Tracking Stem Cell Implants in Cartilage Defects of Minipigs by Using Ferumoxytol-enhanced MRI.

Background Cartilage repair outcomes of matrix-associated stem cell implants (MASIs) in patients have been highly variable. Conventional MRI cannot help distinguish between grafts that will and grafts that will not repair the underlying cartilage defect until many months after the repair. Purpose To determine if ferumoxytol nanoparticle labeling could be used to depict successful or failed MASIs compared with conventional MRI in a large-animal model. Materials and Methods Between January 2016 and December 2017, 10 Göttingen minipigs (n = 5 male; n = 5 female; mean age, 6 months ± 5.1; age range, 4-20 months) received implants of unlabeled (n = 12) or ferumoxytol-labeled (n = 20) viable and apoptotic MASIs in cartilage defects of the distal femur. All MASIs were serially imaged with MRI on a 3.0-T imaging unit at week 1 and weeks 2, 4, 8, 12, and 24, with calculation of T2 relaxation times. Cartilage regeneration outcomes were assessed by using the MR observation of cartilage repair tissue (MOCART) score (scale, 0-100), the Pineda score, and histopathologic quantification of collagen 2 production in the cartilage defect. Findings were compared by using the unpaired Wilcoxon rank sum test, a linear regression model, the Fisher exact test, and Pearson correlation. Results Ferumoxytol-labeled MASIs showed significant T2 shortening (22.2 msec ± 3.2 vs 27.9 msec ± 1.8; P < .001) and no difference in cartilage repair outcomes compared with unlabeled control MASIs (P > .05). At week 2 after implantation, ferumoxytol-labeled apoptotic MASIs showed a loss of iron signal and higher T2 relaxation times compared with ferumoxytol-labeled viable MASIs (26.6 msec ± 4.9 vs 20.8 msec ± 5.3; P = .001). Standard MRI showed incomplete cartilage defect repair of apoptotic MASIs at 24 weeks. Iron signal loss at 2 weeks correlated with incomplete cartilage repair, diagnosed at histopathologic examination at 12-24 weeks. Conclusion Ferumoxytol nanoparticle labeling can accelerate the diagnosis of successful and failed matrix-associated stem cell implants at MRI in a large-animal model. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Sneag and Potter in this issue.

Full biomechanical mapping of the ovine knee joint to determine creep-recovery, stiffness and thickness variation.

BACKGROUND: Clinical cartilage repair strategies can be tested using the sheep model as suggest by the European Medicines Agency. To characterize variation within the joint a full biomechanical mapping is necessary. The aim of this study is to establish a loading model, to map regional differences within the knee and determine reference areas for area specific replacement techniques. METHODS: A porous indenter was selected to evaluate 22 defined test locations (femoral condyles, tibia plateau, patella, femoral groove) on ovine knees (n = 7). A high-dynamic force-controlled micro creep and creep-recovery indentation test system applied five loading (0.11 MPa) and unloading (5.6 kPa) cycles for 60 s each and recorded creep-recovery. Needle indentation was used to measure cartilage thickness and calculate total strain. FINDINGS: Steady state behaviour was observed from the third cycle and further evaluated. Little variation of stiffness in N/mm was found within the patella (4.3SD0.5) and femoral groove (8.1SD0.7) compared to larger variations in the femur (7.9SD2.0) and tibia (7.5SD3.2). Creep indentation showed values of 14.5%(SD2.7%) for the patella and 17.4%(SD3%) for the femoral grove opposed to 13.4%(SD4.3%) for the femoral condyles and 21.8%(SD6.6%) for the tibia plateau. Similar trends were observed analysing creep-recovery. Values were normalized to cartilage thickness which ranged between 0.36 mm and 1.14 mm. INTERPRETATION: Our setup allows a reliable evaluation of zonal differences. Homogenous biomechanical behaviour is found within the patella and femoral groove whereas significant biomechanical variation within the femoral condyles and tibia plateau indicates the need for site-specific cartilage repair products.

Biomarkers for tissue engineering of the tendon-bone interface.

The tendon-bone interface (enthesis) is a highly sophisticated biomaterial junction that allows stress transfer between mechanically dissimilar materials. The enthesis encounters very high mechanical demands and the regenerative capacity is very low resulting in high rupture recurrence rates after surgery. Tissue engineering offers the potential to recover the functional integrity of entheses. However, recent enthesis tissue engineering approaches have been limited by the lack of knowledge about the cells present at this interface. Here we investigated the cellular differentiation of enthesis cells and compared the cellular pattern of enthesis cells to tendon and cartilage cells in a next generation sequencing transcriptome study. We integrated the transcriptome data with proteome data of a previous study to identify biomarkers of enthesis cell differentiation. Transcriptomics detected 34468 transcripts in total in enthesis, tendon, and cartilage. Transcriptome comparisons revealed 3980 differentially regulated candidates for enthesis and tendon, 395 for enthesis and cartilage, and 946 for cartilage and tendon. An asymmetric distribution of enriched genes was observed in enthesis and cartilage transcriptome comparison suggesting that enthesis cells are more chondrocyte-like than tenocyte-like. Integrative analysis of transcriptome and proteome data identified ten enthesis biomarkers and six tendon biomarkers. The observed gene expression characteristics and differentiation markers shed light into the nature of the cells present at the enthesis. The presented markers will foster enthesis tissue engineering approaches by setting a bench-mark for differentiation of seeded cells towards a physiologically relevant phenotype.

