Fluoroscopy Assisted Minimally Invasive Transplantation of Allogenic Mesenchymal Stromal Cells Embedded in HyStem Reduces the Progression of Nucleus Pulposus Degeneration in the Damaged Interverbal Disc: A Preliminary Study in Rabbits

This study was conducted to develop a technique for minimally invasive and accurate delivery of stem cells to augment nucleus pulposus (NP) in damaged intervertebral discs (IVD). IVD damage was created in noncontiguous discs at L4-L5 level; rabbits (N=12) were randomly divided into three groups: group I treated with MSCs in HyStem hydrogel, group II treated with HyStem alone, and group III received no intervention. MSCs and hydrogel were administered to the damaged disc under guidance of fluoroscopy. Augmentation of NP was assessed through histological and MRI T2 mapping of the NP after eight weeks of transplantation. T2 weighted signal intensity was higher in group I than in groups II and III (P<0.05). Disc height index showed maximum disc height in group I compared to groups II and III. Histological score of the degenerative index was significantly (P<0.05) lower in group I (8.6 ± 1.8) than that in groups II (11.6 ± 2.3) and III (18.0 ± 5.7). Immunohistochemistry staining for collagen type II and aggrecan staining were higher in group I as compared to other groups. Our results demonstrate that the minimally invasive administration of MSCs in hyaluronan hydrogel (HyStem) augments the repair of NP in damaged IVD

Factors Influencing the Successful Isolation and Expansion of Aging Human Mesenchymal Stem Cells

Most studies highlight mesenchymal stem cells (MSCs) extracted primarily from bone marrow (BM), very few report the use of peripheral blood (PB), often due to the associated low seeding density and difficulties with extraction techniques. As ageing populations are becoming more predominant globally, together with escalating demands for MSC transplantation and tissue regeneration, obtaining quality MSCs suitable for induced differentiation and biological therapies becomes increasingly important. In this study, BM and PB were obtained from elderly patients and extracted MSCs grown in vitro to determine their successful isolation and expansion. Patients' socio-demographic background and other medical information were obtained from medical records. Successful and failed cultures were correlated with key demographic and medical parameters. A total of 112 samples (BM or PB) were used for this study. Of these, 50 samples (44.6%) were successfully cultured according to standardised criteria with no signs of contamination. Our comparative analyses demonstrated no statistical correlation between successful MSC cultures and any of the six demographic or medical parameters examined, including sample quantity, age, sex, race, habits and underlying comorbidities of sample donors. In conclusion, the present study demonstrates that typical demographics and comorbidities do not influence successful MSC isolation and expansion in culture

Uniaxial Cyclic Tensile Stretching at 8% Strain Exclusively Promotes Tenogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells

The present study was conducted to establish the amount of mechanical strain (uniaxial cyclic stretching) required to provide optimal tenogenic differentiation expression in human mesenchymal stromal cells (hMSCs) in vitro, in view of its potential application for tendon maintenance and regeneration. Methods. In the present study, hMSCs were subjected to 1 Hz uniaxial cyclic stretching for 6, 24, 48, and 72 hours; and were compared to unstretched cells. Changes in cell morphology were observed under light and atomic force microscopy. The tenogenic, osteogenic, adipogenic, and chondrogenic differentiation potential of hMSCs were evaluated using biochemical assays, extracellular matrix expressions, and selected mesenchyme gene expression markers; and were compared to primary tenocytes. Results. Cells subjected to loading displayed cytoskeletal coarsening, longer actin stress fiber, and higher cell stiffness as early as 6 hours. At 8% and 12% strains, an increase in collagen I, collagen III, fibronectin, and N-cadherin production was observed. Tenogenic gene expressions were highly expressed (p<0.05) at 8% (highest) and 12%, both comparable to tenocytes. In contrast, the osteoblastic, chondrogenic, and adipogenic marker genes appeared to be downregulated. Conclusion. Our study suggests that mechanical loading at 8% strain and 1 Hz provides exclusive tenogenic differentiation; and produced comparable protein and gene expression to primary tenocytes

Platelet Rich Concentrate Promotes Early Cellular Proliferation and Multiple Lineage Differentiation of Human Mesenchymal Stromal Cells In Vitro

Platelet rich concentrate (PRC) is a natural adjuvant that aids in human mesenchymal stromal cell (hMSC) proliferation in vitro; however, its role requires further exploration. This study was conducted to determine the optimal concentration of PRC required for achieving the maximal proliferation, and the need for activating the platelets to achieve this effect, and if PRC could independently induce early differentiation of hMSC. The gene expression of markers for osteocytes (ALP, RUNX2), chondrocytes (SOX9, COL2A1), and adipocytes (PPAR-γ) was determined at each time point in hMSC treated with 15% activated and nonactivated PRC since maximal proliferative effect was achieved at this concentration. The isolated PRC had approximately fourfold higher platelet count than whole blood. There was no significant difference in hMSC proliferation between the activated and nonactivated PRC. Only RUNX2 and SOX9 genes were upregulated throughout the 8 days. However, protein expression study showed formation of oil globules from day 4, significant increase in ALP at days 6 and 8 (P≤0.05), and increased glycosaminoglycan levels at all time points (P<0.05), suggesting the early differentiation of hMSC into osteogenic and adipogenic lineages. This study demonstrates that the use of PRC increased hMSC proliferation and induced early differentiation of hMSC into multiple mesenchymal lineages, without preactivation or addition of differentiation medium

Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells.

To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering

Surface analysis of early retrieved polyethylene tibial inserts for both knees in total knee replacement

This study involves the failure analysis of a pair of ultra-high-molecular-weight-polyethylene (UHMWPE) knee tibial inserts from Scorpio® fixed-bearing total knee system by Stryker, which were retrieved from Total Knee Replacements (TKR) that was performed on 64 years old male patient with periprosthetic joint infection detected on both knees. Although the implants were removed due to infection, surface analysis was essential to be studied in order to analyse the surface damage mode of short-term implants. This study reports relevant damage mechanisms seen in early-retrieved UHMWPE tibial inserts (implanted for 6 and 8 months) and further analysis of chemical, physical and mechanical properties that possibly accompanied with failure. The surface characterization was done using a 3D laser microscope and Scanning Electron Microscope (SEM) to evaluate surface damage and dimensional change of both UHMWPE tibial inserts. Nano-indentation is used to measure the hardness and elasticity modulus of the tibial inserts. Attenuated Total Reflection-Fourier Transform Infra-Red (ATR-FTIR), Differential Scanning Calorimetry (DSC) and Gel Permeation Chromatography (GPC) were used to characterize the chemical and physical properties of the inserts. In present study, retrieved polyethylene inserts with short implantation duration was considered to have high-grade wear modes. The high incidence of micro pits (with the average depth of 27.5 μm for 6 months insert and 18 μm for 8 months insert) and scratches as the observed surface defects strengthen the role played by the particles upon defects generation for both tibial inserts. The average surface roughness of 6 and 8 months inserts were 1.6798 μm and 1.2376 μm, respectively. The rough surface (4.207 μm) of region 4 at the lateral compartment proves that the 6 months tibial insert suffered more damage due to loosening defect where the radiolucencies (the gap between bone and cement) were seen below medial and lateral aspects of the tibial tray. Our data demonstrated a strong association between the change of molecular weight and degradation of mechanical properties with wear for both inserts. The oxidation induced wear mechanism was observed on 6 months old insert due to the presence of delamination features with the evidence of in vivo oxidation from IR analysis. © 2019 Elsevier Lt