ABSTRACT
The transcriptional regulators underlying induction and differentiation of dense connective tissues such as tendon and related fibrocartilaginous tissues (meniscus and annulus fibrosus) remain largely unknown. Using an iterative approach informed by developmental cues and single cell RNA sequencing (scRNA-seq), we establish directed differentiation models to generate tendon and fibrocartilage cells from mouse embryonic stem cells (mESCs) by activation of TGFß and hedgehog pathways, achieving 90% induction efficiency. Transcriptional signatures of the mESC-derived cells recapitulate embryonic tendon and fibrocartilage signatures from the mouse tail. scRNA-seq further identify retinoic acid signaling as a critical regulator of cell fate switch between TGFß-induced tendon and fibrocartilage lineages. Trajectory analysis by RNA sequencing define transcriptional modules underlying tendon and fibrocartilage fate induction and identify molecules associated with lineage-specific differentiation. Finally, we successfully generate 3-dimensional engineered tissues using these differentiation protocols and show activation of mechanotransduction markers with dynamic tensile loading. These findings provide a serum-free approach to generate tendon and fibrocartilage cells and tissues at high efficiency for modeling development and disease.
Subject(s)
Fibrocartilage/growth & development , Mouse Embryonic Stem Cells/physiology , Tendons/growth & development , Tissue Engineering/methods , Transcriptional Activation , Animals , Cell Differentiation/genetics , Embryo, Mammalian , Fibrocartilage/cytology , Gene Expression Regulation, Developmental , Hedgehog Proteins/metabolism , Mechanotransduction, Cellular/genetics , Mice , RNA-Seq , Signal Transduction/genetics , Single-Cell Analysis , Tendons/cytology , Transforming Growth Factor beta/metabolism , Tretinoin/metabolismABSTRACT
PURPOSE: This study aimed to investigate how fibroblastic and chondrocytic properties of human meniscal fibrochondrocytes are affected in culture conditions according to the type of meniscal pathology and localization, and to provide basic information for tissue-engineering studies. METHODS: Primary fibrochondrocyte cultures were prepared from meniscus samples of patients who had either traumatic tear or degeneration due to osteoarthritis. Cultures were compared in terms of mRNA expression levels of COL1A1, COL2A1, COMP1, HIF1A, HIF2A, and SOX9 and secreted total collagen and sulfated sGAG levels according to the type of meniscal pathology, anatomical localization, and the number of subcultures. RESULTS: mRNA expression levels of COL1A1, COMP1, HIF1A, HIF2A, and SOX9 were found to be increased in subsequent subcultures in all specimens. COL1A1 mRNA expression levels of both lateral and medial menisci of patients with traumatic tear were significantly higher than in patients with degenerative pathology, indicating a more fibroblastic character. P1 subculture of lateral and P3 or further subculture of medial meniscus showed more fibroblastic characteristics in patients with degenerative pathology. Furthermore, in patients with degenerative pathology, the subcultures of the lateral meniscus (especially on the inner part) presented more chondrocytic characteristics than did those of medial meniscus. CONCLUSIONS: The mRNA expression levels of the cultures showed significant differences according to the anatomical localization and pathology of the meniscus, indicating distinct chondrocytic and fibroblastic features. This fundamental knowledge would help researchers to choose more efficient cell sources for cell-seeding of a meniscus scaffold, and to generate a construct resembling the original meniscus tissue.
