Intrinsic and growth-mediated cell and matrix specialization during murine meniscus tissue assembly.

The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1, and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences in meniscus zones were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.

FOXO1 and FOXO3 transcription factors have unique functions in meniscus development and homeostasis during aging and osteoarthritis.

The objective of this study was to examine FoxO expression and FoxO function in meniscus. In menisci from human knee joints with osteoarthritis (OA), FoxO1 and 3 expression were significantly reduced compared with normal menisci from young and old normal donors. The expression of FoxO1 and 3 was also significantly reduced in mouse menisci during aging and OA induced by surgical meniscus destabilization or mechanical overuse. Deletion of FoxO1 and combined FoxO1, 3, and 4 deletions induced abnormal postnatal meniscus development in mice and these mutant mice spontaneously displayed meniscus pathology at 6 mo. Mice with Col2Cre-mediated deletion of FoxO3 or FoxO4 had normal meniscus development but had more severe aging-related damage. In mature AcanCreERT2 mice, the deletion of FoxO1, 3, and 4 aggravated meniscus lesions in all experimental OA models. FoxO deletion suppressed autophagy and antioxidant defense genes and altered several meniscus-specific genes. Expression of these genes was modulated by adenoviral FoxO1 in cultured human meniscus cells. These results suggest that FoxO1 plays a key role in meniscus development and maturation, and both FoxO1 and 3 support homeostasis and protect against meniscus damage in response to mechanical overuse and during aging and OA.

Maturation of the Meniscal Collagen Structure Revealed by Polarization-Resolved and Directional Second Harmonic Generation Microscopy.

We report Polarization-resolved Second Harmonic Generation (P-SHG) and directional SHG (forward and backward, F/B) measurements of equine foetal and adult collagen in meniscus, over large field-of-views using sample-scanning. Large differences of collagen structure and fibril orientation with maturation are revealed, validating the potential for this novel methodology to track such changes in meniscal structure. The foetal menisci had a non-organized and more random collagen fibrillar structure when compared with adult using P-SHG. For the latter, clusters of homogeneous fibril orientation (inter-fibrillar areas) were revealed, separated by thick fibers. F/B SHG showed numerous different features in adults notably, in thick fibers compared to interfibrillar areas, unlike foetal menisci that showed similar patterns for both directions. This work confirms previous studies and improves the understanding of meniscal collagen structure and its maturation, and makes F/B and P-SHG good candidates for future studies aiming at revealing structural modifications to meniscus due to pathologies.

A novel kartogenin-platelet-rich plasma gel enhances chondrogenesis of bone marrow mesenchymal stem cells in vitro and promotes wounded meniscus healing in vivo.

BACKGROUND: The meniscus tear is one of the most common knee injuries particularly seen in athletes and aging populations. Subchondral bone sclerosis, irreparable joint damage, and the early onset of osteoarthritis make the injured meniscus heal difficultly. METHODS: The study was performed by in vitro and in vivo experiments. The in vitro experiments were carried out using the bone marrow stem cells (BMSCs) isolated from the rabbits, and the stemness of the BMSCs was tested by immunostaining. The BMSCs positively expressed stem cell markers were cultured with various concentrations of kartogenin (KGN) for 2 weeks. The chondrogenesis of BMSCs induced by KGN was examined by histochemical staining and quantitative RT-PCR. The in vivo experiments were completed by a rabbit model. Three holes were created in each meniscus by a biopsy punch. The rabbits were treated with four different conditions in each group. Group 1 was treated with 20 µl of saline (saline); group 2 was treated with 5 µl of 100 µM KGN and 15 µl saline (KGN); group 3 was treated with 5 µl of 100 µM KGN, 5 µl of 10,000 U/ ml thrombin, and 10 µl of PRP (KGN+PRP); group 4 was treated with 10,000 BMSCs in 10 µl of PRP, 5 µl of saline solution, and 5 µl of 10,000 U/ml thrombin (PRP+BMSC); group 5 was treated with 10,000 BMSCs in 10 µl of PRP, 5 µl of 100 µM KGN, and 5 µl of 10,000 U/ml thrombin (KGN+PRP+BMSC). The menisci were collected at day 90 post-surgery for gross inspection and histochemical analysis. RESULTS: The histochemical staining showed that KGN induced chondrogenesis of BMSCs in a concentration-dependent manner. The RT-PCR results indicated that chondrocyte-related genes were also increased in the BMSCs cultured with KGN in a dose-dependent manner. The in vivo results showed that large unhealed wound areas were still found in the wounds treated with saline and KGN groups. The wounds treated with BMSCs-containing PRP gel healed much faster than the wounds treated without BMSCs. Furthermore, the wounds treated with BMSCs-containing KGN-PRP gel have healed completely and formed more cartilage-like tissues than the wounds treated with BMSCs-containing PRP gel. CONCLUSIONS: BMSCs could be differentiated into chondrocytes when they were cultured with KGN-PRP gel in vitro and formed more cartilage-like tissues in the wounded rabbit meniscus when the wounds were treated with BMSCs-containing KGN-PRP gel. The results indicated that the BMSCs-containing KGN-PRP gel is a good substitute for injured meniscus repair and regeneration.

