scholarly journals Effects of chondrogenic priming duration on mechanoregulation of engineered cartilage anlagen

2020 ◽  
Author(s):  
Anna M. McDermott ◽  
Emily A. Eastburn ◽  
Daniel J. Kelly ◽  
Joel D. Boerckel
Keyword(s):  
Dynamic Loading ◽  
Mechanical Loading ◽  
Endochondral Ossification ◽  
Bone Development ◽  
Mechanical Compression ◽  
Dynamic Mechanical ◽  
Matrix Deposition ◽  
Static Conditions

AbstractBone development and repair occur by endochondral ossification of a cartilage anlage, or template. Endochondral ossification is regulated by mechanical cues. Recently, we found that in vivo mechanical loading promoted regeneration of large bone defects through endochondral ossification, in a manner dependent on the timing of load initiation. Here, we have developed an in vitro model of the cartilage anlage to test whether the chondrogenic differentiation state alters the response to dynamic mechanical compression. We cultured human bone marrow stromal cells (hMSCs) at high cell density in fibrin hydrogels under chondrogenic priming conditions for periods of 0, 2, 4, or 6 weeks prior to two weeks of dynamic mechanical loading. Samples were evaluated by biomechanical testing, biochemical analysis of collagen and glycosaminoglycan (GAG) deposition, gene expression analysis, and immunohistological analysis, in comparison to time-matched controls cultured under static conditions. We found that dynamic loading increased the mechanical stiffness of engineered anlagen in a manner dependent on the duration of chondrogenic priming prior to load initiation. For chondrogenic priming times of 2 weeks or greater, dynamic loading enhanced the expression of type II collagen and aggrecan, although no significant changes in overall levels of matrix deposition was observed. For priming periods less than 4 weeks, dynamic loading generally supressed markers of hypertrophy and osteogenesis, although this was not observed if the priming period was extended to 6 weeks, where loading instead enhanced the expression of type X collagen. Taken together, these data demonstrate that the duration of chondrogenic priming regulates the endochondral response to dynamic mechanical compression in vitro, which may contribute to the effects of mechanical loading on endochondral bone development, repair, and regeneration in vivo.

scholarly journals Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair

2019 ◽  
Author(s):  
S. Herberg ◽  
A. M. McDermott ◽  
P. N. Dang ◽  
D. S. Alt ◽  
R. Tang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Bone Formation ◽  
Mechanical Loading ◽  
Endochondral Ossification ◽  
Bone Development ◽  
Human Mesenchymal Stem Cell ◽  
Bone Defects ◽  
Mechanical Environment ◽  
Tgf Β1

AbstractEndochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation and is directed by local morphogen signals and mechanical cues. Here, we aimed to mimic these developmental conditions for regeneration of large bone defects. We hypothesized that engineered human mesenchymal stem cell (hMSC) condensations with in situ presentation of transforming growth factor-β1 (TGF-β1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles would promote endochondral regeneration of critical-sized rat femoral bone defects in a manner dependent on the in vivo mechanical environment. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with dual BMP-2 + TGF-β1 fully restoring mechanical bone function by week 12. In vivo ambulatory mechanical loading, initiated at week 4 by delayed unlocking of compliant fixation plates, significantly enhanced the bone formation rate in the four weeks after load initiation in the dual morphogen group. In vitro, local presentation of either BMP-2 alone or BMP-2 + TGF-β1 initiated endochondral lineage commitment of mesenchymal condensations, inducing both chondrogenic and osteogenic gene expression through SMAD3 and SMAD5 signaling. In vivo, however, endochondral cartilage formation was evident only in the BMP-2 + TGF-β1 group and was enhanced by mechanical loading. The degree of bone formation was comparable to BMP-2 soaked on collagen but without the ectopic bone formation that limits the clinical efficacy of BMP-2/collagen. In contrast, mechanical loading had no effect on autograft-mediated repair. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.One Sentence SummaryMimicking aspects of the cellular, biochemical, and mechanical environment during early limb development, chondrogenically-primed human mesenchymal stem cell condensations promoted functional healing of critical-sized femoral defects via endochondral ossification, and healing rate and extent was a function of the in vivo mechanical environment.

scholarly journals Sodium hyaluronate supplemented culture medium combined with joint-simulating mechanical loading improves chondrogenic differentiation of human mesenchymal stem cells