Subject(s)
Fibrocartilage , Joints/injuries , Meniscus , Osteoarthritis/pathology , Transcriptome , Adolescent , Adult , Aged , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Chondrocytes/pathology , Female , Fibrocartilage/cytology , Fibrocartilage/metabolism , Fibrocartilage/pathology , Gene Expression Profiling , Humans , Joints/metabolism , Joints/pathology , Male , Meniscus/cytology , Meniscus/injuries , Meniscus/metabolism , Meniscus/pathology , Middle Aged , Osteoarthritis/genetics , Osteoarthritis/metabolism , Primary Cell Culture/methods , Rupture/genetics , Rupture/metabolism , Rupture/pathology , Young AdultABSTRACT
OBJECTIVE: This study was aimed to identify the residence of human fibrocartilage stem cells (hFCSCs), characterize their stem cell properties and investigate the functional mechanisms which regulate fibrocartilage stem cells (FCSCs) toward chondrogenic differentiation during cartilage homeostasis and repairing. METHODS: Cytological characteristics of hFCSCs and human orofacial mesenchymal stem cells (hOFMSCs) were analyzed. Chondrogenic potential of hFCSCs was compared with hOFMSCs both in vitro and in vivo. Regulatory role of SOX9 during FCSCs chondrogenesis was studied by shRNA interference in vitro, and by GFP+ FCSCs treatment in rat condylar cartilage defect model. SOX9 expression was also examined in temporomandibular joint osteoarthritis (TMJOA) patients' cartilage surface. RESULTS: hFCSCs exhibited typical mesenchymal stem cell characteristics, with significantly stronger chondrogenic capability compared to hOFMSCs. Moreover, hFCSCs showed remarkably increased expression of SOX9. During cartilage pellet culture, there was stronger SOX9 expression in hFCSCs than hOFMSCs. SOX9 shRNA interference downregulated chondrogenic capability of hFCSCs in vitro, as well as disrupting migration and chondrogenic differentiation of GFP+ FCSCs toward mature chondrocytes in rat condylar cartilage defect. Of note, SOX9 expression was also found suppressed in the condylar superficial zone of TMJOA patients. CONCLUSION: We found the existence of FCSCs in human TMJ cartilage, and characterized their distinct stem cell features. SOX9 is essential for hFCSCs chondrogenic differentiation, and a comprehensive understanding of the regulatory role of SOX9 in hFCSCs would be important for exploring potential intervention strategy of condylar cartilage degradation during TMJ disorders.
Subject(s)
Cell Differentiation , Chondrogenesis , Fibrocartilage/cytology , Stem Cells/cytology , Stem Cells/physiology , Temporomandibular Joint , Animals , Cells, Cultured , Humans , Male , Mice , RatsABSTRACT
Articular cartilage injury and degeneration causing pain and loss of quality-of-life has become a serious problem for increasingly aged populations. Given the poor self-renewal of adult human chondrocytes, alternative functional cell sources are needed. Direct reprogramming by small molecules potentially offers an oncogene-free and cost-effective approach to generate chondrocytes, but has yet to be investigated. Here, we directly reprogrammed mouse embryonic fibroblasts into PRG4+ chondrocytes using a 3D system with a chemical cocktail, VCRTc (valproic acid, CHIR98014, Repsox, TTNPB, and celecoxib). Using single-cell transcriptomics, we revealed the inhibition of fibroblast features and activation of chondrogenesis pathways in early reprograming, and the intermediate cellular process resembling cartilage development. The in vivo implantation of chemical-induced chondrocytes at defective articular surfaces promoted defect healing and rescued 63.4% of mechanical function loss. Our approach directly converts fibroblasts into functional cartilaginous cells, and also provides insights into potential pharmacological strategies for future cartilage regeneration.
Subject(s)
Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibrocartilage/cytology , Animals , Cellular Reprogramming , Chondrocytes/cytology , Chondrocytes/metabolism , Chondrogenesis , Fibroblasts/metabolism , Mice , Organoids/cytology , Regeneration , Tissue Scaffolds/chemistry , Transcriptome/geneticsABSTRACT
BACKGROUND: A detailed evaluation focusing on the fibrocartilage layers in the anterior cruciate ligament (ACL) insertion is necessary to consider regeneration of the insertion. This study examined the development of the fibrocartilage layers in the ACL tibial insertion in rabbits by quantitative morphometric evaluations based on histological and immunohistochemical analyses. METHODS: Male Japanese white rabbits were used because of their history of use for histomorphometric analyses of the ACL insertion and to eliminate the influence of female hormones on the ACL. Six animals were euthanized at each age (1 day and 1, 2, 4, 6, 8, 12, and 24 weeks); in total, 48 animals were used. Proliferation rate, apoptosis rate, Sox9-positive rate, and chondrocyte number were evaluated. Safranin O-stained glycosaminoglycan (GAG) areas, tidemark length, ACL insertion width, and ACL length were also evaluated. All parameters were compared with those at age 24 weeks of age. RESULTS: High levels of chondrocyte proliferation and Sox9 expression continued until 4 and 8 weeks of age, respectively, and then gradually decreased. Chondrocyte apoptosis increased up to 8 weeks. The chondrocyte number, ACL insertion width, ACL length, safranin O-stained GAG areas, and tidemark length gradually increased up to 12 weeks. CONCLUSION: Chondrocytes that displayed chondrocyte proliferation and Sox9 expression increased until 12 weeks of age, in accordance with development of the ACL length and its insertion width. The GAG production and tidemark length also increased until 12 weeks of age. The development of fibrocartilage layers in the ACL insertion was complete at 12 weeks of age.
