Time course of 3D fibrocartilage formation by expanded human meniscus fibrochondrocytes in hypoxia.

Adult human meniscus fibrocartilage is avascular and nonhealing after injury. Meniscus tissue engineering aims to replace injured meniscus with lab-grown fibrocartilage. Dynamic culture systems may be necessary to generate fibrocartilage of sufficient mechanical properties for implantation; however, the optimal static preculture conditions before initiation of dynamic culture are unknown. This study thus investigated the time course of fibrocartilage formation by human meniscus fibrochondrocytes on a three-dimensional biomaterial scaffold under various static conditions. Human meniscus fibrochondrocytes from partial meniscectomy were expanded to passage 1 (P1) or P2 (3.0 ± 0.4 and 6.5 ± 0.6 population doublings), seeded onto type I collagen scaffolds, and grown in hypoxia (HYP, 3% O2 ) or normoxia (NRX, 20% O2 ) for 3, 6, and 9 weeks. Mechanical properties were not different between P1 and P2 cell-based constructs. Mechanical properties were lower in HYP, increased continually in NRX only, and were positively correlated with glycosaminoglycan content and accumulation of hyaline cartilage-like matrix components. The most mechanically competent tissues (NRX/9 weeks) reached 1/5 of the native meniscus instantaneous compression modulus but had an increasingly hypertrophic matrix-forming phenotype. HYP consistently suppressed the hypertrophic phenotype. The results provide baselines of engineered meniscus fibrocartilage properties under static conditions, which can be used to select a preculture strategy for dynamic culture depending on the desired combination of mechanical properties, hyaline cartilage-like matrix abundance, and hypertrophic phenotype.

TEMPO-Oxidized Cellulose Nanofiber-Alginate Hydrogel as a Bioink for Human Meniscus Tissue Engineering.

Objective: The avascular inner regions of the knee menisci cannot self-heal. As a prospective treatment, functional replacements can be generated by cell-based 3D bioprinting with an appropriate cell source and biomaterial. To that end, human meniscus fibrochondrocytes (hMFC) from surgical castoffs of partial meniscectomies as well as cellulose nanofiber-alginate based hydrogels have emerged as a promising cell source and biomaterial combination. The objectives of the study were to first find the optimal formulations of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)-oxidized cellulose nanofiber/alginate (TCNF/ALG) precursors for bioprinting, and then to use them to investigate redifferentiation and synthesis of functional inner meniscus-like extracellular matrix (ECM) components by expanded hMFCs. Methods: The rheological properties including shear viscosity, thixotropic behavior recovery, and loss tangent of selected TCNF/ALG precursors were measured to find the optimum formulations for 3D bioprinting. hMFCs were mixed with TCNF/ALG precursors with suitable formulations and 3D bioprinted into cylindrical disc constructs and crosslinked with CaCl2 after printing. The bioprinted constructs then underwent 6 weeks of in vitro chondrogenesis in hypoxia prior to analysis with biomechanical, biochemical, molecular, and histological assays. hMFCs mixed with a collagen I gel were used as a control. Results: The TCNF/ALG and collagen-based constructs had similar compression moduli. The expression of COL2A1 was significantly higher in TCNF/ALG. The TCNF/ALG constructs showed more of an inner meniscus-like phenotype while the collagen I-based construct was consistent with a more outer meniscus-like phenotype. The expression of COL10A1 and MMP13 were lower in the TCNF/ALG constructs. In addition, the immunofluorescence of human type I and II collagens were evident in the TCNF/ALG, while the bovine type I collagen constructs lacked type II collagen deposition but did contain newly synthesized human type I collagen.

Mechano-Hypoxia Conditioning of Engineered Human Meniscus.

