular matrix protein-coated culture plates and transwell membranes. While these initial studies have provided invaluable insight into the process of megakaryopoiesis, the goal of the present project was to create a bone marrow model that mimics the vascular niche for functional in vitro platelet production. We hypothesized that a silk-based in vitro tissue model would allow the effects of substrate surface properties and endothelial co-culture on megakaryopoiesis to be studied in a holistic manner, thereby enabling further elucidation of the mechanisms involved in the process of platelet production. Results In order to more closely mimic the bone marrow vascular niche structure, a porous silk sponge was assembled around the silk vessel-like tubes. Megakarycytes seeded in the porous silk sponge migrated toward the silk tube and released platelets into the tube lumen. The perfusion bioreactor moved the platelets into the platelet collecting bags. After perfusion the platelets were collected and analyzed by flow cytometry. The bioreactor platelets exhibited similar morphology, CD41 positive staining, and activation compared to peripheral blood platelet controls. Megakaryocyte attachment and proplatelet formation through the silk vascular wall were improved by altering the silk properties. Silk functionalized by entrapping extracellular matrix proteins within the tube membrane resulted in increased megakaryocyte attachment and proplatelet compared to unfunctionalized silk tube controls. Silk surface roughness improved megakaryocyte attachment compared to the control but did not affect proplatelets. Decreasing the silk stiffness improved proplatelets, but did not significantly affect megakaryocyte attachment. Co-culture with endothelial cells improved megakaryocyte attachment while maintaining a high level of proplatelet formation. Additionally, megakaryocyte and endothelial cell co-culture on the silk vessel model resulted in an icreased platelet production compared to megakaryocytes cultured alone. Conclusions The goal of this project was to develop an in vitro model of megakaryopoiesis using a tissue engineering approach. Using human megakaryocytes and endothelial cells, we demonstrate the following advanced features of the silk-based model: (1) immobilization of extracellular matrix components within the membrane, (2) tunable surface topography, (3) tunable mechanical properties, (4) physiologically relevant thickness for appropriate proplatelet extension, and (5) controlled localization of a vascular endothelium. Thus, by functionalizing silk, we can control megakaryocyte function on silk. The broader impact of this work offers a versatile new tool for studying megakaryocyte development and platelet production in vitro. Disclosures: No relevant conflicts of interest to declare.

Natural Scaffold, from Bovine Bone Marrow, Reproduces Native Microenvironment and Supports CD34+ and Stromal Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2400-2400
Author(s):  
Renata Giardini Rosa ◽  
Juares E. Romero Bianco ◽  
Gabriela Pereira dos Santos ◽  
Stephen D. Waldman ◽  
Joanna Weber ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Tissue Regeneration ◽  
Stromal Cells ◽  
Bovine Bone ◽  
Ecm Proteins ◽  
Histological Staining

Abstract Background: The idea of studying bone marrow outside its native environment is attractive and ideal. Due to the many functions of extracellular matrix (ECM), currently there is an interest in creating an environment that mimics the ECM present in the tissue, similar to the microenvironment in vivo. Molds replacing the ECM (scaffolds) have a porous structure and may assist the tissue regeneration by forming a suitable environment for adhesion, migration, proliferation and cellular differentiation. The appropriate ECM is a key factor as ECM proteins are site-specific and provide protein 'footprints' of previous resident cells. Because ECM proteins are among the most conserved proteins, the removal of xenogenic/allogenic cellular contents via decellularization could theoretically produce an essentially minimally immunogenic scaffold with a native intact structure for new tissue regeneration. Thus, the search for a scaffold that could be used to assess the behavior of cells and their interactions with the ECM in vitro/in vivo, and has different niches in its composition is highly desirable. Aims: In recent years, a large number of molecular and cytogenetic abnormalities have been identified in AML, MDS and multiple myeloma, many of these defects can serve as markers for diagnosis/prognosis or as therapeutic targets. However, there are still many unknown molecular factors involved in genetic abnormalities or signaling pathways that contribute to the pathogenesis of the disease. Another very important aspect of these diseases is that they all are related to the mutual interaction of neoplastic cells and the microenvironment of bone marrow. In the absence of an ideal model or even the difficulty in reproduce a native environment, we proposed the characterization of a natural scaffold, from bovine bone marrow, which can be used as a study model, previously patented by our laboratory. Materials and Methods: Bone marrow was decellularized by one or more incubations in an enzymatic digestion solution and polar solvent extractions, comprising an extracellular matrix with well-preserved 3D structure. Scaffolds were analyzed after the decelularization process for potential changes in structure (TEM, SEM, Histological staining, and immunohistochemistry for collagen III, IV, fibronectin) and mechanical properties. To verify if the scaffold would hold and support cell survival and extracellular matrix production, an in vitro study was performed using CD34+ (non-stromal) and HS-5 (stromal) cells. Cell-seeded decellularized scaffolds were cultured for 7-14 days and analyzed for Histological staining. Results: Histology sections (H&E staining), TEM and SEM demonstrated the structure and ultrastructure of the processed matrix and confirmed both cellular extraction and preservation of the macroscopic 3-D architecture of the collagen fibers, blood vessels, and preservation of an organized matrix. Also, the decellularized scaffold was quite comparable to the native tissue in terms of its mechanical properties. Immunohistochemistry of the scaffold showed that the main components of the ECM were preserved. The in vitro experiments of both stromal cells (HS-5) and non-stromal cells (CD34+) demonstrated that they were able to adhere and in the HS-5 case also produce ECM during 7-14 days of culture. In both cases, an increase in cell number was observed and CD34+ overtime formed cluster and with 14 days of culture the cluster formation increased in size. Conclusions: The results demonstrated that the decellularization process was efficient in keeping a 3-D structure and mechanical properties with a well-organized-preserved ECM. In vitro experiments showed that both CD34+ and HS-5 were able to proliferate and adhere in specific sites of the scaffold, suggesting that they were able to recognize their native environment. HS-5 produced ECM indicating that the scaffold worked as an optimal microenvironment. In conclusion, the scaffold could be used as a model, which has the potential to mimic the native microenvironment to enable research/studies of factors that are involved in self-renewal and maintenance of neoplastic cells in bone marrow. Also, this model could be very useful for pharmacological testing of bone marrow in vitro. Disclosures No relevant conflicts of interest to declare.