Subject(s)
Anterior Cruciate Ligament/physiology , Chondrocytes/physiology , Fibrocartilage/physiology , Regeneration , Animals , Anterior Cruciate Ligament/transplantation , Anterior Cruciate Ligament Injuries/surgery , Apoptosis/physiology , Cell Proliferation/physiology , Fibrocartilage/cytology , Male , Models, Animal , Rabbits , SOX9 Transcription Factor/metabolismABSTRACT
BACKGROUND: To evaluate synchrotron radiation-based Fourier transform infrared microspectroscopy (SR-FTIR) as a tool for quantitative mapping of the content and distribution of the extracellular matrix in decellularized fibrocartilage bioscaffolds, and to provide a new platform for quantitatively characterizing bioscaffolds for tissue engineering. METHODS: Fibrocartilage was harvested and cut into book-shape bioscaffolds (N = 54), which were then decellularized. The structures and distribution of collagen fibrous and intrinsic ultrastructure in decellularized fibrocartilage bioscaffolds were evaluated by histological staining and scanning electron microscopy (SEM), respectively. The content of collagen and proteoglycan in the cellularized or decellularized bioscaffolds were also measured by SR-FTIR and biochemical assay. RESULTS: Book-shape fibrocartilage decellularized bioscaffolds were successfully obtained. Histological examination revealed that the structure of extracellular matrix endured during decellularization. Histology and DNA quantification analysis confirmed substantial removal of cells during decellularization. SEM demonstrated that intrinsic ultrastructure of the fibrocartilage bioscaffold was also well preserved. SR-FTIR quantitative analysis confirmed that decellularization had a significant effect on the content and distribution of collagen and proteoglycan in fibrocartilage bioscaffolds, these results are confirmed with the biochemical assay results. CONCLUSION: SR-FTIR imaging can capture the histological morphology of decellularized bioscaffolds. Moreover, it can be used for quantitative mapping of the content and distribution of collagen in the bioscaffolds.
Subject(s)
Extracellular Matrix , Fibrocartilage/cytology , Synchrotrons , Tissue Scaffolds , Animals , Extracellular Matrix/metabolism , Female , Fibrocartilage/metabolism , Rabbits , Spectroscopy, Fourier Transform Infrared/methods , Tissue Engineering/methodsABSTRACT
The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.
Subject(s)
Bone Remodeling/physiology , Fibrocartilage/physiology , Mandibular Condyle/physiology , Temporomandibular Joint/physiology , Animals , Fibrocartilage/cytology , Mandibular Condyle/cytology , Mice , Models, Animal , Temporomandibular Joint/cytologyABSTRACT
Angiogenesis is a complex, multicellular process that is critical for bone development and generation. Endochondral ossification depends on an avascular cartilage template that completely remodels into vascularized bone and involves a dynamic interplay among chondrocytes, osteoblasts, and endothelial cells. We have discovered fibrocartilage stem cells (FCSCs) derived from the temporomandibular joint (TMJ) mandibular condyle that generates cartilage anlagen, which is subsequently remodeled into vascularized bone using an ectopic transplantation model. Here we explore FCSC and endothelial cell interactions during vascularized bone formation. We found that a single FCSC colony formed transient cartilage and host endothelial cells may participate in bone angiogenesis upon subcutaneous transplantation in a nude mouse. FCSCs produced an abundance of the proangiogenic growth factor vascular endothelial growth factor A and promoted the proliferation of human umbilical vein endothelial cells (HUVECs). Using a fibrinogen gel bead angiogenesis assay experiment, FCSC cell feeder layer induced HUVECs to form significantly shorter and less sprouts than D551 fibroblast controls, suggesting that FCSCs may initially inhibit angiogenesis to allow for avascular cartilage formation. Conversely, direct FCSC-HUVEC contact significantly enhanced the osteogenic differentiation of FCSCs. To corroborate this idea, upon transplantation of FCSCs into a bone defect microenvironment, FCSCs engrafted and regenerated intramembranous bone. Taken together, we demonstrate that the interactions between FCSCs and endothelial cells are essential for FCSC-derived vascularized bone formation. A comprehensive understanding of the environmental cues that regulate FCSC fate decisions may contribute to deciphering the mechanisms underlying the role of FCSCs in regulating bone formation.