Efficient decellularization for tissue engineering of the tendon-bone interface with preservation of biomechanics.

Interfaces between tendon/ligament and bone ("entheses") are highly specialized tissues that allow for stress transfer between mechanically dissimilar materials. Entheses show very low regenerative capacity resulting in high incidences of failure after surgical repair. Tissue engineering is a promising approach to recover functionality of entheses. Here, we established a protocol to decellularize porcine entheses as scaffolds for enthesis tissue engineering. Chemical detergents as well as physical treatments were investigated with regard to their efficiency to decellularize 2 mm thick porcine Achilles tendon entheses. A two-phase approach was employed: study 1 investigated the effect of various concentrations of sodium dodecyl sulfate (SDS) and t-octylphenoxypolyethoxy-ethanol (Triton X-100) as decellularization agents. The most efficient combination of SDS and Triton was then carried forward into study 2, where different physical methods, including freeze-thaw cycles, ultrasound, perfusion, and hydrostatic washing were used to enhance the decellularization effect. Cell counts, DNA quantification, and histology showed that washing with 0.5% SDS + 1% Triton X-100 for 72 h at room temperature could remove ~ 98% cells from the interface. Further investigation of physical methods proved that washing under 200 mmHg hydrostatic pressure shortened the detergent exposing time from 72 h to 48 h. Biomechanical tensile testing showed that the biomechanical features of treated samples were preserved. Washing under 200 mmHg hydrostatic pressure with 0.5% SDS + 1% Triton X-100 for 48 h efficiently decellularized entheses with preservation of matrix structure and biomechanical features. This protocol can be used to efficiently decellularize entheses as scaffolds for tissue engineering.

Methylation of the ER-alpha Promoter Is Influenced by its Ligand Estrogen in Osteosarcoma Cells SAOS-2 In Vitro.

BACKGROUND/AIM: The aggressive fast-growing osteosarcoma is the most common primary malignant bone tumor. The relevance of estrogen as a key player in bone metabolism and bone tumor is well-known. At the molecular level, estrogen activates the estrogen receptor α (ERα) as a natural ligand of this receptor. ERα acts as a transcription factor by binding to the "estrogen response element" (ERE) and regulates the expression of a various number of genes. Epigenetic processes, e.g. the methylation of the "cytosine-phosphatidyl-guanine (CpG) islands" can change the transcription of target genes and subsequently the protein expression. As DNA methylation is generally associated with gene transcription repression, up until now little is known about the ERα methylation in osteosarcoma cells. The aim of the present pilot study was to evaluate the methylation status of ERα in osteosarcoma cells SAOS-2 and MG 63 after stimulation with estrogen. MATERIALS AND METHODS: SAOS-2 and MG 63 cells were cultured in DMEM. After treatment with 10 nmol estrogen (E2) for 24 h, the expression of ERα was detected by immunocytochemistry (ICC). As controls we used untreated cells. Staining was evaluated semi-quantitatively by the immunoreactive score of Remmele and Stegner (IRS). To determine mRNA gene expression, extracted RNA was transcribed into c-DNA and a quantitative real-time-PCR (qRT-PCR) was carried out. The semi quantitative evaluation of the ERα mRNA was based on the 2(-ΔΔct) method using untreated cells as reference control. One microgram of each extracted genomic DNA sample was converted with bisulfite and a real-time methylation-specific PCR (rt-MSP) was performed. RESULTS: The estrogen-stimulated SAOS-2 cells showed a significant increase of ERα expression. A 7-fold up-regulation of ERα mRNA confirmed the results of immunocytochemistry. Methylation of the ERα promoter was not detected in treated cells. In contrast, we identified methylation of the ERα promoters in untreated cells. The staining of MG 63 cells showed a weak gain of ERα expression in the stimulated cells, as well as a weak increase of the ER-α mRNA (2-fold). Methylation of the ERα promoters was not detectable in either treated or untreated cells. CONCLUSION: The methylation status of ERα in osteosarcoma cells is affected by estrogen. These findings indicate that epigenetic changes of genomic DNA regulate ERα synthesis. Taken together, our results suggest that SAOS-2 cells can be an interesting model for further investigating ERα synthesis. In addition, the evaluation of ERα methylation in osteosarcoma specimens is in progress.