Short-term response of primary human meniscus cells to simulated microgravity.

BACKGROUND: Mechanical unloading of the knee articular cartilage results in cartilage matrix atrophy, signifying the osteoarthritic-inductive potential of mechanical unloading. In contrast, mechanical loading stimulates cartilage matrix production. However, little is known about the response of meniscal fibrocartilage, a major mechanical load-bearing tissue of the knee joint, and its functional matrix-forming fibrochondrocytes to mechanical unloading events. METHODS: In this study, primary meniscus fibrochondrocytes isolated from the inner avascular region of human menisci from both male and female donors were seeded into porous collagen scaffolds to generate 3D meniscus models. These models were subjected to both normal gravity and mechanical unloading via simulated microgravity (SMG) for 7 days, with samples collected at various time points during the culture. RESULTS: RNA sequencing unveiled significant transcriptome changes during the 7-day SMG culture, including the notable upregulation of key osteoarthritis markers such as COL10A1, MMP13, and SPP1, along with pathways related to inflammation and calcification. Crucially, sex-specific variations in transcriptional responses were observed. Meniscus models derived from female donors exhibited heightened cell proliferation activities, with the JUN protein involved in several potentially osteoarthritis-related signaling pathways. In contrast, meniscus models from male donors primarily regulated extracellular matrix components and matrix remodeling enzymes. CONCLUSION: These findings advance our understanding of sex disparities in knee osteoarthritis by developing a novel in vitro model using cell-seeded meniscus constructs and simulated microgravity, revealing significant sex-specific molecular mechanisms and therapeutic targets.

Mechano-bioengineering of the knee meniscus.

The meniscus is a fibrocartilaginous structure of the knee joint that serves a crucial role in joint health and biomechanics. Degeneration or removal of the meniscus is known to lead to a chronic and debilitating disease known as knee osteoarthritis, whose prevalence is expected to increase in the next few decades. Meniscus bioengineering has been developed as a potential alternative to current treatment methods, wherein meniscus-like tissues are engineered using cells, materials, and biomechanical stimuli. The application of mechanical stimulation in meniscus bioengineering has presented varied results but, for the most part, it has been shown to enhance meniscus-like tissue formation. In this review, we summarized literature over the last 10 years of various mechanical stimuli applied in bioengineering meniscus tissues. The role of individual loading types is examined, and the effects on engineered meniscus are evaluated on both molecular and tissue levels. In addition, simulated microgravity is highlighted as a new area of interest in meniscus engineering, and its potential use as a disease-driving platform is discussed. Taken together, with the increased understanding of the effects of mechanical stimulation on bioengineered meniscus tissues, the most suitable loading regime could be developed for meniscus tissue engineering and osteoarthritis modeling.

Mechanotransduction in meniscus fibrochondrocytes: What about caveolae?

Meniscus fibrochondrocytes (MFCs) are an important cell population responsible for regulating the biomechanical properties of the knee meniscus. Despite their significance, not much is known about them, including how they sense and respond to mechanical stimuli. Due to the mechanical nature of the knee joint, it is therefore paramount to our understanding of the meniscus that its mechanotransductive mechanism be elucidated. In this review, we will summarize the current knowledge on mechanotransduction in MFCs and highlight the relevance of caveolae in lieu of a recent discovery. Additionally, we will discuss the importance of future studies in this area to help advance the field of meniscus research.

Bioprinting of human nasoseptal chondrocytes-laden collagen hydrogel for cartilage tissue engineering.

Skin cancer patients often have tumorigenic lesions on their noses. Surgical resection of the lesions often results in nasal cartilage removal. Cartilage grafts taken from other anatomical sites are used for the surgical reconstruction of the nasal cartilage, but donor-site morbidity is a common problem. Autologous tissue-engineered nasal cartilage grafts can mitigate the problem, but commercially available scaffolds define the shape and sizes of the engineered grafts during tissue fabrication. Moreover, the engineered grafts suffer from the inhomogeneous distribution of the functional matrix of cartilage. Advances in 3D bioprinting technology offer the opportunity to engineer cartilages with customizable dimensions and anatomically shaped configurations without the inhomogeneous distribution of cartilage matrix. Here, we report the fidelity of Freeform Reversible Embedding of Suspended Hydrogel (FRESH) bioprinting as a strategy to generate customizable and homogenously distributed functional cartilage matrix engineered nasal cartilage. Using FRESH and in vitro chondrogenesis, we have fabricated tissue-engineered nasal cartilage from combining bovine type I collagen hydrogel and human nasoseptal chondrocytes. The engineered nasal cartilage constructs displayed molecular, biochemical and histological characteristics akin to native human nasal cartilage.