Adult Human Bone Marrow– and Adipose Tissue–Derived Stromal Cells Support the Formation of Prevascular-like Structures from Endothelial Cells In Vitro

2010 ◽  
Vol 16 (1) ◽  
pp. 101-114 ◽  
Author(s):  
Femke Verseijden ◽  
Sandra J. Posthumus-van Sluijs ◽  
Predrag Pavljasevic ◽  
Stefan O.P. Hofer ◽  
Gerjo J.V.M van Osch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Adipose Tissue ◽  
Stromal Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Adult Human

scholarly journals Perturbation of human endothelial cells by thrombin or PMA changes the reactivity of their extracellular matrix towards platelets.

1987 ◽  
Vol 104 (3) ◽  
pp. 697-704 ◽  
Author(s):  
P G de Groot ◽  
J H Reinders ◽  
J J Sixma
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
De Novo ◽  
The Matrix ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets ◽  
De Novo Protein Synthesis

In this study we have examined the influence of perturbation of endothelial cells on the amounts of fibronectin and von Willebrand factor in their extracellular matrix and the consequences of a changed composition of the matrix on platelet adhesion. For this purpose, we have used an in vitro perfusion system with which we can investigate the interactions of platelets in flowing blood with cultured endothelial cells and their extracellular matrix (Sakariassen, K. S., P. A. M. M. Aarts, P. G. de Groot, W. P. M. Houdgk, and J. J. Sixma, 1983, J. Lab. Clin Med. 102:522-535). Treatment of endothelial cells with 0.1-1.0 U/ml thrombin for 2 h increased the reactivity of the extracellular matrix, isolated after the thrombin treatment, towards platelets by approximately 50%. The increased reactivity did not depend on de novo protein synthesis but was inhibited by 3-deazaadenosine, an inhibitor of phospholipid methylation, which also inhibits the stimulus-induced instantaneous release of von Willebrand factor from endothelial cells. However, no changes in the amounts of von Willebrand factor and fibronectin in the matrix were detected. Thrombin may change the organization of the matrix proteins, not the composition. When endothelial cells were perturbed with the phorbol ester PMA or thrombin for 3 d, the adhesion of platelets to the extracellular matrix of treated cells was strongly impaired. This impairment coincided with a decrease in the amounts of von Willebrand factor and fibronectin present in the matrix. These results indicate that, after perturbation, endothelial cells regulate the composition of their matrix, and that this regulation has consequences for the adhesion of platelets.

Sustained Alterations In Bone Marrow Stromal Cells From Patients With Myeloproliferative Neoplasms (MPN) Contribute To Remodelling Of The Bone Marrow Microenvironment Prior To The Manifestation Of Myelofibrosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4102-4102 ◽  
Author(s):  
Rebekka K. Schneider ◽  
Isabelle Leisten ◽  
Susanne Ziegler ◽  
Anne Schumacher ◽  
Björn Rath ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Board Of Directors ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Myeloproliferative Neoplasms ◽  
Bone Marrow Microenvironment ◽  
Marrow Stromal Cells ◽  
Advisory Committees