Meniscus fibrochondrocytes (MFCs) experience simultaneous hypoxia and mechanical loading in the knee joint. Experimental conditions based on these aspects of the native MFC environment may have promising applications in human meniscus tissue engineering. We hypothesized that in vitro "mechano-hypoxia conditioning" with mechanical loading such as dynamic compression (DC) and cyclic hydrostatic pressure (CHP) would enhance development of human meniscus fibrocartilage extracellular matrix in vitro. MFCs from inner human meniscus surgical discards were pre-cultured on porous type I collagen scaffolds with TGF-ß3 supplementation to form baseline tissues with newly formed matrix that were used in a series of experiments. First, baseline tissues were treated with DC or CHP under hypoxia (HYP, 3% O2) for 5 days. DC was the more effective load regime in inducing gene expression changes, and combined HYP/DC enhanced gene expression of fibrocartilage precursors. The individual treatments of DC and HYP regulated thousands of genes, such as chondrogenic markers SOX5/6, in an overwhelmingly additive rather than synergistic manner. Similar baseline tissues were then treated with a short course of DC (5 vs 60 min, 10-20% vs 30-40% strain) with different pre-culture duration (3 vs 6 weeks). The longer course of loading (60 min) had diminishing returns in regulating mechano-sensitive and inflammatory genes such as c-FOS and PTGS2, suggesting that as few as 5 min of DC was adequate. There was a dose-effect in gene regulation by higher DC strains, whereas outcomes were inconsistent for different MFC donors in pre-culture durations. A final set of baseline tissues was then cultured for 3 weeks with mechano-hypoxia conditioning to assess mechanical and protein-level outcomes. There were 1.8-5.1-fold gains in the dynamic modulus relative to baseline in HYP/DC, but matrix outcomes were equal or inferior to static controls. Long-term mechano-hypoxia conditioning was effective in suppressing hypertrophic markers (e.g., COL10A1 10-fold suppression vs static/normoxia). Taken together, these results indicate that appropriately applied mechano-hypoxia conditioning can support meniscus fibrocartilage development in vitro and may be useful as a strategy for developing non-hypertrophic articular cartilage using mesenchymal stem cells.

Inability of Low Oxygen Tension to Induce Chondrogenesis in Human Infrapatellar Fat Pad Mesenchymal Stem Cells.

OBJECTIVE: Articular cartilage of the knee joint is avascular, exists under a low oxygen tension microenvironment, and does not self-heal when injured. Human infrapatellar fat pad-sourced mesenchymal stem cells (IFP-MSC) are an arthroscopically accessible source of mesenchymal stem cells (MSC) for the repair of articular cartilage defects. Human IFP-MSC exists physiologically under a low oxygen tension (i.e., 1-5%) microenvironment. Human bone marrow mesenchymal stem cells (BM-MSC) exist physiologically within a similar range of oxygen tension. A low oxygen tension of 2% spontaneously induced chondrogenesis in micromass pellets of human BM-MSC. However, this is yet to be demonstrated in human IFP-MSC or other adipose tissue-sourced MSC. In this study, we explored the potential of low oxygen tension at 2% to drive the in vitro chondrogenesis of IFP-MSC. We hypothesized that 2% O2 will induce stable chondrogenesis in human IFP-MSC without the risk of undergoing endochondral ossification at ectopic sites of implantation. METHODS: Micromass pellets of human IFP-MSC were cultured under 2% O2 or 21% O2 (normal atmosphere O2) in the presence or absence of chondrogenic medium with transforming growth factor-ß3 (TGFß3) for 3 weeks. Following in vitro chondrogenesis, the resulting pellets were implanted in immunodeficient athymic nude mice for 3 weeks. RESULTS: A low oxygen tension of 2% was unable to induce chondrogenesis in human IFP-MSC. In contrast, chondrogenic medium with TGFß3 induced in vitro chondrogenesis. All pellets were devoid of any evidence of undergoing endochondral ossification after subcutaneous implantation in athymic mice.

Correction: Engineered human meniscus' matrix-forming phenotype is unaffected by low strain dynamic compression under hypoxic conditions.

[This corrects the article DOI: 10.1371/journal.pone.0248292.].

Engineered human meniscus' matrix-forming phenotype is unaffected by low strain dynamic compression under hypoxic conditions.

Low oxygen and mechanical loading may play roles in regulating the fibrocartilaginous phenotype of the human inner meniscus, but their combination in engineered tissues remains unstudied. Here, we investigated how continuous low oxygen ("hypoxia") combined with dynamic compression would affect the fibrocartilaginous "inner meniscus-like" matrix-forming phenotype of human meniscus fibrochondrocytes (MFCs) in a porous type I collagen scaffold. Freshly-seeded MFC scaffolds were cultured for 4 weeks in either 3 or 20% O2 or pre-cultured for 2 weeks in 3% O2 and then dynamically compressed for 2 weeks (10% strain, 1 Hz, 1 h/day, 5 days/week), all with or without TGF-ß3 supplementation. TGF-ß3 supplementation was found necessary to induce matrix formation by MFCs in the collagen scaffold regardless of oxygen tension and application of the dynamic compression loading regime. Neither hypoxia under static culture nor hypoxia combined with dynamic compression had significant effects on expression of specific protein and mRNA markers for the fibrocartilaginous matrix-forming phenotype. Mechanical properties significantly increased over the two-week loading period but were not different between static and dynamic-loaded tissues after the loading period. These findings indicate that 3% O2 applied immediately after scaffold seeding and dynamic compression to 10% strain do not affect the fibrocartilaginous matrix-forming phenotype of human MFCs in this type I collagen scaffold. It is possible that a delayed hypoxia treatment and an optimized pre-culture period and loading regime combination would have led to different outcomes.

