scholarly journals Short-Term Evaluation of Cellular Fate in an Ovine Bone Formation Model

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1776
Author(s):  
Hareklea Markides ◽  
Nicola C. Foster ◽  
Jane S. McLaren ◽  
Timothy Hopkins ◽  
Cameron Black ◽  
...  
Keyword(s):  
Magnetic Fields ◽  
Stromal Cells ◽  
Bone Fractures ◽  
Femoral Condyle ◽  
Preclinical Model ◽  
Defect Model ◽  
Cell Dose ◽  
Initial Surgery ◽  
Post Implantation ◽  
Critical Sized Defect

The ovine critical-sized defect model provides a robust preclinical model for testing tissue-engineered constructs for use in the treatment of non-union bone fractures and severe trauma. A critical question in cell-based therapies is understanding the optimal therapeutic cell dose. Key to defining the dose and ensuring successful outcomes is understanding the fate of implanted cells, e.g., viability, bio-distribution and exogenous infiltration post-implantation. This study evaluates such parameters in an ovine critical-sized defect model 2 and 7 days post-implantation. The fate of cell dose and behaviour post-implantation when combined with nanomedicine approaches for multi-model tracking and remote control using external magnetic fields is also addressed. Autologous STRO-4 selected mesenchymal stromal cells (MSCs) were labelled with a fluorescent lipophilic dye (CM-Dil), functionalised magnetic nanoparticles (MNPs) and delivered to the site within a naturally derived bone extracellular matrix (ECM) gel. Encapsulated cells were implanted within a critical-sized defect in an ovine medial femoral condyle and exposed to dynamic gradients of external magnetic fields for 1 h per day. Sheep were sacrificed at 2 and 7 days post-initial surgery where ECM was harvested. STRO-4-positive (STRO-4+) stromal cells expressed osteocalcin and survived within the harvested gels at day 2 and day 7 with a 50% loss at day 2 and a further 45% loss at 7 days. CD45-positive leucocytes were also observed in addition to endogenous stromal cells. No elevation in serum C-reactive protein (CRP) or non-haem iron levels was observed following implantation in groups containing MNPs with or without magnetic field gradients. The current study demonstrates how numbers of therapeutic cells reduce substantially after implantation in the repair site. Cell death is accompanied by enhanced leucocyte invasion, but not by inflammatory blood marker levels. Crucially, a proportion of implanted STRO-4+ stromal cells expressed osteocalcin, which is indicative of osteogenic differentiation. Furthermore, MNP labelling did not alter cell number or result in a further deleterious impact on stromal cells following implantation.

scholarly journals Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Weichao Zhai ◽  
Jerome Tan ◽  
Tobias Russell ◽  
Sixun Chen ◽  
Dennis McGonagle ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Preclinical Model ◽  
Label Free ◽  
Current Standard ◽  
Differentiation Potential ◽  
Human Mesenchymal Stromal Cells ◽  
Endogenous Fluorophores

AbstractHuman mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical $$\upbeta $$ β -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-$$\upbeta $$ β -galactosidase activity using the fluorogenic substrate, C$${_{12}}$$ 12 FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in $$\upbeta $$ β -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p $$\le $$ ≤ 0.001 for the fluorogenic C$${_{12}}$$ 12 FDG method, and r = 0.72, p $$\le $$ ≤ 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

scholarly journals Ex vivo MRI cell tracking of autologous mesenchymal stromal cells in an ovine osteochondral defect model

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Hareklea Markides ◽  
Karin J. Newell ◽  
Heike Rudorf ◽  
Lia Blokpoel Ferreras ◽  
James E. Dixon ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Cell Tracking ◽  
Osteochondral Defect ◽  
Ex Vivo ◽  
Defect Model

Hard Tissue Augmentation of Aged Bone by Means of a Tin-Free PLLA-PCL Co-Polymer Exhibiting in vivo Anergy and Long-Term Structural Stability

Gerontology ◽  
2019 ◽  
Vol 65 (2) ◽  
pp. 174-185 ◽  
Author(s):  
Magdalena M. Schimke ◽  
Swaraj Paul ◽  
Katharina Tillmann ◽  
Günter Lepperdinger ◽  
Robert G. Stigler
Keyword(s):  
Stromal Cells ◽  
Perfusion Culture ◽  
Autologous Bone Marrow ◽  
Porous Scaffolds ◽  
Sheep Model ◽  
Physicochemical Stability ◽  
Space Filler ◽  
Post Implantation