Electrospun PCL nanofibers blended with Wattakaka volubilis active phytochemicals for bone and cartilage tissue engineering.

In the present study, the polycaprolactone (PCL) nanofibers were investigated as a carrier to deliver phytochemicals for bone and cartilage tissue engineering. The PCL nanofibers was blended with phytochemicals hexadecanoic acid, octadecanoic acid and N,N-diisopropyl (2,2,3,3,3-pentafluoropropyl) amine isolated from a medicinal plant, Wattakaka volubilis. The scaffolds were characterized using scanning electron microscope (SEM) and Fourier transform infrared (FTIR) spectroscopy. The average diameter of control and phytochemical loaded nanofiber was 208 ±â€¯9.6 nm and 316 ±â€¯7.0 nm respectively. Biodegradation rate of nanofibers, impact of nanofiber on meniscus and osteoblast cell growth was analyzed using 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay, DNA content and extra cellular matrix secretion. Hoechst stain and SEM images were used to visualize and monitor the cell growth on PCL scaffold. The phytochemicals incorporated PCL nanofibers enhanced the growth and proliferation of primary human meniscus and osteoblast like cells and hence may be suitable scaffold for bone and cartilage tissue engineering applications.

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.

Maturation State and Matrix Microstructure Regulate Interstitial Cell Migration in Dense Connective Tissues.

Few regenerative approaches exist for the treatment of injuries to adult dense connective tissues. Compared to fetal tissues, adult connective tissues are hypocellular and show limited healing after injury. We hypothesized that robust repair can occur in fetal tissues with an immature extracellular matrix (ECM) that is conducive to cell migration, and that this process fails in adults due to the biophysical barriers imposed by the mature ECM. Using the knee meniscus as a platform, we evaluated the evolving micromechanics and microstructure of fetal and adult tissues, and interrogated the interstitial migratory capacity of adult meniscal cells through fetal and adult tissue microenvironments with or without partial enzymatic digestion. To integrate our findings, a computational model was implemented to determine how changing biophysical parameters impact cell migration through these dense networks. Our results show that the micromechanics and microstructure of the adult meniscus ECM sterically hinder cell mobility, and that modulation of these ECM attributes via an exogenous matrix-degrading enzyme permits migration through this otherwise impenetrable network. By addressing the inherent limitations to repair imposed by the mature ECM, these studies may define new clinical strategies to promote repair of damaged dense connective tissues in adults.

Meniscus, articular cartilage and nucleus pulposus: a comparative review of cartilage-like tissues in anatomy, development and function.

The degradation of cartilage in the human body is impacted by aging, disease, genetic predisposition and continued insults resulting from daily activity. The burden of cartilage defects (osteoarthritis, rheumatoid arthritis, intervertebral disc damage, knee replacement surgeries, etc.) is daunting in light of substantial economic and social stresses. This review strives to broaden the scope of regenerative medicine and tissue engineering approaches used for cartilage repair by comparing and contrasting the anatomical and functional nature of the meniscus, articular cartilage (AC) and nucleus pulposus (NP). Many review papers have provided detailed evaluations of these cartilages and cartilage-like tissues individually but none have comprehensively examined the parallels and inconsistencies in signaling, genetic expression and extracellular matrix composition between tissues. For the first time, this review outlines the importance of understanding these three tissues as unique entities, providing a comparative analysis of anatomy, ultrastructure, biochemistry and function for each tissue. This novel approach highlights the similarities and differences between tissues, progressing research toward an understanding of what defines each tissue as distinctive. The goal of this paper is to provide researchers with the fundamental knowledge to correctly engineer the meniscus, AC and NP without inadvertently developing the wrong tissue function or biochemistry.