Subject(s)
Bone Regeneration/physiology , Fibrocartilage/cytology , Human Umbilical Vein Endothelial Cells/cytology , Mandibular Condyle/cytology , Neovascularization, Physiologic/physiology , Stem Cells/cytology , Temporomandibular Joint/cytology , Animals , Coculture Techniques , Female , Flow Cytometry , Humans , Immunohistochemistry , Male , Mice , Mice, Nude , Rats , Rats, Sprague-Dawley , Skull/surgeryABSTRACT
Tissue engineering offers promising perspectives in the therapy of osteoarthritis. In the context of cell-based therapy, chondrogenic progenitor cells (CPCs) may be used to regenerate defects in cartilage tissue. An in-depth characterization of the secretome of CPCs is a prerequisite to this approach. In this study, a method was developed for the qualitative and quantitative analysis of the secretome of undifferentiated and differentiated CPCs. Secreted proteins from cells grown in two-dimensional as well as three-dimensional alginate cultures were extracted and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Quantitation was achieved by internal standardization using stable isotope-labeled amino acids in cell culture (SILAC). Qualitative analysis of CPC secretomes revealed ECM-components, signal proteins and growth factors most of which were also found in healthy cartilage. A quantitative comparison revealed significantly upregulated proteins with regenerative potential during differentiation, while proteins involved in catabolic metabolism were significantly downregulated. The development of methods for qualitative and quantitative analysis of the secretome of CPCs by mass spectrometry provides a foundation for the investigation of progenitor or stem cells from other sources.
Subject(s)
Cartilage, Articular/pathology , Osteoarthritis/pathology , Proteins/metabolism , Proteome/chemistry , Stem Cells/metabolism , Adult , Animals , Cartilage, Articular/anatomy & histology , Cartilage, Articular/cytology , Cattle , Cells, Cultured , Chemical Precipitation , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Extracellular Space/chemistry , Fibrocartilage/cytology , Humans , Osteoarthritis/therapy , Proteins/analysis , Proteins/isolation & purification , Proteome/metabolism , Stem Cells/chemistry , Tandem Mass SpectrometryABSTRACT
A nearly avascular tissue, the knee meniscus relies on diffusive transport for nutritional supply to cells. Nutrient transport depends on solute partitioning in the tissue, which governs the amount of nutrients that can enter a tissue. The purpose of the present study was to investigate the effects of mechanical strain, tissue region, and tissue composition on the partition coefficient of glucose in meniscus fibrocartilage. A simple partitioning experiment was employed to measure glucose partitioning in porcine meniscus tissues from two regions (horn and central), from both meniscal components (medial and lateral), and at three levels of compression (0%, 10%, and 20%). Partition coefficient values were correlated to strain level, water volume fraction, and glycosaminoglycan (GAG) content of tissue specimens. Partition coefficient values ranged from 0.47 to 0.91 (n = 48). Results show that glucose partition coefficient is significantly (p < 0.001) affected by compression, decreasing with increasing strain. Furthermore, we did not find a statistically significant effect of tissue when comparing medial versus lateral (p = 0.181) or when comparing central and horn regions (p = 0.837). There were significant positive correlations between tissue water volume fraction and glucose partitioning for all groups. However, the correlation between GAG content and partitioning was only significant in the lateral horn group. Determining how glucose partitioning is affected by tissue composition and loading is necessary for understanding nutrient availability and related tissue health and/or degeneration. Therefore, this study is important for better understanding the transport and nutrition-related mechanisms of meniscal degeneration.