Background: Due to aging, tissue regeneration gradually declines. Contemporary strategies to promote tissue-specific regeneration, in particular in elderly patients, often include synthetic material apt for implantation primarily aiming at upholding body functions and regaining appropriate anatomical and functional integrity. Objective: Biomaterials suitable for complex reconstruction surgical procedures have to exert high physicochemical stability and biocompatibility. Method: A polymer made of poly-L-lactic acid and poly-ε-caprolactone was synthesized by means of a novel tin-free catalytic process. The material was tested in a bioreactor-assisted perfusion culture and implanted in a sheep model for lateral augmentation of the mandible. Histological and volumetric evaluation was performed 3 and 6 months post-implantation. Results: After synthesis the material could be further refined by cryogrinding and sintering, thus yielding differently porous scaffolds that exhibited a firm and stable appearance. In perfusion culture, no disintegration was observed for extended periods of up to 7 weeks, while mesenchymal stromal cells readily attached to the material, steadily proliferated, and deposited extracellular calcium. The material was tested in vivo together with autologous bone marrow-derived stromal cells. Up to 6 months post-implantation, the material hardly changed in shape with composition also refraining from foreign body reactions. Conclusion: Given the long-term shape stability in vivo, featuring imperceptible degradation and little scarring as well as exerting good compatibility to cells and surrounding tissues, this novel biomaterial is suitable as a space filler in large anatomical defects.

scholarly journals A Rabbit Femoral Condyle Defect Model for Assessment of Osteochondral Tissue Regeneration

2020 ◽  
Vol 26 (11) ◽  
pp. 554-564
Author(s):  
Jason L. Guo ◽  
Yu Seon Kim ◽  
Elysse A. Orchard ◽  
Jeroen J.J.P. van den Beucken ◽  
John A. Jansen ◽  
...  
Keyword(s):  
Tissue Regeneration ◽  
Femoral Condyle ◽  
Defect Model ◽  
Osteochondral Tissue

Mesenchymal stromal cells improve the osteogenic capabilities of mineralized agarose gels in a rat full-thickness cranial defect model

2012 ◽  
Vol 7 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Norihiko Mizuta ◽  
Koji Hattori ◽  
Yoshika Suzawa ◽  
Soichi Iwai ◽  
Tomohiro Matsumoto ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Full Thickness ◽  
Defect Model ◽  
Agarose Gels ◽  
Cranial Defect

Comparing the osteogenic potential of bone marrow and tendon-derived stromal cells to repair a critical-sized defect in the rat femur

2015 ◽  
Vol 11 (7) ◽  
pp. 2014-2023 ◽  
Author(s):  
Nadja Kunkel ◽  
Andrea Wagner ◽  
Renate Gehwolf ◽  
Patrick Heimel ◽  
Herbert Tempfer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Rat Femur ◽  
Osteogenic Potential ◽  
Critical Sized Defect

scholarly journals The Development of Gelatin/Hyaluronate Copolymer Mixed with Calcium Sulfate, Hydroxyapatite, and Stromal-Cell-Derived Factor-1 for Bone Regeneration Enhancement

Polymers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1454 ◽  
Author(s):  
Yun-Liang Chang ◽  
Chia-Ying Hsieh ◽  
Chao-Yuan Yeh ◽  
Feng-Huei Lin
Keyword(s):  
Bone Regeneration ◽  
Bone Defect ◽  
Calcium Sulfate ◽  
Stromal Cell ◽  
Femoral Condyle ◽  
Bone Defects ◽  
Blood Analysis ◽  
Control Group ◽  
Defect Model ◽  
Stromal Cell Derived Factor

In clinical practice, bone defects still remain a challenge. In recent years, apart from the osteoconductivity that most bone void fillers already provide, osteoinductivity has also been emphasized to promote bone healing. Stromal-cell-derived factor-1 (SDF-1) has been shown to have the ability to recruit mesenchymal stem cells (MSCs), which play an important role in the bone regeneration process. In this study, we developed a gelatin–hyaluronate (Gel-HA) copolymer mixed with calcium sulfate (CS), hydroxyapatite (HAP), and SDF-1 in order to enhance bone regeneration in a bone defect model. The composites were tested in vitro for biocompatibility and their ability to recruit MSCs after material characterization. For the in vivo test, a rat femoral condyle bone defect model was used. Micro computed tomography (Micro-CT), two-photon excitation microscopy, and histology analysis were performed to assess bone regeneration. As expected, enhanced bone regeneration was well observed in the group filled with Gel-HA/CS/HAP/SDF-1 composites compared with the control group in our animal model. Furthermore, detailed blood analysis of rats showed no obvious systemic toxicity or side effects after material implantation. In conclusion, the Gel-HA/CS/HAP/SDF-1 composite may be a safe and applicable material to enhance bone regeneration in bone defects.

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