Histological and molecular characterization of the growing nasal septum in mice.

The nasal septum is a cartilaginous structure that serves as a pacemaker for the development of the midface. The septum is a hyaline cartilage which is surrounded by a perichondrium and epithelium. It remains cartilaginous anteriorly, but posteriorly it undergoes endochondral ossification to form the perpendicular plate of the ethmoid. Understanding of hyaline cartilage differentiation stems predominantly from investigations of growth plate cartilage. It is currently unclear if the morphological and molecular properties of the differentiating nasal septum align with what is known from the growth plate. In this study, we describe growth, molecular, and cellular characteristics of the nasal septum with reference to hyaline cartilage differentiation. The nasal septum grows asynchronous across its length with phases of rapid growth interrupted by more stagnant growth. Growth appears to be driven predominantly by acquisition of chondrocyte hypertrophy. Similarly, cellular differentiation is asynchronous, and differentiation observed in the anterior part precedes posterior differentiation. Overall, the nasal septum is structurally and molecularly heterogeneous. Early and extensive chondrocyte hypertrophy but no ossification is observed in the anterior septum. Onset of hypertrophic chondrocyte differentiation coincided with collagen fiber deposition along the perichondrium. Sox9, Col2, Col10, Mmp13, Sp7, and Runx2 expression was heterogeneous and did not always follow the expected pattern established from chondrocyte differentiation in the growth plate. The presence of hypertrophic chondrocytes expressing bone-related proteins early on in regions where the nasal septum does not ossify displays incongruities with current understanding of hyaline cartilage differentiation. Runx2, Collagen II, Collagen X, and Sp7 commonly used to mark distinct stages of chondrocyte maturation and early bone formation show wider expression than expected and do not align with expected cellular characteristics. Thus, the hyaline cartilage of the nasal septum is quite distinct from growth plate hyaline cartilage, and caution should be taken before assigning cartilage properties to less well-defined cartilage structures using these commonly used markers. Beyond the structural description of the nasal cartilage, this study also provides important information for cartilage tissue engineering when using nasal septal cartilage for tissue regeneration.

Effect of cell seeding density on matrix-forming capacity of meniscus fibrochondrocytes and nasal chondrocytes in meniscus tissue engineering.

The presence of intact menisci is imperative for the proper function of the knee joint. Meniscus injuries are often treated by the surgical removal of the damaged tissue, which increases the likelihood of post-traumatic osteoarthritis. Tissue engineering holds great promise in producing viable engineered meniscal tissue for implantation using the patient's own cells; however, the cell source for producing the engineered tissue is unclear. Nasal chondrocytes (NC) possess many attractive features for engineering meniscus. However, in order to validate the use of NC for engineering meniscus fibrocartilage, a thorough comparison of NC and meniscus fibrochondrocytes (MFC) must be considered. Our study presents an analysis of the relative features of NC and MFC and their respective chondrogenic potential in a pellet culture model. We showed considerable differences in the cartilage tissue formed by the two different cell types. Our data showed that NC were more proliferative in culture, deposited more extracellular matrix, and showed higher expression of chondrogenic genes than MFC. Overall, our data suggest that NC produce superior cartilage tissue to MFC in a pellet culture model. In addition, NCs produce higher quality cartilage tissue at higher cell seeding densities during cell expansion.

Current concepts on structure-function relationships in the menisci.

The menisci are intricately organized structures that perform many tasks in the knee. We review their structure and function and introduce new data about their tibial and femoral surfaces. As the femur and tibia approach each other when the knee is bearing load, circumferential tension develops in the menisci, enabling the transmission of compressive load between the femoral and tibial cartilage layers. A low shear modulus is necessary for the tissue to adapt its shape to the changing radius of the femur as that bone moves relative to the tibia during joint articulation. The organization of the meniscus facilitates its functions. In the outer region of the menisci, intertwined collagen fibrils, fibers, and fascicles with predominantly circumferential orientation are prevalent; these structures are held together by radial tie fibers and sheets. Toward the inner portion of the menisci, there is more proteoglycan and the structure becomes more cartilage-like. The transition between these structural forms is gradual and seamless. The flexible roots, required for rigid body motion of the menisci, meld with both the tibia and the outer portion of the menisci to maintain continuity for resistance to the circumferential tension. Our new data demonstrate that the femoral and tibial surfaces of the menisci are structurally analogous to the surfaces of articular cartilage, enabling consistent modes of lubrication and load transfer to occur at the interfacing surfaces throughout motion. The structure and function of the menisci are thus shown to be strongly related to one another: form clearly complements function.

