Search

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 TGFβ 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 TGFβ-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., (© 2021. The Author(s).)

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.

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., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

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.

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.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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).

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., (Copyright © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

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., (© 2015. Published by The Company of Biologists Ltd.)

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., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (© 2014 The Author(s).)

Meniscal injury is a common cause of osteoarthritis, pain, and disability in dogs and humans, but tissue-engineered bioscaffolds could be a treatment option for meniscal deficiency. The objective of this study was to compare meniscus-like matrix histology, composition, and biomechanical properties of autologous tensioned synoviocyte neotissues (TSN) treated with fetal bovine serum (TSNfbs) or three chondrogenic growth factors (TSNgf). Fourth passage canine synoviocytes from 10 dogs were grown in hyperconfluent monolayer culture, formed into TSN, and then cultured for 3 weeks with 17.7% FBS or three human recombinant TSNgf (bFGF, TGF-β1, and IGF-1). Cell viability was determined with laser microscopy. Histological architecture and the composition of fibrocartilage matrix were evaluated in TSN by staining tissues for glycosaminoglycan (GAG), α-smooth muscle actin, and collagen 1 and 2; quantifying the content of GAG, DNA, and hydroxyproline; and measuring the gene expression of collagens type 1α and 2α, the GAG aggrecan, and transcription factor Sry-type Homeobox Protein-9 (SOX9). Biomechanical properties were determined by materials testing force-deformation curves. The TSN contained components and histological features of mensical fibrocartilage extracellular matrix. Growth factor-treated TSN had higher DNA content but lower cell viability than TSNfbs. TSNgf had greater fibrocartilage-like matrix content (collagen 2 and GAG content with increased collagen 2α and SOX9 gene expression). Additionally, TSNgf collagen was more organized histologically and so had greater tensile biomechanical properties. The results indicate the potential of TSN when cultured with growth factors as implantable bioscaffolds for the treatment of canine meniscal deficiency., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Meniscal injury is a common cause of canine lameness. Tissue engineered bioscaffolds may be a treatment option for dogs suffering from meniscal damage. The aim of this study was to compare in vitro meniscal-like matrix formation and biomechanical properties of porcine intestinal submucosa sheets (SIS), used in canine meniscal regenerative medicine, to synoviocyte-seeded SIS bioscaffold (SSB), cultured with fetal bovine serum (SSBfbs) or chondrogenic growth factors (SSBgf). Synoviocytes from nine dogs were seeded on SIS and cultured for 30days with 17.7% fetal bovine serum or recombinant chondrogenic growth factors (IGF-1, TGFβ1 and bFGF). The effect on fibrochondrogenesis was determined by comparing mRNA expression of collagen types Iα and IIα, aggrecan, and Sry-type homeobox protein-9 (SOX9) as well as protein expression of collagens I and II, glycosaminoglycan (GAG), and hydroxyproline. The effect of synoviocyte seeding and culture conditions on biochemical properties was determined by measuring peak load, tensile stiffness, resilience, and toughness of bioscaffolds. Pre-culture SIS contained 13.6% collagen and 2.9% double-stranded DNA. Chondrogenic growth factor treatment significantly increased SOX9, collagens I and IIα, aggrecan gene expression (P<0.05), and histological deposition of fibrocartilage extracellular matrix (GAG and collagen II). Culture with synoviocytes increased SIS tensile peak load at failure, resilience, and toughness of bioscaffolds (P<0.05). In conclusion, culturing SIS with synoviocytes prior to implantation might provide biomechanical benefits, and chondrogenic growth factor treatment of cultured synoviocytes improves in vitro axial meniscal matrix formation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Meniscal injury is a common cause of lameness in the dog. Tissue engineered bioscaffolds may be a treatment option for meniscal incompetency, and ideally would possess meniscus- like extracellular matrix (ECM) and withstand meniscal tensile hoop strains. Synovium may be a useful cell source for meniscal tissue engineering because of its natural role in meniscal deficiency and its in vitro chondrogenic potential. The objective of this study is to compare meniscal -like extracellular matrix content of hyperconfluent synoviocyte cell sheets ("HCS") and hyperconfluent synoviocyte sheets which have been tensioned over wire hoops (tensioned synoviocyte bioscaffolds, "TSB") and cultured for 1 month., Results: Long term culture with tension resulted in higher GAG concentration, higher chondrogenic index, higher collagen concentration, and type II collagen immunoreactivity in TSB versus HCS. Both HCS and TSB were immunoreactive for type I collagen, however, HCS had mild, patchy intracellular immunoreactivity while TSB had diffuse moderate immunoreactivity over the entire bisocaffold. The tissue architecture was markedly different between TSB and HCS, with TSB containing collagen organized in bands and sheets. Both HCS and TSB expressed alpha smooth muscle actin and displayed active contractile behavior. Double stranded DNA content was not different between TSB and HCS, while cell viability decreased in TSB., Conclusions: Long term culture of synoviocytes with tension improved meniscal- like extra cellular matrix components, specifically, the total collagen content, including type I and II collagen, and increased GAG content relative to HCS. Future research is warranted to investigate the potential of TSB for meniscal tissue engineering.