Subject(s)
Fibrocartilage/cytology , Fibrocartilage/metabolism , Glucose/metabolism , Menisci, Tibial/cytology , Stress, Mechanical , Animals , Biomechanical Phenomena , Menisci, Tibial/metabolism , SwineABSTRACT
Tissue regeneration using stem cell-based transplantation faces many hurdles. Alternatively, therapeutically exploiting endogenous stem cells to regenerate injured or diseased tissue may circumvent these challenges. Here we show resident fibrocartilage stem cells (FCSCs) can be used to regenerate and repair cartilage. We identify FCSCs residing within the superficial zone niche in the temporomandibular joint (TMJ) condyle. A single FCSC spontaneously generates a cartilage anlage, remodels into bone and organizes a haematopoietic microenvironment. Wnt signals deplete the reservoir of FCSCs and cause cartilage degeneration. We also show that intra-articular treatment with the Wnt inhibitor sclerostin sustains the FCSC pool and regenerates cartilage in a TMJ injury model. We demonstrate the promise of exploiting resident FCSCs as a regenerative therapeutic strategy to substitute cell transplantation that could be beneficial for patients suffering from fibrocartilage injury and disease. These data prompt the examination of utilizing this strategy for other musculoskeletal tissues.
Subject(s)
Fibrocartilage/cytology , Regeneration , Stem Cell Transplantation , Stem Cells/cytology , Temporomandibular Joint/pathology , Temporomandibular Joint/physiopathology , Wound Healing , Adaptor Proteins, Signal Transducing , Animals , Bone and Bones/pathology , Cell Differentiation , Chondrocytes/pathology , Glycoproteins/metabolism , Homeostasis , Humans , Intercellular Signaling Peptides and Proteins , Male , Mice, Nude , Models, Biological , Rabbits , Rats, Sprague-Dawley , Stem Cell Niche , Wnt Proteins/metabolism , Wnt Signaling PathwayABSTRACT
The purpose of the present study was to investigate the effects of mechanical strain, anisotropy, and tissue region on electrical conductivity and ion diffusivity in meniscus fibrocartilage. A one-dimensional, 4-wire conductivity experiment was employed to measure the electrical conductivity in porcine meniscus tissues from two tissue regions (horn and central), for two tissue orientations (axial and circumferential), and for three levels of compressive strain (0%, 10%, and 20%). Conductivity values were then used to estimate the relative ion diffusivity in meniscus. The water volume fraction of tissue specimens was determined using a buoyancy method. A total of 135 meniscus samples were measured; electrical conductivity values ranged from 2.47mS/cm to 4.84mS/cm, while relative ion diffusivity was in the range of 0.235 to 0.409. Results show that electrical conductivity and ion diffusion are significantly anisotropic (p<0.001), both being higher in the circumferential direction than in the axial direction. Additionally, the findings show that compression significantly affects the electrical conductivity with decreasing conductivity levels corresponding to increased compressive strain (p<0.001). Furthermore, there was no statistically significant effect of tissue region when comparing axial conductivity in the central and horn regions of the tissue (p=0.999). There was a positive correlation between tissue water volume fraction and both electrical conductivity and relative ion diffusivity for all groups investigated. This study provides important insight into the electromechanical and transport properties in meniscus fibrocartilage, which is essential in developing new strategies to treat and/or prevent tissue degeneration.
Subject(s)
Electric Conductivity , Meniscus/metabolism , Stress, Mechanical , Animals , Anisotropy , Diffusion , Fibrocartilage/cytology , Fibrocartilage/metabolism , Humans , Meniscus/cytology , SwineABSTRACT
Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.