Ethylene glycol and glycerol loading and unloading in porcine meniscal tissue.

The development of a long-term storage method for meniscus, a complex tissue of the knee prone to injury, would improve the procedure and outcomes of meniscus transplantation. Cryopreservation uses cryoprotective agents (CPAs) including ethylene glycol (EG) and glycerol to preserve a variety of live tissues, and understanding of the CPA permeation kinetics will be critical in designing a vitrification protocol for meniscus. The purpose of this preliminary study was to understand the loading and unloading behaviours of EG and glycerol in meniscus by observing their efflux. For the main experiment, lateral and medial porcine menisci were incubated with CPA for 24 h at three temperatures (i.e., 4, 22, and 37 °C). Then, the menisci were immersed in 25 ml of X-VIVO™10 and CPA efflux was recorded by monitoring the molality of two consecutive washout solutions at different time points. In a subsequent experiment, menisci were incubated in the CPA solutions for 48 h at 22 °C, and the results were compared to those obtained at 22 °C in the main experiment. Results showed a rapid efflux of CPA from meniscus at the beginning of each wash. With increasing temperature, the amount of CPA efflux (and hence loading) increased. Using 24 h incubation, EG loaded the menisci more completely than glycerol. But after 48 h of incubation, both EG and glycerol achieved approximately the same degree of meniscus loading. This study provides preliminary data that will facilitate future design of experiments aimed at development of meniscus permeation studies.

The structural and compositional transition of the meniscal roots into the fibrocartilage of the menisci.

The meniscal roots, or insertional ligaments, firmly attach the menisci to tibial plateau. These strong attachments anchor the menisci and allow for the generation of hoop stress in the tissue. The meniscal roots have a ligament-like structure that transitions into the fibrocartilagenous structure of the meniscal body. The purpose of this study was to carry out a complete analysis of the structure and tissue organization from the body of the meniscus through the transition region and into the insertional roots. Serial sections were obtained from the meniscal roots into the meniscal body in fixed juvenile bovine menisci. Sections were stained for collagen and proteoglycans (PG) using fast green and safranin-o staining protocols. Unstained sections were imaged used a backlit stereo microscope. Optical projection tomography (OPT) was employed to evaluate the three-dimensional collagen architecture of the root-meniscus transition in lapine menisci. Tie-fibres were observed in the sections of the ligaments furthest from the bovine meniscal body. Blood vessels were observed to be surrounded by these tie-fibres and a PG-rich region within the ligaments. Near the tibial insertion, the roots contained large ligament-like collagen fascicles. In sections approaching the meniscus, there was an increase in tie-fibre size and density. Small tie-fibres extended into the ligament from the epiligamentous structure in the outermost sections of the meniscal roots, while large tie-fibre bundles were apparent at the meniscus transition. The staining pattern indicates that the root may continue into the outer portion of the meniscus where it then blends with the more fibrocartilage-like inner portions of the tissue. In unstained sections it was observed that the femoral side of the epiligamentous structure surrounding the root becomes more fibrous and thickens in the inferior inner portion of the posterior medial root. This thickening changes the shape of the root to more closely resemble the meniscus wedge shape. These observations support the concept of root continuity with the outer portion of the meniscus, thereby connecting with the hoop-like structure of the peripheral meniscus. OPT identified continuous collagen organization from the root into the meniscal body in longitudinal sections. In the radial direction, the morphology of the root continues into the meniscal body consistent with the serially sectioned bovine menisci. Blood vessels were prevalent on the periphery of the root. These blood vessels then arborized to cover the anterior femoral surface of the meniscus. This is the first study of the structural transition between the insertional ligaments (roots) and the fibrocartilagenous body of the menisci. These new structural details are important to understanding the meniscal load-bearing mechanism in the knee.

Stem Cell Therapy: Current Applications and Potential for Urology.

Stem cell therapy holds the potential to revolutionize the treatment of a number of chronic conditions. Stem cells ability to home in on injured sites of the body, stimulate angiogenesis, tissue regeneration, immunomodulation, anti-inflammatory, and anti-fibrotic factors have attracted their use in the treatment of many conditions. Urology has registered one of the highest experimental successes using stem cell therapy. However, the rate of clinical applications is comparatively lower. This review takes a look at our efforts so far and what needs to be done in order to maximize the clinical benefit we can derive from stem cells.

