Elevated Lipid Metabolites in Stored Clinical OCA Media Correlate With Chondrocyte Death.

BACKGROUND: A major limitation of osteochondral allografts (OCA) is the deterioration of cartilage health associated with cell death during prolonged storage. However, little is known about the mechanisms that contribute to chondrocyte death during storage. PURPOSE/HYPOTHESIS: This study aimed to determine whether bioactive lipid metabolites accumulate in the storage media of OCA and whether they are associated with a loss of chondrocyte viability during prolonged storage. It was hypothesized that free fatty acids (FFAs) would accumulate over time in the storage media of OCA and adversely affect cartilage health during storage. STUDY DESIGN: Controlled laboratory study. METHODS: A group of 21 (n = 6-8 OCA/treatment group) fresh human hemicondylar OCA tissues and media were analyzed after 7, 28, and 68 days of prolonged cold (4°C) storage. Targeted mass spectrometry analysis was used to quantify bioactive FFAs, as well as primary (lipid hydroperoxide [ROOH]) and secondary (malondialdehyde) lipid oxidation products. Chondrocyte viability was measured using a fluorescence-based live/dead assay and confocal microscopy. RESULTS: The concentration of all targeted fatty acid metabolites in storage media was significantly increased with increased cold storage time (P < .05). ROOH was significantly higher on day 28 of cold storage. No difference in secondary ROOH products in storage media was observed. Chondrocyte viability significantly declined in both the en face and the vertical cross-sectional analysis with increased cold storage time and inversely correlated with fatty acid metabolites (P < .05). CONCLUSION: It is well established that elevated levels of certain FFAs and lipid oxidation products can alter cell function and cause cell death via lipotoxicity and other mechanisms. This work is the first to identify elevated levels of FFA metabolites and primary oxidation lipid products in the storage media from clinical OCA. The concentrations of FFA metabolites were measured at levels (>100 µM) known to induce cell death and were directly correlated with chondrocyte viability. CLINICAL RELEVANCE: These findings provide important targets for understanding why cartilage health declines during cold storage, which can be used to optimize media formulations and improve graft health.

ALK-5 Inhibitors for Efficient Derivation of Mesenchymal Stem Cells from Human Embryonic Stem Cells.

Objectives: Successful tissue regeneration requires a clinically viable source of mesenchymal stem cells (MSCs). We explored activin receptor-like kinase (ALK)-5 inhibitors to rapidly derive an MSC-like phenotype with high cartilage forming capacity from a xeno-free human embryonic cell line. Methods: Embryonic stem cell (ESC) lines (H9 and HADC100) were treated with the ALK-5 inhibitor SB431542; HADC100 cells were additionally treated with ALK-5 inhibitors SB525334 or GW788388. Cells were then seeded upon human fibronectin in the presence of fibroblast growth factor 2 (FGF2) in a serum-free medium. Flow cytometry was used to assess MSC markers (positive for CD73, CD90, and CD105; negative for CD34 and CD45). Differentiation status was assessed through quantitative polymerase chain reaction. Cartilage forming capacity was determined in high-density pellet cultures, in fibrin gels containing extracellular matrix (fibrin-ECM), and after implantation in ex vivo human osteoarthritic cartilage. Gene expression, histology, and immunostaining were used to assess cartilage phenotype, tissue regeneration, and integration. Results: Exposure to all three ALK-5 inhibitors lead to expression of mesodermal gene markers and differentiation into MSC-like cells (embryonic stem cell-derived mesenchymal stem cells [ES-MSCs]) based on surface marker expression. ES-MSC in pellet cultures or in fibrin-ECM gels expressed high levels of chondrogenic genes: COL2A1, ACAN, and COMP; and low levels of COL1A1 and RUNX2. Cell pellets or fibrin constructs implanted into ex vivo human osteoarthritic cartilage defects produced GAG-rich (safranin O positive) and collagen type II-positive neocartilage tissues that integrated well with native diseased tissue. Conclusions: We developed a protocol for rapid differentiation of xeno-free ESC into MSC-like cells with high cartilage forming capacity with potential for clinical applications. Impact statement Osteoarthritis (OA) is a common disease resulting in significant disability and no approved disease modifying treatment (other than total joint replacement). Embryonic stem cell-derived cell therapy has the potential to benefit patients with cartilage lesions leading to OA and may prevent or delay the need for total joint replacement.