Bone Marrow Mesenchymal Stem Cell-Derived Tissues are Mechanically Superior to Meniscus Cells.

Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to form the mechanically responsive matrices of joint tissues, including the menisci of the knee joint. The purpose of this study is to assess BMSC's potential to engineer meniscus-like tissue relative to meniscus fibrochondrocytes (MFCs). MFCs were isolated from castoffs of partial meniscectomy from nonosteoarthritic knees. BMSCs were developed from bone marrow aspirates of the iliac crest. All cells were of human origin. Cells were cultured in type I collagen scaffolds under normoxia (21% O2) for 2 weeks followed by hypoxia (3% O2) for 3 weeks. The structural and functional assessment of the generated meniscus constructs were based on glycosaminoglycan (GAG) content, histological appearance, gene expression, and mechanical properties. The tissues formed by both cell types were histologically positive for Safranin O stain and appeared more intense in the BMSC constructs. This observation was confirmed by a 2.7-fold higher GAG content. However, there was no significant difference in collagen I (COL1A2) expression in BMSC- and MFC-based constructs (p = 0.17). The expression of collagen II (COL2A1) and aggrecan (ACAN) were significantly higher in BMSCs than MFC (p ≤ 0.05). Also, the gene expression of the hypertrophic marker collagen X (COL10A1) was 199-fold higher in BMSCs than MFC (p < 0.001). Moreover, relaxation moduli were significantly higher in BMSC-based constructs at 10-20% strain step than MFC-based constructs. BMSC-based constructs expressed higher COL2A1, ACAN, COL10A1, contained higher GAG content, and exhibited higher relaxation moduli at 10-20% strain than MFC-based construct. Impact statement Cell-based tissue engineering (TE) has the potential to produce functional tissue replacements for irreparably damaged knee meniscus. But the source of cells for the fabrication of the tissue replacements is currently unknown and of research interest in orthopedic TE. In this study, we fabricated tissue-engineered constructs using type I collagen scaffolds and two candidate cell sources in meniscus TE. We compared the mechanical properties of the tissues formed from human meniscus fibrochondrocytes and bone marrow-derived mesenchymal stem cells (BMSCs). Our data show that the tissues engineered from the BMSC are mechanically superior in relaxation modulus.

Re-Differentiation of Human Meniscus Fibrochondrocytes Differs in Three-Dimensional Cell Aggregates and Decellularized Human Meniscus Matrix Scaffolds.

Decellularized matrix (DCM) derived from native tissues may be a promising supporting material to induce cellular differentiation by sequestered bioactive factors. However, no previous study has investigated the use of human meniscus-derived DCM to re-differentiate human meniscus fibrochondrocytes (MFCs) to form meniscus-like extracellular matrix (ECM). We expanded human MFCs and seeded them upon a cadaveric meniscus-derived DCM prepared by physical homogenization under hypoxia. To assess the bioactivity of the DCM, we used conditions with and without chondrogenic factor TGF-ß3 and set up a cell pellet culture model as a biomaterial-free control. We found that the DCM supported chondrogenic re-differentiation and ECM formation of MFCs only in the presence of exogenous TGF-ß3. Chondrogenic re-differentiation was more robust at the protein level in the pellet model as MFCs on the DCM appeared to favour a more proliferative phenotype. Interestingly, without growth factors, the DCM tended to promote expression of hypertrophic differentiation markers relative to the pellet model. Therefore, the human meniscus-derived DCM prepared by physical homogenization contained insufficient bioactive factors to induce appreciable ECM formation by human MFCs.

Mimicking "J-Shaped" and Anisotropic Stress-Strain Behavior of Human and Porcine Aorta by Fabric-Reinforced Elastomer Composites.