2021 ◽  
Vol 41 ◽  
pp. 616-632
Author(s):  
G Monaco ◽  
◽  
AJ El Haj ◽  
M Alini ◽  
MJ Stoddart
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Mechanical Loading ◽  
Chondrogenic Differentiation ◽  
Culture Medium ◽  
In Vitro Models ◽  
Matrix Deposition ◽  
Sulphated Glycosaminoglycan

In vitro models aim to recapitulate the in vivo situation. To more closely mimic the knee joint environment, current in vitro models need improvements to reflect the complexity of the native tissue. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules in healthy synovial fluid, while shear and dynamic compression are two joint-relevant mechanical forces. The present study aimed at investigating the concomitant effect of joint-simulating mechanical loading (JSML) and hMwt HA-supplemented culture medium on the chondrogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBM-MSCs). hBM-MSC chondrogenesis was investigated over 28 d at the gene expression level and total DNA, sulphated glycosaminoglycan, TGF-β1 production and safranin O staining were evaluated. The concomitant effect of hMwt HA culture medium and JSML significantly increased cartilage-like matrix deposition and sulphated glycosaminoglycan synthesis, especially during early chondrogenesis. A stabilisation of the hBM-MSC-derived chondrocyte phenotype was observed through the reduced upregulation of the hypertrophic marker collagen X and an increase in the chondrogenic collagen type II/X ratio. A combination of JSML and hMwt HA medium better reflects the complexity of the in vivo synovial joint environment. Thus, JSML and hMwt HA medium will be two important features for joint-related culture models to more accurately predict the in vivo outcome, therefore reducing the need for animal studies. Reducing in vitro artefacts would enable a more reliable prescreening of potential cartilage repair therapies.

scholarly journals Recapitulating bone development for tissue regeneration through engineered mesenchymal condensations and mechanical cues

2017 ◽  
Author(s):  
Anna M. McDermott ◽  
Samuel Herberg ◽  
Devon E. Mason ◽  
Hope B. Pearson ◽  
James H. Dawahare ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Bone Formation ◽  
Mechanical Loading ◽  
Endochondral Ossification ◽  
Bone Development ◽  
Bone Defects ◽  
Limb Bud ◽  
Large Bone ◽  
Large Bone Defects

ABSTRACTLarge bone defects cannot heal without intervention and have high complication rates even with the best treatments available. In contrast, bone fractures naturally healing with high success rates by recapitulating the process of bone development through endochondral ossification.1 Endochondral tissue engineering may represent a promising paradigm, but large bone defects are unable to naturally form a callus. We engineered mesenchymal condensations featuring local morphogen presentation (TGF-β1) to mimic the cellular organization and lineage progression of the early limb bud. As mechanical forces are 2,3 critical for proper endochondral ossification during bone morphogenesis2,3 and fracture healing, we hypothesized that mechanical cues would be important for endochondral regeneration.4,5 Here, using fixation plates that modulate ambulatory load transfer through dynamic tuning of axial compliance, we found that in vivo mechanical loading was necessary to restore bone function to large bone defects through endochondral ossification. Endochondral regeneration produced zonal cartilage and primary spongiosa mimetic of the native growth plate. Live human chondrocytes contributed to endochondral regeneration in vivo, while cell devitalization prior to condensation transplantation abrogated bone formation. Mechanical loading induced regeneration comparable to high-dose BMP-2 delivery, but without heterotopic bone formation and with order-of-magnitude greater mechanosensitivity.6–8In vitro, mechanical loading promoted chondrogenesis, and upregulated pericellular collagen 6 deposition and angiogenic gene expression. Consistently, in vivo mechanical loading regulated cartilage formation and neovascular invasion dependent on load timing. Together, this study represents the first demonstration of the effects of mechanical loading on transplanted cell-mediated bone defect regeneration, and provides a new template for recapitulating developmental programs for tissue engineering.

scholarly journals Tenascin is associated with chondrogenic and osteogenic differentiation in vivo and promotes chondrogenesis in vitro.

