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Compression Bioreactor-Based Mechanical Loading Induces Mobilization of Human Bone Marrow-Derived Mesenchymal Stromal Cells into Collagen Scaffolds In Vitro.

ABSTRACT: Articular cartilage (AC) is an avascular tissue composed of scattered chondrocytes embedded in a dense extracellular matrix, in which nourishment takes place via the synovial fluid at the surface. AC has a limited intrinsic healing capacity, and thus mainly surgical techniques have been used to relieve pain and improve function. Approaches to promote regeneration remain challenging. The microfracture (MF) approach targets the bone marrow (BM) as a source of factors and progenitor cells to heal chondral defects in situ by opening small holes in the subchondral bone. However, the original function of AC is not obtained yet. We hypothesize that mechanical stimulation can mobilize mesenchymal stromal cells (MSCs) from BM reservoirs upon MF of the subchondral bone. Thus, the aim of this study was to compare the counts of mobilized human BM-MSCs (hBM-MSCs) in alginate-laminin (alginate-Ln) or collagen-I (col-I) scaffolds upon intermittent mechanical loading. The mechanical set up within an established bioreactor consisted of 10% strain, 0.3 Hz, breaks of 10 s every 180 cycles for 24 h. Contrary to previous findings using porcine MSCs, no significant cell count was found for hBM-MSCs into alginate-Ln scaffolds upon mechanical stimulation (8 ± 5 viable cells/mm3 for loaded and 4 ± 2 viable cells/mm3 for unloaded alginate-Ln scaffolds). However, intermittent mechanical stimulation induced the mobilization of hBM-MSCs into col-I scaffolds 10-fold compared to the unloaded col-I controls (245 ± 42 viable cells/mm3 vs. 22 ± 6 viable cells/mm3, respectively; p-value < 0.0001). Cells that mobilized into the scaffolds by mechanical loading did not show morphological changes. This study confirmed that hBM-MSCs can be mobilized in vitro from a reservoir toward col-I but not alginate-Ln scaffolds upon intermittent mechanical loading, against gravity.

SUBMITTER: Gamez C

PROVIDER: S-EPMC7672606 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Compression Bioreactor-Based Mechanical Loading Induces Mobilization of Human Bone Marrow-Derived Mesenchymal Stromal Cells into Collagen Scaffolds In Vitro.

Gamez Carolina C Schneider-Wald Barbara B Bieback Karen K Schuette Andy A Büttner Sylvia S Hafner Mathias M Gretz Norbert N Schwarz Markus L ML

International journal of molecular sciences 20201104 21

Articular cartilage (AC) is an avascular tissue composed of scattered chondrocytes embedded in a dense extracellular matrix, in which nourishment takes place via the synovial fluid at the surface. AC has a limited intrinsic healing capacity, and thus mainly surgical techniques have been used to relieve pain and improve function. Approaches to promote regeneration remain challenging. The microfracture (MF) approach targets the bone marrow (BM) as a source of factors and progenitor cells to heal c ...[more]

PMID: 33158020

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Project description:INTRODUCTION:Articular cartilage (AC) is a viscoelastic tissue with a limited regenerative capability because of the lack of vasculature. Mechanical stimulation contributes to the homeostasis of functional AC since it promotes the delivery of nutrients, cytokines and growth factors between the distant chondrocytes. We hypothesized that biomechanical stimulation might enhance mobilization of endogenous mesenchymal stem/stromal cells (MSCs) from neighboring niches as the bone marrow. AIM:This study aimed to introduce a bioreactor for inducing mobilization of MSCs from one compartment to another above by mechanical stimulation in vitro. METHODS:A novel mechanical system for evaluating mobilization of cells in a 3D context in vitro is presented. The system consists of a compression bioreactor able to induce loading on hydrogel-based scaffolds, custom-made software for settings management and data recording, and image based biological evaluation. Intermittent load was applied under a periodic regime with frequency of 0.3 Hz and unload phases of 10 seconds each 180 cycles over 24 hours. The mechanical stimulation acted on an alginate scaffold and a cell reservoir containing MSCs below it. The dynamic compression exerted amplitude of 200 ?m as 10% strain regarding the original height of the scaffold. RESULTS:The bioreactor was able to stimulate the scaffolds and the cells for 24.4 (±1.7) hours, exerting compression with vertical displacements of 185.8 (±17.8) ?m and a force-amplitude of 1.87 (±1.37; min 0.31, max 4.42) N. Our results suggest that continuous mechanical stimulation hampered the viability of the cells located at the cell reservoir when comparing to intermittent mechanical stimulation (34.4 ± 2.0% vs. 66.8 ± 5.9%, respectively). Functionalizing alginate scaffolds with laminin-521 (LN521) seemed to enhance the mobilization of cells from 48 (±21) to 194 (±39) cells/mm3 after applying intermittent mechanical loading. CONCLUSION:The bioreactor presented here was able to provide mechanical stimulation that seemed to induce the mobilization of MSCs into LN521-alginate scaffolds under an intermittent loading regime.