Subject(s)
Calcium Signaling , Chondrocytes/metabolism , Fibrocartilage/metabolism , Mechanotransduction, Cellular , Proteoglycans/metabolism , Stress, Mechanical , Adult , Aged , Animals , Cattle , Cells, Cultured , Female , Fibrocartilage/cytology , Humans , Male , Mesenchymal Stem Cells/metabolism , Middle Aged , Tissue Engineering , Weight-BearingABSTRACT
PURPOSE: This study investigated the direct and continuous attachment of the labrum to the glenoid fossa, including the fibrocartilaginous tissue, using image-analysis software and histology. METHODS: Twenty-six cadaveric shoulders (11 male, 15 female; mean age 80.1 years; age range 36-103 years) were used. The glenoid of each specimen was divided into six pie-slice-shaped pieces from the center perpendicular to the articular surface by radial incisions at the 2, 4, 6, 8, 10, and 12 o'clock positions. The general distribution of the labrum, including the fibrocartilage, was assessed in hematoxylin and eosin-, Safranin O- and Azan-Mallory-stained sections. The continuous length of attachment of the labrum to the glenoid was measured using image-analysis software. The width of attachment to the articular surface of the glenoid was assessed in each position. RESULTS: The labrum attached to both the articular surface and the neck of the glenoid in all shoulders (100 %) in the 4 and 6 o'clock positions. The mean length of the entire attachment to the glenoid was 4.6 mm (range 3.2-6.1 mm). The width of attachment from the bony edge of the glenoid to the edge of the labrum on the articular surface ranged from 0 to 4.3 mm. The length of the entire attachment of the labrum was shortest in the 2 o'clock position (p = 0.229). Additionally, the length of the entire attachment of the labrum was longest in the 4 o'clock position. The width of attachment to the articular surface of the glenoid was greatest in the 4 o'clock position (p < 0.01). CONCLUSION: In the 4 and 6 o'clock positions, the labrum attached to both the articular surface and neck of the glenoid in all of the shoulders (100 %). The length of the entire attachment to the labrum, including the fibrocartilage, was shortest in the 2 o'clock position. The width of attachment to the articular surface of the glenoid was greatest in the 4 o'clock position (p < 0.01).
Subject(s)
Glenoid Cavity/anatomy & histology , Scapula/anatomy & histology , Shoulder Joint/anatomy & histology , Adult , Aged , Aged, 80 and over , Cadaver , Female , Fibrocartilage/cytology , Humans , Male , Middle AgedABSTRACT
The sequence of events that leads to the formation of a functionally graded enthesis is not clearly defined. The current study demonstrates that clonal expansion of Gdf5 progenitors contributes to linear growth of the enthesis. Prior to mineralization, Col1+ cells in the enthesis appose Col2+ cells of the underlying primary cartilage. At the onset of enthesis mineralization, cells at the base of the enthesis express alkaline phosphatase, Indian hedgehog, and ColX as they mineralize. The mineralization front then extends towards the tendon midsubstance as cells above the front become encapsulated in mineralized fibrocartilage over time. The hedgehog (Hh) pathway regulates this process, as Hh-responsive Gli1+ cells within the developing enthesis mature from unmineralized to mineralized fibrochondrocytes in response to activated signaling. Hh signaling is required for mineralization, as tissue-specific deletion of its obligate transducer Smoothened in the developing tendon and enthesis cells leads to significant reductions in the apposition of mineralized fibrocartilage. Together, these findings provide a spatiotemporal map of events - from expansion of the embryonic progenitor pool to synthesis of the collagen template and finally mineralization of this template - that leads to the formation of the mature zonal enthesis. These results can inform future tendon-to-bone repair strategies to create a mechanically functional enthesis in which tendon collagen fibers are anchored to bone through mineralized fibrocartilage.
Subject(s)
Fibrocartilage/cytology , Growth Differentiation Factor 5/metabolism , Hedgehog Proteins/metabolism , Minerals/metabolism , Signal Transduction , Stem Cells/cytology , Animals , Bone Marrow/pathology , Bone Resorption/pathology , Bone Resorption/physiopathology , Bone and Bones/physiology , Calcification, Physiologic , Cell Differentiation , Chondrocytes/metabolism , Clone Cells , Collagen/metabolism , Epiphyses/pathology , Integrases/metabolism , Kruppel-Like Transcription Factors/metabolism , Mice , Models, Biological , Osteoclasts/metabolism , Patella/physiology , Staining and Labeling , Stem Cells/metabolism , Tendons/physiology , Zinc Finger Protein GLI1ABSTRACT
We aimed to build a culture model of chondrocytes in vitro, and to study the differential properties between fibrochondrocytes and hyaline chondrocytes. Histological sections were stained with haematoxylin and eosin so that we could analyse the histological structure of the fibrocartilage and hyaline cartilage. Condylar fibrochondrocytes and femoral hyaline chondrocytes were cultured from four, 4-week-old, New Zealand white rabbits. The production of COL2A1, COL1OA1, SOX9 and aggrecan was detected by real time-q polymerase chain reaction (RT-qPCR) and immunoblotting and the differences between them were compared statistically. Histological structures obviously differed between fibrocartilage and hyaline cartilage. COL2A1 and SOX9 were highly expressed within cell passage 2 (P2) of both fibrochondrocytes and hyaline chondrocytes, and reduced significantly after cell passage 4 (P4). The mRNA expressions of COL2A1 (p=0.05), COL10A1 (p=0.04), SOX9 (p=0.03), and aggrecan (p=0.04) were significantly higher in hyaline chondrocytes than in fibrochondrocytes, whereas the expression of COL1A1 (p=0.02) was the opposite. Immunoblotting showed similar results. We have built a simple and effective culture model of chondrocytes in vitro, and the P2 of chondrocytes is recommended for further studies. Condylar fibrocartilage and femoral hyaline cartilage have unique biological properties, and the regulatory mechanisms of endochondral ossification for the condyle should be studied independently in the future.