An ex vivo heart perfusion device preserves the donor heart in a warm beating state during transfer between extraction and implantation surgeries. One of the current challenges includes the use of rigid and noncompliant plastic tubes, which causes injuries to the heart at the junction between the tissue and the tube. The compliant and rapidly strain-stiffening mechanical property that generates a "J-shaped" stress-strain behavior is necessary for producing the Windkessel effect, which ensures continuous flow of blood through the aorta. In this study, we mimic the J-shaped and anisotropic stress-strain behavior of human aorta in synthetic elastomers to replace the problematic noncompliant plastic tube. First, we assess the mechanical properties of human (n = 1) and porcine aorta (n = 14) to quantify the nonlinear and anisotropic behavior under uniaxial tensile stress from five different regions of the aorta. Second, fabric-reinforced elastomer composites were prepared by reinforcing silicone elastomers with embedded fabrics in a trilayer geometry. The knitted structures of the fabric provide strain-stiffening as well as anisotropic mechanical properties of the resulting composite in a deterministic manner. By optimizing the combination between different elastomers and fabrics, the resulting composites matched the J-shaped and anisotropic stress-strain behavior of natural human and porcine aorta. Finally, improved analytical constitutive models based on Gent's and Mooney-Rivlin's constitutive model (to describe the elastomer matrix) combined with Holzapfel-Gasser-Ogden's model (to represent the stiffer fabrics) were developed to describe the J-shaped behavior of the natural aortas and the fabric-reinforced composites. We anticipate that the suggested fabric-reinforced silicone elastomer composite design concept can be used to develop complex soft biomaterials, as well as in emerging engineering fields such as soft robotics and microfluidics, where the Windkessel effect can be useful in regulating the flow of fluids.

Hypoxia and TGF-ß3 Synergistically Mediate Inner Meniscus-Like Matrix Formation by Fibrochondrocytes.

IMPACT STATEMENT: The interactions of hypoxia and TGF-ß3 in aggregates of human meniscus fibrochondrocytes are synergistic in nature, suggesting combinatorial strategies using these factors are promising for tissue engineering the inner meniscus regions. Hypoxia alone in the absence of TGF-ß supplementation may be insufficient to initiate an inner meniscus-like extracellular matrix-forming response in this model.

Chondrogenic differentiation of synovial fluid mesenchymal stem cells on human meniscus-derived decellularized matrix requires exogenous growth factors.

The objective of this study was to investigate whether meniscus-derived decellularized matrix (DCM) has the capacity to induce differentiation of synovial fluid-derived mesenchymal stem cells (SF-MSCs) towards a meniscus fibrochondrocyte (MFC) phenotype. The potential roles of transforming growth factor beta-3 (TGF-ß3) and insulin-like growth factor 1 (IGF-1) in the differentiation of SF-MSCs towards an MFC phenotype were also investigated. SF-MSCs were isolated via plastic adherence cell culture from the synovial fluid of five donors (5 male, average age 34 years). Porous DCM was generated by homogenizing and freeze-drying fresh normal human cadaveric meniscus tissue. SF-MSCs were seeded and cultured on the DCM scaffold in a defined serum-free media (SFM) supplemented with or without the combination of TGF-ß3 and IGF-1. Cell pellets of SF-MSCs were cultured in SFM with either TGF-ß3 or IGF-1 or their combination as controls. The duration of culture was 3 weeks for both experimental configurations. We assessed newly-formed tissues by biochemical assays, scanning electron microscopy (SEM), immunofluorescence and quantitative real-time PCR (qPCR). The combination of TGF-ß3 and IGF-1 induced production of the cartilaginous matrix in DCM and upregulated the expression of aggrecan, collagens I and II. Moreover, the SF-MSCs exhibited a round morphology in the DCM scaffolds in the presence of the growth factors. In pellets, combined TGF-ß3 and IGF-1 synergistically enhanced cartilaginous matrix production. In contrast to bone marrow mesenchymal stem cells (BM-MSCs), the differentiated SF-MSCs showed little evidence of the expression of the hypertrophic differentiation marker, collagen X. In conclusion, meniscus-derived DCM appears to require exogenous growth factor supplementation to direct differentiation of SF-MSCs. STATEMENT OF SIGNIFICANCE: Meniscus tears are the most common injury of the knee joint. These tears pose a major risk factor for the early development of knee osteoarthritis. Unfortunately, the majority of these tears occur in the inner region of the meniscus and lacks blood supply with no reparative or regenerative capacity. The goal of this study was to determine if the native extracellular matrix (ECM) of human meniscus has the capacity to differentiate human knee synovial fluid resident mesenchymal stem cells (SF-MSCs) towards a meniscus phenotype as a potential strategy to repair avascular meniscal tears. Our findings show that the human meniscus-derived ECM without supplementation with growth factors (TGF-ß3 and IGF-1) cannot differentiate SF-MSCs towards a meniscus phenotype. The use of meniscus-derived scaffolds as a material to stimulate endogenous repair of meniscus tears via differentiation of SF-MSCs may require supplementation with TGF-ß3 and IGF-1.