Double crosslinked hyaluronic acid and collagen as a potential bioink for cartilage tissue engineering.

The avascular nature of hyaline cartilage results in limited spontaneous self-repair and regenerative capabilities when damaged. Recent advances in three-dimensional bioprinting have enabled the precise dispensing of cell-laden biomaterials, commonly referred to as 'bioinks', which are emerging as promising solutions for tissue regeneration. An effective bioink for cartilage tissue engineering needs to create a micro-environment that promotes cell differentiation and supports neocartilage tissue formation. In this study, we introduced an innovative bioink composed of photocurable acrylated type I collagen (COLMA), thiol-modified hyaluronic acid (THA), and poly(ethylene glycol) diacrylate (PEGDA) for 3D bioprinting cartilage grafts using human nasal chondrocytes. Both collagen and hyaluronic acid, being key components of the extracellular matrix (ECM) in the human body, provide essential biological cues for tissue regeneration. We evaluated three formulations - COLMA, COLMA+THA, and COLMA+THA+PEGDA - for their printability, cell viability, structural integrity, and capabilities in forming cartilage-like ECM. The addition of THA and PEGDA significantly enhanced these properties, showcasing the potential of this bioink in advancing applications in cartilage repair and reconstructive surgery.

Effect of interleukin-1ß treatment on co-cultures of human meniscus cells and bone marrow mesenchymal stromal cells.

BACKGROUND: Interleukin-1ß (IL-1ß) is a major mediator of local inflammation present in injured joints. In this study, we aimed at comparing the effect of IL-1ß on engineered tissues from MCs, BMSCs and co-cultured MCs and BMSCs. METHODS: We compared the effect of IL-1ß in 3 groups: (1) MCs, (2) BMSCs and, (3) co-cultures of MCs and BMSCs. We selected 1 to 3 ratio of MCs to BMSCs for the co-cultures. Passage two (P2) human BMSCs were obtained from two donors. Human MCs were isolated from menisci of 4 donors. Mono-cultures of MCs and BMSCs, and co-cultures of MCs and BMSCs were cultured in chondrogenic medium with TGFß3, as cell pellets for 14 days. Thereafter, pellets were cultured for 3 more days in same medium as before with or without IL-1ß (500 pg/ml). Pellets were assessed histologically, biochemically and by RT-PCR for gene expression of aggrecan, sox9, MMP-1, collagens I and II. Statistics was performed using one-way ANOVA with Tukey's post-tests. RESULTS: Co-cultured pellets were the most intensely stained with safranin O and collagen II. Co-cultured pellets had the highest expression of sox9, collagen I and II. IL-1ß treatment slightly reduced the GAG/DNA of co-cultured pellets but still exceeded the sum of the GAG/DNA from the proportion of MCs and BMSCs in the co-cultured pellets. After IL-1ß treatment, the expression of sox9, collagen I and II in co-cultured pellets was higher compared to their expression in pure pellets. IL-1ß induced MMP-1 expression in mono-cultures of MCs but not significantly in mono-cultures of BMSCs or in co-cultured pellets. IL-1ß induced MMP-13 expression in mono-cultured pellets of BMSCs and in co-cultured pellets. CONCLUSIONS: Co-cultures of MCs and BMSCs resulted in a synergistic production of cartilaginous matrix compared to mono-cultures of MCs and BMSCs. IL-1ß did not abrogate the accumulated GAG matrix in co-cultures but mediated a decreased mRNA expression of aggrecan, collagen II and Sox9. These results strengthen the combinatorial use of primary MCs and BMSCs as a cell source for meniscus tissue engineering by demonstrating retention of fibrochondrogenic phenotype after exposure to IL-1ß.

3D Bioprinting of Hyaline Cartilage Using Nasal Chondrocytes.

Due to the limited self-repair capacity of the hyaline cartilage, the repair of cartilage remains an unsolved clinical problem. Tissue engineering strategy with 3D bioprinting technique has emerged a new insight by providing patient's personalized cartilage grafts using autologous cells for hyaline cartilage repair and regeneration. In this review, we first summarized the intrinsic property of hyaline cartilage in both maxillofacial and orthopedic regions to establish the requirement for 3D bioprinting cartilage tissue. We then reviewed the literature and provided opinion pieces on the selection of bioprinters, bioink materials, and cell sources. This review aims to identify the current challenges for hyaline cartilage bioprinting and the directions for future clinical development in bioprinted hyaline cartilage.