The knee meniscus, intervertebral disc, and temporomandibular joint (TMJ) disc all possess complex geometric shapes and anisotropic matrix organization. While these characteristics are imperative for proper tissue function, they are seldom recapitulated following injury or disease. Thus, this study's objective was to engineer fibrocartilages that capture both gross and molecular structural features of native tissues. Self-assembled TMJ discs were selected as the model system, as the disc exhibits a unique biconcave shape and functional anisotropy. To drive anisotropy, 50:50 co-cultures of meniscus cells and articular chondrocytes were grown in biconcave, TMJ-shaped molds and treated with two exogenous stimuli: biomechanical (BM) stimulation via passive axial compression and bioactive agent (BA) stimulation via chondroitinase-ABC and transforming growth factor-β1. BM + BA synergistically increased Col/WW, Young's modulus, and ultimate tensile strength 5.8-fold, 14.7-fold, and 13.8-fold that of controls, respectively; it also promoted collagen fibril alignment akin to native tissue. Finite element analysis found BM stimulation to create direction-dependent strains within the neotissue, suggesting shape plays an essential role toward driving in vitro anisotropic neotissue development. Methods used in this study offer insight on the ability to achieve physiologic anisotropy in biomaterials through the strategic application of spatial, biomechanical, and biochemical cues., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The weak intrinsic meniscus healing response and technical challenges associated with meniscus repair contribute to a high rate of repair failures and meniscectomies. Given this limited healing response, the development of biologically active adjuncts to meniscal repair may hold the key to improving meniscal repair success rates. This study demonstrates the development of a bone marrow (BM) adhesive that binds, stabilizes, and stimulates fusion at the interface of meniscus tissues. Hydrogels containing several chondroitin sulfate (CS) adhesive levels (30, 50, and 70 mg/mL) and BM levels (30%, 50%, and 70%) were formed to investigate the effects of these components on hydrogel mechanics, bovine meniscal fibrochondrocyte viability, proliferation, matrix production, and migration ability in vitro. The BM content positively and significantly affected fibrochondrocyte viability, proliferation, and migration, while the CS content positively and significantly affected adhesive strength (ranged from 60±17 kPa to 335±88 kPa) and matrix production. Selected material formulations were translated to a subcutaneous model of meniscal fusion using adhered bovine meniscus explants implanted in athymic rats and evaluated over a 3-month time course. Fusion of adhered meniscus occurred in only the material containing the highest BM content. The technology can serve to mechanically stabilize the tissue repair interface and stimulate tissue regeneration across the injury site.

Tendons are typically composed of two histologically different regions: the midsubstance and insertion site. We previously showed that Gli1, a downstream effector of the hedgehog (Hh) signaling pathway, is expressed only in the insertion site of the mouse patellar tendon during its differentiation. To test for a functional role of Hh signaling, we targeted the Smoothened (Smo) gene in vivo using a Cre/Lox system. Constitutive activation of the Hh pathway in the mid-substance caused molecular markers of the insertion site, e.g. type II collagen, to be ectopically expressed or up-regulated in the midsubstance. This was confirmed using a novel organ culture method in vitro. Conversely, when Smo was excised in the scleraxis-positive cell population, the development of the fibrocartilaginous insertion site was affected. Whole transcriptome analysis revealed that the expression of genes involved in chondrogenesis and mineralization was down-regulated in the insertion site, and expression of insertion site markers was decreased. Biomechanical testing of murine adult patellar tendon, which developed in the absence of Hh signaling, showed impairment of tendon structural properties (lower linear stiffness and greater displacement) and material properties (greater strain), although the linear modulus of the mutant group was not significantly lower than controls. These studies provide new insights into the role of Hh signaling during tendon development.