Abstract Myeloproliferative neoplasms (MPN) are characterized by the loss of normal hematopoiesis and the excessive production and accumulation of non-lymphoid cells and platelets in the BM. Clonal hematopoiesis in MPN is assumed to generate factors which induce profound changes in the non-clonal BM microenvironment. Alterations include massive deposition of extracellular matrix proteins (ECM), progression to bone marrow (BM)-fibrosis and osteosclerosis. We hypothesize that particularly in MPN, alterations in the cross talk between hematopoietic and bone marrow stromal cells (BMSC) play a critical role for an impaired bone marrow microenvironment long before overt signs of myelofibrosis can be detected by conventional methods. To dissect the hematopoiesis supporting capacity and extracellular matrix remodelling of BMSC from patients with MPN, we isolated BMSC from BM of patients with essential thrombocytemia (ET, n=5), polycythemia vera (PV, n=5), chronic myeloid leukemia (CML, n=5) and control BM (n=6). BMSC isolates were taken only from pre-fibrotic MPN patients (bone marrow trephine biopsy reticulin staining graded 0 or 1) and the resulting expansion cultures fullfilled MSC criteria according to the common consensus (Dominici et al., Cytotherapy, 2006; 8(4):315-317). When subjected to myeloid colony forming unit assays, MPN-BMSC conditioned supernatants showed a significantly reduced capacity to stimulate a CFU-GM/G/M growth of non-malignant hematopoietic stem and progenitor cells as compared to control BMSC (control (n=6) vs CML (n=5): p= 0.0032; control vs PV (n=5): p=0.016, control vs ET (n=4) not significant; student«s T- test ). BMSC-dependent matrix remodelling was analysed in a previously established robust matrix remodelling assay in vitro. MPN-BMSC displayed a pronounced increased matrix remodelling capacity compared to control BMSC. Interestingly, among the different MPN subtypes, this effect was highly significant in BMSC derived from patients with ET (Control (n=6) vs. ET (n=5): p<0.001). Furthermore, in vitro ECM production by MPN-BMSC was paralleled by ECM changes observed in matched bone marrow punches as shown by fibronectin immunohistochemistry. Co-expression of the stroma marker CD271 and fibronectin -as shown by confocal microscopy- points towards stroma-mediated ECM production in vivo. As upregulation of fibronectin expression was also detected in reticulin 0 graded BM punches, we hypothesized that fibronectin staining might be a potential marker for pre-fibrotic ECM changes in –so far- reticulin-negative MPN biopsies. To validate this hypothesis, we stained fibronectin in a tissue microarray (TMA), containing primary BM biopsies from patients with ET (n=14), PV (n=14), CML (n=14), MF (n=11) and controls (Non-Hodgkin's lymphoma without bone marrow involvement, n=17). Interestingly, within the reticulin-negative subcohort of pre-fibrotic MPN (n=34), fibronectin stained positive (grade 1 or higher) in 5/7 cases with ET (71%), 6/9 cases with PV (66%) and in 14/14 cases with CML (100%) as well as in all cases with PMF (100%). Furthermore, fibronectin staining correlated significantly with patients' decreased haemoglobin levels as shown by ANOVA analysis of routine clinical parameters (F=5.71; Prob> F 0.0037). We conclude that prior to the manifestation of fibrosis in MPN, BMSC loose their capacity to support non-malignant hematopoiesis and increase their matrix remodelling potential. As to our surprise, these effects are stably maintained in the absence of the malignant hematopoietic clone and since BMSC cultures develop over numerous population doublings in vitro from proliferating mesenchymal precursors, we hypothesize that cell intrinsic effects in BMSC from patients with MPN contribute to the development of myelofibrosis in MPN.Figure 1reticulin-negative subcohort of pre-fibrotic MPNFigure 1. reticulin-negative subcohort of pre-fibrotic MPN Disclosures: Bruemmendorf: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria.

Biomimetic titanium implant coated with extracellular matrix enhances and accelerates osteogenesis

Nanomedicine ◽  
2020 ◽  
Vol 15 (18) ◽  
pp. 1779-1793
Author(s):  
Yu Wu ◽  
Haikuo Tang ◽  
Lin Liu ◽  
Qianting He ◽  
Luodan Zhao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Stromal Cells ◽  
Titanium Surface ◽  
Titanium Implant ◽  
Titanium Implants ◽  
In Vivo Studies ◽  
Calcium Deposition

Aim: To evaluate the biological function of titanium implants coated with cell-derived mineralized extracellular matrix, which mimics a bony microenvironment. Materials & methods: A biomimetic titanium implant was fabricated primarily by modifying the titanium surface with TiO2 nanotubes or sand-blasted, acid-etched topography, then was coated with mineralized extracellular matrix constructed by culturing bone marrow mesenchymal stromal cells. The osteogenic ability of biomimetic titanium surface in vitro and in vivo were evaluated. Results: In vitro and in vivo studies revealed that the biomimetic titanium implant enhanced and accelerated osteogenesis of bone marrow stromal cells by increasing cell proliferation and calcium deposition. Conclusion: By combining surface topography modification with biological coating, the results provided a valuable method to produce biomimetic titanium implants with excellent osteogenic ability.

scholarly journals Neutrophils Inhibit Synthesis of Mineralized Extracellular Matrix by Human Bone Marrow-Derived Stromal Cells In Vitro

2018 ◽  
Vol 9 ◽  
Author(s):  
Okan W. Bastian ◽  
Michiel Croes ◽  
Jacqueline Alblas ◽  
Leo Koenderman ◽  
Luke P. H. Leenen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Stromal Cells ◽  
Human Bone ◽  
Human Bone Marrow
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