The Effect of Crosslinking Density on Nasal Chondrocytes' Redifferentiation.

Hydrogels appear to be an attractive class of biomaterial for cartilage tissue engineering due to their high water content, excellent biocompatibility, tunable stiffness, etc. The crosslinking density of the hydrogel can affect their viscoelastic property, and therefore potentially impact the chondrogenic phenotype of re-differentiated chondrocytes in a 3D microenvironment through physical cues. To understand the effect of crosslinking densities on chondrocytes phenotype and cellular interaction with the hydrogel, this study utilized a clinical grade thiolate hyaluronic acid and thiolate gelatin (HA-Gel) hydrogel, crosslinked with poly(ethylene glycol) diacrylate to create various crosslinking densities. The HA-Gel hydrogels were then mixed with human nasal chondrocytes to generate neocartilage in vitro. The influence of the hydrogel crosslinking density and the viscoelastic property on the cell behaviours on the gene and matrix levels were evaluated using biochemistry assays, histology, quantitative polymerase chain reaction (qPCR) and next-generation sequencing (RNA seq). In general, the differences in the storage modulus of the HA-Gel hydrogel are not enough to alter the cartilaginous gene expression of chondrocytes. However, a positively correlated trend of PPAR-γ gene expression to the crosslinking density was measured by qPCR. The RNA-seq results have shown that 178 genes are significantly negatively correlated and 225 genes are positively correlated to the crosslinking density, which is worth investigating in the future studies.

Non-hypertrophic chondrogenesis of mesenchymal stem cells through mechano-hypoxia programing.

Cartilage tissue engineering aims to generate functional replacements to treat cartilage defects from damage and osteoarthritis. Human bone marrow-derived mesenchymal stem cells (hBM-MSC) are a promising cell source for making cartilage, but current differentiation protocols require the supplementation of growth factors like TGF-ß1 or -ß3. This can lead to undesirable hypertrophic differentiation of hBM-MSC that progress to bone. We have found previously that exposing engineered human meniscus tissues to physiologically relevant conditions of the knee (mechanical loading and hypoxia; hence, mechano-hypoxia conditioning) increased the gene expression of hyaline cartilage markers, SOX9 and COL2A1, inhibited hypertrophic marker COL10A1, and promoted bulk mechanical property development. Adding further to this protocol, we hypothesize that combined mechano-hypoxia conditioning with TGF-ß3 growth factor withdrawal will promote stable, non-hypertrophic chondrogenesis of hBM-MSC embedded in an HA-hydrogel. We found that the combined treatment upregulated many cartilage matrix- and development-related markers while suppressing many hypertrophic- and bone development-related markers. Tissue level assessments with biochemical assays, immunofluorescence, and histochemical staining confirmed the gene expression data. Further, mechanical property development in the dynamic compression treatment shows promise toward generating functional engineered cartilage through more optimized and longer culture conditions. In summary, this study introduced a novel protocol to differentiate hBM-MSC into stable, cartilage-forming cells.

Engineered Human Meniscus in Modeling Sex Differences of Knee Osteoarthritis in Vitro.

Background: Osteoarthritis (OA) primarily affects mechanical load-bearing joints. The knee joint is the most impacted by OA. Knee OA (KOA) occurs in almost all demographic groups, but the prevalence and severity are disproportionately higher in females. The molecular mechanism underlying the pathogenesis and progression of KOA is unknown. The molecular basis of biological sex matters of KOA is not fully understood. Mechanical stimulation plays a vital role in modulating OA-related responses of load-bearing tissues. Mechanical unloading by simulated microgravity (SMG) induced OA-like gene expression in engineered cartilage, while mechanical loading by cyclic hydrostatic pressure (CHP), on the other hand, exerted a pro-chondrogenic effect. This study aimed to evaluate the effects of mechanical loading and unloading via CHP and SMG, respectively, on the OA-related profile changes of engineered meniscus tissues and explore biological sex-related differences. Methods: Tissue-engineered menisci were made from female and male meniscus fibrochondrocytes (MFCs) under static conditions of normal gravity in chondrogenic media and subjected to SMG and CHP culture. Constructs were assayed via histology, immunofluorescence, GAG/DNA assays, RNA sequencing, and testing of mechanical properties. Results: The mRNA expression of ACAN and COL2A1, was upregulated by CHP but downregulated by SMG. COL10A1, a marker for chondrocyte hypertrophy, was downregulated by CHP compared to SMG. Furthermore, CHP increased GAG/DNA levels and wet weight in both female and male donors, but only significantly in females. From the transcriptomics, CHP and SMG significantly modulated genes related to the ossification, regulation of ossification, extracellular matrix, and angiogenesis Gene Ontology (GO) terms. A clear difference in fold-change magnitude and direction was seen between the two treatments for many of the genes. Furthermore, differences in fold-change magnitudes were seen between male and female donors within each treatment. SMG and CHP also significantly modulated genes in OA-related KEGG pathways, such as mineral absorption, Wnt signalling pathway, and HIF-1 signalling pathway. Conclusion: Engineered menisci responded to CHP and SMG in a sex-dependent manner. SMG may induce an OA-like profile, while CHP promotes chondrogenesis. The combination of SMG and CHP could serve as a model to study the early molecular events of KOA and potential drug-targetable pathways.