Subject(s)
Chondrocytes/physiology , Fibrocartilage/cytology , Hyaline Cartilage/cytology , Aggrecans/analysis , Animals , Cartilage, Articular/chemistry , Cartilage, Articular/cytology , Cell Culture Techniques , Cell Proliferation , Cells, Cultured , Chondrocytes/chemistry , Collagen Type I/analysis , Collagen Type II/analysis , Collagen Type X/analysis , Female , Femur/chemistry , Femur/cytology , Fibrocartilage/chemistry , Hyaline Cartilage/chemistry , Hypertrophy , Mandibular Condyle/chemistry , Mandibular Condyle/cytology , Rabbits , SOX9 Transcription Factor/analysisABSTRACT
Tendon attaches to bone across a specialized tissue called the enthesis. This tissue modulates the transfer of muscle forces between two materials, i.e. tendon and bone, with vastly different mechanical properties. The enthesis for many tendons consists of a mineralized graded fibrocartilage that develops postnatally, concurrent with epiphyseal mineralization. Although it is well described that the mineralization and development of functional maturity requires muscle loading, the biological factors that modulate enthesis development are poorly understood. By genetically demarcating cells expressing Gli1 in response to Hedgehog (Hh) signaling, we discovered a unique population of Hh-responsive cells in the developing murine enthesis that were distinct from tendon fibroblasts and epiphyseal chondrocytes. Lineage-tracing experiments revealed that the Gli1 lineage cells that originate in utero eventually populate the entire mature enthesis. Muscle paralysis increased the number of Hh-responsive cells in the enthesis, demonstrating that responsiveness to Hh is modulated in part by muscle loading. Ablation of the Hh-responsive cells during the first week of postnatal development resulted in a loss of mineralized fibrocartilage, with very little tissue remodeling 5 weeks after cell ablation. Conditional deletion of smoothened, a molecule necessary for responsiveness to Ihh, from the developing tendon and enthesis altered the differentiation of enthesis progenitor cells, resulting in significantly reduced fibrocartilage mineralization and decreased biomechanical function. Taken together, these results demonstrate that Hh signaling within developing enthesis fibrocartilage cells is required for enthesis formation.
Subject(s)
Fibrocartilage/cytology , Hedgehog Proteins/metabolism , Muscles/physiology , Animals , Animals, Newborn , Biomechanical Phenomena , Botulinum Toxins/toxicity , Calcification, Physiologic , Integrases/metabolism , Mice, Transgenic , Models, Biological , Paralysis/chemically induced , Paralysis/pathology , Signal Transduction , Weight-Bearing , X-Ray MicrotomographyABSTRACT
A recent development in the field of tissue engineering is the rise of all-biologic, scaffold-free engineered tissues. Since these biomaterials rely primarily upon cells, investigation of initial seeding densities constitutes a particularly relevant aim for tissue engineers. In this study, a scaffold-free method was used to create fibrocartilage in the shape of the rabbit knee meniscus. The objectives of this study were to: (i) determine the minimum seeding density, normalized by an area of 44 mm(2), necessary for the self-assembling process of fibrocartilage to occur; (ii) examine relevant biomechanical properties of engineered fibrocartilage, such as tensile and compressive stiffness and strength, and their relationship to seeding density; and (iii) identify a reduced, or optimal, number of cells needed to produce this biomaterial. It was found that a decreased initial seeding density, normalized by the area of the construct, produced superior mechanical and biochemical properties. Collagen per wet weight, glycosaminoglycans per wet weight, tensile properties and compressive properties were all significantly greater in the 5 million cells per construct group as compared to the historical 20 million cells per construct group. Scanning electron microscopy demonstrated that a lower seeding density results in a denser tissue. Additionally, the translational potential of the self-assembling process for tissue engineering was improved though this investigation, as fewer cells may be used in the future. The results of this study underscore the potential for critical seeding densities to be investigated when researching scaffold-free engineered tissues.