1987 ◽  
Vol 105 (6) ◽  
pp. 2569-2579 ◽  
Author(s):  
E J Mackie ◽  
I Thesleff ◽  
R Chiquet-Ehrismann
Keyword(s):  
Endochondral Ossification ◽  
Culture Medium ◽  
Bone Development ◽  
Bone Matrix ◽  
Immunofluorescent Staining ◽  
Tissue Culture Plastic ◽  
Osteogenic Cells ◽  
Wing Bud

The tissue distribution of the extracellular matrix glycoprotein, tenascin, during cartilage and bone development in rodents has been investigated by immunohistochemistry. Tenascin was present in condensing mesenchyme of cartilage anlagen, but not in the surrounding mesenchyme. In fully differentiated cartilages, tenascin was only present in the perichondrium. In bones that form by endochondral ossification, tenascin reappeared around the osteogenic cells invading the cartilage model. Tenascin was also present in the condensing mesenchyme of developing bones that form by intramembranous ossification and later was present around the spicules of forming bone. Tenascin was absent from mature bone matrix but persisted on periosteal and endosteal surfaces. Immunofluorescent staining of wing bud cultures from chick embryos showed large amounts of tenascin in the forming cartilage nodules. Cultures grown on a substrate of tenascin produced more cartilage nodules than cultures grown on tissue culture plastic. Tenascin in the culture medium inhibited the attachment of wing bud cells to fibronectin-coated substrates. We propose that tenascin plays an important role in chondrogenesis by modulating fibronectin-cell interactions and causing cell rounding and condensation.

Analysis of the Dynamics of the Electric Capacitance of a Cell Monolayer at the Late Stages of Caco-2 cell Differentiation

2019 ◽  
Vol 35 (6) ◽  
pp. 87-90
Author(s):  
S.V. Nikulin ◽  
V.A. Petrov ◽  
D.A. Sakharov
Keyword(s):  
Impedance Spectroscopy ◽  
Cell Monolayer ◽  
Microfluidic Chips ◽  
Russian Science ◽  
Electric Capacitance ◽  
A Cell ◽  
Static Conditions ◽  
Late Stages

The real-time monitoring of electric capacitance (impedance spectroscopy) allowed obtaining evidence that structures which look like intestinal villi can be formed during the cultivation under static conditions as well as during the cultivation in microfluidic chips. It was shown in this work via transcriptome analysis that the Hh signaling pathway is involved in the formation of villus-like structures in vitro, which was previously shown for their formation in vivo. impedance spectroscopy, intestine, villi, electric capacitance, Hh The study was funded by the Russian Science Foundation (Project 16-19-10597).

In Vitro Stain Transport in Canaliculi of Rat Femora Under Cyclic Loading

2000 ◽  
Author(s):  
Bixia Li ◽  
Timothy L. Norman
Keyword(s):  
Fluid Flow ◽  
Cyclic Loading ◽  
Confocal Microscopy ◽  
Mechanical Loading ◽  
Cyclic Load ◽  
Flow Behavior ◽  
Load Level ◽  
Static Conditions ◽  
Rat Bone

Abstract In this study, rat femurs were used to test the diffusion and mechanical transport properties of a fluroscein stain tracer in microvessels of bone. Fluroscein was used as a tracer to visualize the fluid flow behavior using confocal microscopy. It was found that stain transport occurs due to diffusion under static conditions and due to mechanical loading. The transport increased with cyclic load level and frequency. Our results also show that stain transport at the canaliculi level occurs rapidly in rat bone.

scholarly journals Impact of Dynamic Loading on the Implant-abutment Interface Using a Gas-enhanced Permeation Test In Vitro

2015 ◽  
Vol 9 (1) ◽  
pp. 112-119
Author(s):  
Anas Al-Jadaa ◽  
Thomas Attin ◽  
Timo Peltomäki ◽  
Christian Heumann ◽  
Patrick Roger Schmidlin
Keyword(s):  
Dynamic Loading ◽  
Group Testing ◽  
Pressure Change ◽  
Leakage Resistance ◽  
Implant Abutment ◽  
Bacterial Leakage ◽  
Static Conditions ◽  
Implant System ◽  
Permeation Test

Purpose : To assess implant leakage under static conditions as well as during and after dynamic loading. Materials and methods : Implants (Astra Tech (A), Biomet 3i (B) and Nobel Biocare (C)) were evaluated for leakage (n=8/group). Testing to assess the gas pressure change over time (hPa/min) and infiltrated fluid volume, was performed in a Gas Enhanced Permeation Test (GEPT) to qualify embedding. Implant apexes were then drilled, abutments were mounted and resin build-ups were fabricated. GEPT was reassessed. Samples were afterward mounted in a computer-controlled masticator while tested to bacterial leakage, they were daily observed for turbidity. Samples were then reassessed using GEPT. Dunnett's and Fisher's exact tests were utilized to compare implant and to analyze bacterial leakage. Results : Significant differences in GEPT values were shown after loading (p=0.034). Leakage resistance was best for B when compared to C (p=0.023). Samples with higher GEPT values demonstrated earlier bacterial leakage, occurring after 1 or 2 days (A=4, B=0, C=6) and showing favorability for implant system B (p=0.009). Conclusion : Implants leaking under static conditions had increased potential for bacterial leakage under dynamic conditions. As strongly correlating to sophisticated analytical methods, GEPT is a promising technique for assessing the overall implant system leakage resistance.