Mechanical Unloading of Engineered Human Meniscus Models Under Simulated Microgravity: A Transcriptomic Study.

Osteoarthritis (OA) primarily affects mechanical load-bearing joints, with the knee being the most common. The prevalence, burden and severity of knee osteoarthritis (KOA) are disproportionately higher in females, but hormonal differences alone do not explain the disproportionate incidence of KOA in females. Mechanical unloading by spaceflight microgravity has been implicated in OA development in cartilaginous tissues. However, the mechanisms and sex-dependent differences in OA-like development are not well explored. In this study, engineered meniscus constructs were generated from healthy human meniscus fibrochondrocytes (MFC) seeded onto type I collagen scaffolds and cultured under normal gravity and simulated microgravity conditions. We report the whole-genome sequences of constructs from 4 female and 4 male donors, along with the evaluation of their phenotypic characteristics. The collected data could be used as valuable resources to further explore the mechanism of KOA development in response to mechanical unloading, and to investigate the molecular basis of the observed sex differences in KOA.

In vitro maturation and in vivo stability of bioprinted human nasal cartilage.

The removal of skin cancer lesions on the nose often results in the loss of nasal cartilage. The cartilage loss is either surgically replaced with autologous cartilage or synthetic grafts. However, these replacement options come with donor-site morbidity and resorption issues. 3-dimensional (3D) bioprinting technology offers the opportunity to engineer anatomical-shaped autologous nasal cartilage grafts. The 3D bioprinted cartilage grafts need to embody a mechanically competent extracellular matrix (ECM) to allow for surgical suturing and resistance to contraction during scar tissue formation. We investigated the effect of culture period on ECM formation and mechanical properties of 3D bioprinted constructs of human nasal chondrocytes (hNC)-laden type I collagen hydrogel in vitro and in vivo. Tissue-engineered nasal cartilage constructs developed from hNC culture in clinically approved collagen type I and type III semi-permeable membrane scaffold served as control. The resulting 3D bioprinted engineered nasal cartilage constructs were comparable or better than the controls both in vitro and in vivo. This study demonstrates that 3D bioprinted constructs of engineered nasal cartilage are feasible options in nasal cartilage reconstructive surgeries.

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.

Nasal Septum Deviation as the Consequence of BMP-Controlled Changes to Cartilage Properties.

The nasal septum cartilage is a specialized hyaline cartilage important for normal midfacial growth. Abnormal midfacial growth is associated with midfacial hypoplasia and nasal septum deviation (NSD). However, the underlying genetics and associated functional consequences of these two anomalies are poorly understood. We have previously shown that loss of Bone Morphogenetic Protein 7 (BMP7) from neural crest (BMP7 ncko ) leads to midfacial hypoplasia and subsequent septum deviation. In this study we elucidate the cellular and molecular abnormalities underlying NSD using comparative gene expression, quantitative proteomics, and immunofluorescence analysis. We show that reduced cartilage growth and septum deviation are associated with acquisition of elastic cartilage markers and share similarities with osteoarthritis (OA) of the knee. The genetic reduction of BMP2 in BMP7 ncko mice was sufficient to rescue NSD and suppress elastic cartilage markers. To our knowledge this investigation provides the first genetic example of an in vivo cartilage fate switch showing that this is controlled by the relative balance of BMP2 and BMP7. Cellular and molecular changes similar between NSD and knee OA suggest a related etiology underlying these cartilage abnormalities.