Project description:We developed a new method to manufacture dense, aligned, and porous collagen scaffolds using biaxial plastic compression of type I collagen gels. Using a novel compression apparatus that constricts like an iris diaphragm, low density collagen gels were compressed to yield a permanently densified, highly aligned collagen material. Micro-porosity scaffolds were created using hydrophilic elastomer porogens that can be selectively removed following biaxial compression, with porosity modulated by using different porogen concentrations. The resulting scaffolds exhibit collagen densities that are similar to native connective tissues (?10% collagen by weight), pronounced collagen alignment across multiple length scales, and an interconnected network of pores, making them highly relevant for use in tissue culture, the study of physiologically relevant cell-matrix interactions, and tissue engineering applications. The scaffolds exhibited highly anisotropic material behavior, with the modulus of the scaffolds in the fiber direction over 100 times greater than the modulus in the transverse direction. Adipose-derived mesenchymal stem cells were seeded onto the biaxially compressed scaffolds with minimal cell death over seven days of culture, along with cell proliferation and migration into the pore spaces. This fabrication method provides new capabilities to manufacture structurally and mechanically relevant cytocompatible scaffolds that will enable more physiologically relevant cell culture studies. Further improvement of manufacturing techniques has the potential to produce engineered scaffolds for direct replacement of dense connective tissues such as meniscus and annulus fibrosus. STATEMENT OF SIGNIFICANCE:In vitro studies of cell-matrix interactions and the engineering of replacement materials for collagenous connective tissues require biocompatible scaffolds that replicate the high collagen density (15-25%/wt), aligned fibrillar organization, and anisotropic mechanical properties of native tissues. However, methods for creating scaffolds with these characteristics are currently lacking. We developed a new apparatus and method to create high density, aligned, and porous collagen scaffolds using a biaxial compression with porogens technique. These scaffolds have a highly directional structure and mechanical properties, with the tensile strength and modulus up to 100 times greater in the direction of alignment. We also demonstrated that the scaffolds are a suitable material for cell culture, promoting cell adhesion, viability, and an aligned cell morphology comparable to the cell morphology observed in native aligned tissues.