scholarly journals Airway Basal Cells show a dedifferentiated KRT17highPhenotype and promote Fibrosis in Idiopathic Pulmonary Fibrosis

2020 ◽  
Author(s):  
Benedikt Jaeger ◽  
Jonas Christian Schupp ◽  
Linda Plappert ◽  
Oliver Terwolbeck ◽  
Gian Kayser ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
De Novo ◽  
3D Culture ◽  
Xenograft Model ◽  
Basal Cells ◽  
Matrix Deposition ◽  
Mouse Xenograft Model

ABSTRACTIdiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study we focus on the profibrotic properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17high PTENlow dedifferentiated cell type. In the 3D organoid model, compared to ABC obtained from healthy volunteers, IPF-ABC give rise to more bronchospheres, de novo bronchial structures resembling lung developmental processes, induce fibroblast proliferation and extracellular matrix deposition in co-culture. Intratracheal application of IPF-ABC into minimally injured lungs of Rag2-/- or NRG mice causes severe fibrosis, remodeling of the alveolar compartment, and formation of honeycomb cyst-like structures. Connectivity MAP analysis of scRNA seq of bronchial brushings suggested that gene expression changes in IPF-ABC can be reversed by SRC inhibition. After demonstrating enhanced SRC expression and activity in these cells, and in IPF lungs, we tested the effects of saracatinib, a potent SRC inhibitor previously studied in humans. We demonstrated that saracatinib modified in-vitro and in-vivo the profibrotic changes observed in our 3D culture system and novel mouse xenograft model.

scholarly journals Bioreactor Processed Stromal Cell Seeding and Cultivation on Decellularized Pericardium Patches for Cardiovascular Use

2020 ◽  
Vol 10 (16) ◽  
pp. 5473
Author(s):  
Roman Matějka ◽  
Miroslav Koňařík ◽  
Jana Štěpanovská ◽  
Jan Lipenský ◽  
Jaroslav Chlupáč ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Stromal Cells ◽  
Subcutaneous Fat ◽  
Adipose Derived Stromal Cells ◽  
Pulsatile Pressure ◽  
Dynamic Cultivation ◽  
Decellularized Tissue ◽  
Static Conditions

(1) Background: Decellularized xenogeneic tissues are promising matrices for developing tissue-engineered cardiovascular grafts. In vitro recellularization of these tissues with stromal cells can provide a better in vivo remodelling and a lower thrombogenicity of the graft. The process of recellularization can be accelerated using a cultivation bioreactor simulating physiological conditions and stimuli. (2) Methods: Porcine pericardium was decellularized using a custom-built decellularization system with an optimized protocol. Autologous porcine adipose-derived stromal cells (PrASCs), isolated from the subcutaneous fat tissue, were used for recellularizing the decellularized pericardium. A custom cultivation bioreactor allowing the fixing of the decellularized tissue into a special cultivation chamber was created. The bioreactor maintained micro-perfusion and pulsatile pressure stimulation in order to promote the ingrowth of PrASCs inside the tissue and their differentiation. (3) Results: The dynamic cultivation promoted the ingrowth of cells into the decellularized tissue. Under static conditions, the cells penetrated only to the depth of 50 µm, whereas under dynamic conditions, the tissue was colonized up to 250 µm. The dynamic cultivation also supported the cell differentiation towards smooth muscle cells (SMCs). In order to ensure homogeneous cell colonization of the decellularized matrices, the bioreactor was designed to allow seeding of the cells from both sides of the tissue prior to the stimulation. In this case, the decellularized tissue was recolonized with cells within 5 days of dynamic cultivation. (4) Conclusions: Our newly designed dynamic bioreactor markedly accelerated the colonization of decellularized pericardium with ASCs and cell differentiation towards the SMC phenotype.

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