Cartilage tissue engineering combining microspheroid building blocks and microneedle arrays.

Purpose: Scaffold-free cartilage tissue engineering circumvents issues with scaffold seeding, potential toxicity response, and impaired host integration. However, precisely controlling and maintaining a scaffold-free construct shape have been challenging. We explored the feasibility of microneedle arrays to print tissue using cellular microspheroids as building blocks.Materials and Methods: Human embryonic-derived mesenchymal stem cells or infrapatellar fat pad mesenchymal stem cells were used to create microspheroids of 500 µm in diameter, which were assembled on microneedle arrays in a predefined arrangement using a robotic system under computer vision. Microspheroids on microneedles were cultured to permit fusion into a tissue construct. Infrapatellar fat pad mesenchymal stem cell constructs were either implanted into chondral defects created in human osteoarthritic cartilage explants or maintained on the microneedle array for 3 weeks. Embryonic-derived mesenchymal stem cell constructs were designed to be press-fit into 3 mm subchondral defects in New Zealand White rabbits and maintained for up to 8 weeks to assess retention, early tissue repair, and more mature cartilage regeneration.Results: Microspheroids of both cell types fused together in culture to form neotissues of predefined shape and size. Infrapatellar fat pad mesenchymal stem cell neotissues expressed high levels of chondrogenic genes and integrated with the surrounding osteoarthritic host cartilage. Embryonic-derived mesenchymal stem cell constructs generated chondrogenic neotissue in vivo as early as 2 weeks and more mature tissue by 8 weeks with increased glycosaminoglycan deposition.Conclusions: We constructed defined scaffold-free shapes by bioprinting and fusing microspheroids. Proof of concept was shown in the repair of ex vivo osteoarthritic human cartilage and in vivo rabbit osteochondral (OC) defects.

Repair of Avascular Meniscus Tears with Electrospun Collagen Scaffolds Seeded with Human Cells.

The self-healing capacity of an injured meniscus is limited to the vascularized regions and is especially challenging in the inner avascular regions. As such, we investigated the use of human meniscus cell-seeded electrospun (ES) collagen type I scaffolds to produce meniscal tissue and explored whether these cell-seeded scaffolds can be implanted to repair defects created in meniscal avascular tissue explants. Human meniscal cells (derived from vascular and avascular meniscal tissue) were seeded on ES scaffolds and cultured. Constructs were evaluated for cell viability, gene expression, and mechanical properties. To determine potential for repair of meniscal defects, human meniscus avascular cells were seeded and cultured on aligned ES collagen scaffolds for 4 weeks before implantation. Surgical defects resembling "longitudinal tears" were created in the avascular zone of bovine meniscus and implanted with cell-seeded collagen scaffolds and cultured for 3 weeks. Tissue regeneration and integration were evaluated by histology, immunohistochemistry, mechanical testing, and magentic resonance imaging. Ex vivo implantation with cell-seeded collagen scaffolds resulted in neotissue that was significantly better integrated with the native tissue than acellular collagen scaffolds or untreated defects. Human meniscal cell-seeded ES collagen scaffolds may therefore be useful in facilitating meniscal repair of avascular meniscus tears.

Proton density water fraction as a biomarker of bone marrow cellularity: validation in ex vivo spine specimens.

The purpose of this study was to evaluate a magnetic resonance imaging (MRI) technique for quantifying the proton density water fraction (PDWF) as a biomarker of bone marrow cellularity. Thirty-six human bone marrow specimens from 18 donors were excised and subjected to different measurements of tissue composition: PDWF quantification using a multiple gradient echo MRI technique, three biochemical assays (triglyceride, total lipid and water content) and a histological assessment of cellularity. Results showed a strong correlation between PDWF and bone marrow cellularity from histology (r=0.72). A strong correlation was also found between PDWF and the biochemical assay of water content (r=0.76). These results suggest the PDWF is a predictor of bone marrow cellularity in tissues and can provide a non-invasive assessment of bone marrow changes in clinical patients undergoing radiotherapy.