Subject(s)
Cell Culture Techniques/methods , Chondrocytes/metabolism , Fibrocartilage/metabolism , Menisci, Tibial , Tissue Engineering/methods , Animals , Cattle , Cells, Cultured , Chondrocytes/cytology , Fibrocartilage/cytology , RabbitsABSTRACT
BACKGROUND: A combined magnetic field (CMF) is a composite of a dynamic sinusoidal magnetic field and a magnetostatic field. Stimuli from CMFs has proved to be an effective tool for healing problem fractures and spinal fusion procedures. HYPOTHESIS: Combined magnetic field technology will enhance healing of bone-tendon junction repair via endochondral ossification for regeneration of the fibrocartilage zone. STUDY DESIGN: Controlled laboratory study. METHODS: Forty-eight mature rabbits were randomly divided into CMF-treated and placebo-treated (control) groups. A partial patellectomy model was created. The CMF-treated group was subjected to CMF stimulation from the third postoperative day for 30 minutes per day up to weeks 8 or 16. At each time point, tissue samples were harvested and evaluated biomechanically and histomorphologically. The area of newly formed bone and the thickness of fibrocartilage were measured in hematoxylin and eosin-stained sections and toluidine blue-stained sections, respectively, while the density of fibrocartilage cells and the amount of proteoglycans were calculated using safranin O-stained sections. A biomechanical analysis was carried out to ascertain tensile strength. RESULTS: Quantitative histological measurements showed that the newly formed bone and regenerated fibrocartilage zone in the CMF-treated group increased by a respective 99.2% and 41.9% compared with the control group at week 8 and a respective 97.8% and 22.8% at week 16. In the CMF-treated group at postoperative week 16, the amount of proteoglycans was 36.9% more than that of the control group, but the density of fibrocartilage cells was just 71.4% of the control group; there were no significant differences at week 8. Mechanical test results showed that energy to failure was not significantly different between the 2 groups at week 8. Yet, at week 16, load to failure, ultimate strength, and energy to failure in the CMF-treated group (311.0 ± 59.4 N, 8.46 ± 1.41 MPa, and 0.87 ± 0.17 J, respectively) were significantly higher than those in the control group (247.1 ± 65.6 N, 6.84 ± 1.12 MPa, and 0.52 ± 0.15 J, respectively). CONCLUSION: Biophysical stimulation with CMFs enhances healing after bone-tendon junction injuries in a rabbit model. CLINICAL RELEVANCE: These results demonstrate the feasibility of using CMFs for stimulating bone-tendon healing after repair.
Subject(s)
Magnetic Field Therapy , Patella/pathology , Patellar Ligament/pathology , Wound Healing/physiology , Animals , Cell Count , Extracellular Matrix/pathology , Female , Fibrocartilage/cytology , Fibrocartilage/physiology , Microscopy , Models, Animal , Osteogenesis/physiology , Osteotomy , Patella/injuries , Patellar Ligament/injuries , Proteoglycans/metabolism , Rabbits , Random Allocation , Regeneration , Tensile Strength/physiologyABSTRACT
Tissue engineering is a promising field of study toward curing the meniscal deficient stifle; however the ideal cell type for this task is not known. We describe here the extraction of synoviocytes and meniscal fibrochondrocytes from arthroscopic debris from six dogs, which were cultured as tensioned bioscaffolds to synthesize meniscal-like fibrocartilage sheets. Despite the diseased status of the original tissues, synoviocytes and meniscal fibrochondrocytes had high viability at the time of removal from the joint. Glycosaminoglycan and collagen content of bioscaffolds did not differ. Meniscal fibrochondrocyte bioscaffolds contained more type II collagen, but collagen deposition was disorganized, with only 30-40% of cells viable. The collagen of synoviocyte bioscaffolds was organized into sheets and bands and 80-90% of cells were viable. Autologous, diseased meniscal fibrochondrocytes and synoviocytes are plausible cell sources for future meniscal tissue engineering research, however cell viability of meniscal fibrochondrocytes in the tensioned bioscaffolds was low.