Project description:Osteoinduction and proliferation of bone-marrow stromal cells (BMSCs) in three-dimensional (3D) poly(?-caprolactone) (PCL) scaffolds have not been studied throughly and are technically challenging. This study aimed to optimize nanocomposites of 3D PCL scaffolds to provide superior adhesion, proliferation and differentiation environment for BMSCs in this scenario.BMSCs were isolated and cultured in a novel 3D tissue culture poly(?-caprolactone) (PCL) scaffold coated with poly-lysine, hydroxyapatite (HAp), collagen and HAp/collagen. Cell morphology was observed and BMSC biomarkers for osteogenesis, osteoblast differentiation and activation were analyzed.Scanning Electron Microscope (SEM) micrographs showed that coating materials were uniformly deposited on the surface of PCL scaffolds and BMSCs grew and aggregated to form clusters during 3D culture. Both mRNA and protein levels of the key players of osteogenesis and osteoblast differentiation and activation, including runt-related transcription factor 2 (Runx2), alkaline phosphates (ALP), osterix, osteocalcin, and RANKL, were significantly higher in BMSCs seeded in PCL scaffolds coated with HAp or HAp/collagen than those seeded in uncoated PCL scaffolds, whereas the expression levels were not significantly different in collagen or poly-lysine coated PCL scaffolds. In addition, poly-lysine, collagen, HAp/collagen, and HAp coated PCL scaffolds had significantly more viable cells than uncoated PCL scaffolds, especially scaffolds with HAp/collagen and collagen-alone coatings. That BMSCs in HAp or HAp/collagen PCL scaffolds had remarkably higher ALP activities than those in collagen-coated alone or uncoated PCL scaffolds indicating higher osteogenic differentiation levels of BMSCs in HAp or HAp/collagen PCL scaffolds. Moreover, morphological changes of BMSCs after four-week of 3D culture confirmed that BMSCs successfully differentiated into osteoblast with spread-out phenotype in HAp/collagen coated PCL scaffolds.This study showed a proof of concept for preparing biomimetic 3D poly (?-caprolactone)/ hydroxyapatite/collagen scaffolds with excellent osteoinduction and proliferation capacity for bone regeneration.

Project description:The quality of cartilaginous tissue derived from bone marrow mesenchymal stromal stem cell (BMSC) transplantation has been correlated with clinical outcome. Therefore, culture conditions capable of modulating tissue phenotype, such as oxygen tension and scaffold composition, are under investigation. The objective of this study was to assess the effect of hypoxia on in vitro BMSC chondrogenesis within clinically approved porous scaffolds composed of collagen and hyaluronic acid (HA). It was hypothesized that hypoxic isolation/expansion and differentiation would improve BMSC chondrogenesis in each construct.Ovine BMSCs were isolated and expanded to passage 2 under hypoxia (3% oxygen) or normoxia (21% oxygen). Cell proliferation and colony-forming characteristics were assessed. BMSCs were seeded at 10 million cells per cubic centimeter on cylindrical scaffolds composed of either collagen I sponge or esterified HA non-woven mesh. Chondrogenic differentiation was performed in a defined medium under hypoxia or normoxia for 14 days. Cultured constructs were assessed for gene expression, proteoglycan staining, glycosaminoglycan (GAG) quantity, and diameter change.Isolation/expansion under hypoxia resulted in faster BMSC population doublings per day (P <0.05), whereas cell and colony counts were not significantly different (P?=?0.60 and 0.30, respectively). Collagen and HA scaffolds seeded with BMSCs that were isolated, expanded, and differentiated under hypoxia exhibited superior aggrecan and collagen II mRNA expressions (P <0.05), GAG quantity (P <0.05), and proteoglycan staining in comparison with normoxia. GAG/DNA was augmented with hypoxic isolation/expansion in all constructs (P <0.01). Comparison by scaffold composition indicated increased mRNA expressions of hyaline cartilage-associated collagen II, aggrecan, and SOX9 in collagen scaffolds, although expression of collagen X, which is related to hypertrophic cartilage, was also elevated (P <0.05). Proteoglycan deposition was not significantly improved in collagen scaffolds unless culture involved normoxic isolation/expansion followed by hypoxic differentiation. During chondrogenesis, collagen-based constructs progressively contracted to 60.1%?±?8.9% of the initial diameter after 14 days, whereas HA-based construct size was maintained (109.7%?±?4.2%).Hypoxic isolation/expansion and differentiation enhance in vitro BMSC chondrogenesis within porous scaffolds. Although both collagen I and HA scaffolds support the creation of hyaline-like cartilaginous tissue, variations in gene expression, extracellular matrix formation, and construct size occur during chondrogenesis.

Dataset Information

Compression Bioreactor-Based Mechanical Loading Induces Mobilization of Human Bone Marrow-Derived Mesenchymal Stromal Cells into Collagen Scaffolds In Vitro.

Publications

Compression Bioreactor-Based Mechanical Loading Induces Mobilization of Human Bone Marrow-Derived Mesenchymal Stromal Cells into Collagen Scaffolds In Vitro.

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