Objective: To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro., Methods: SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 +/- 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining., Results: SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells., Conclusion: It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

Articular cartilage and the menisci of the knee joint lack intrinsic repair capacity; thus, injuries to these tissues result in eventual osteoarthrotic changes to the joint. Tissue engineering offers the potential to replace damaged cartilage and mitigate long-term debilitating changes to the joint. In an attempt to enhance the ability of adult articular chondrocytes (ACs) and meniscus cells (MCs) to produce robust scaffoldless neocartilage, the effects of passage number, cryopreservation, and redifferentiation prior to construct formation were studied. By increasing passage number, smaller donor biopsies could be used to generate sufficient cells for tissue engineering and, in this study, no detrimental effects were observed when employing passage-4 versus passage-3 cells. Cryopreservation of cells would enable the generation of a cell bank thus reducing lead time and enhancing consistency of cell-based therapies. Interestingly, cryopreservation was shown to enhance the biomechanical properties of the resultant self-assembled constructs. With regard to redifferentiation prior to construct formation, aggregate redifferentiation was shown to enhance the biochemical and biomechanical properties of self-assembled constructs. By increasing passaging number, cryopreserving cells, and applying aggregate redifferentiation prior to neotissue formation, the utility of ACs and MCs in tissue engineering can be enhanced., (Copyright © 2013 S. Karger AG, Basel.)

We determined the precise role of relaxin family peptide (RXFP) receptors-1 and -2 in the regulation of MMP-9 and -13 by relaxin, and delineated the signaling cascade that contributes to relaxin's modulation of MMP-9 in fibrocartilaginous cells. Relaxin treatment of cells in which RXFP1 was silenced resulted in diminished induction of MMP-9 and -13 by relaxin, whereas overexpression of RXFP1 potentiated the relaxin-induced expression of these proteinases. Suppression or overexpression of RXFP2 resulted in no changes in the relaxin-induced MMP-9 and -13. Studies using chemical inhibitors and siRNAs to signaling molecules showed that PI3K, Akt, ERK and PKC-ζ and the transcription factors Elk-1, c-fos and, to a lesser extent, NF-κB are involved in relaxin's induction of MMP-9. Our findings provide the first characterization of signaling cascade involved in the regulation of any MMP by relaxin and offer mechanistic insights on how relaxin likely mediates extracellular matrix turnover., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Aquaporins (AQPs) are a family of hydrophobic membrane channel proteins. The expression of several AQP isoforms has been investigated in different human tissues, including the orofacial region. However, information on the role and localization of AQP1 in joints is limited, and no data are available on aquaporins in the normal temporomandibular joint (TMJ) disc. Sixteen human TMJ discs without degenerative changes were taken from fresh cadavers to investigate the presence and distribution of AQP1 by immunohistochemistry. The aim of the study was to gain additional insights into the biomolecular composition of aquaporins and their role in homeostasis of the TMJ. Porcine TMJ discs were also studied by Western blotting for comparison. Scattered AQP1 immunoexpression was detected in human disc cells, documenting its constitutive expression, but differences amongst the three disc regions were not significant. AQP1 expression was demonstrated in porcine TMJ disc by Western blotting. Our findings suggest that AQP1 is normally expressed in the TMJ disc and confirm a role for it in the maintenance of TMJ homeostasis. Further studies are needed to elucidate expression patterns of aquaporins in diseased TMJ discs., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The ligament-bone interface is a complex structure that comprises ligament, fibrocartilage, and bone. We hypothesize that mesenchymal stem cells cocultured in between ligament and bone cells, on a hybrid silk scaffold with sections suitable for each cell type, would differentiate into fibrocartilage. The section of scaffold for osteoblast seeding was coated with hydroxyapatite. A trilineage coculture system (osteoblasts-BMSCs-fibroblasts) on a hybrid silk scaffold was established. RT-PCR results and immunohistochemistry results demonstrated that BMSCs cocultured between fibroblasts and osteoblasts had differentiated into the fibrocartilaginous lineage. The morphological change was also observed by SEM observation. A gradual transition from the uncalcified to the calcified region was formed in the cocultured BMSCs from the region that directly interacted with fibroblasts to the region that directly interacted with osteoblasts. The role of transforming growth factor β3 (TGF-β3) in this trilineage coculture model was also investigated by supplementing the coculture system with 10 ng/mL TGF-β3. The TGF-treated group showed similar results of fibrocartilaginous differentiation of BMSCs with coculture group without TGF-β3 supplement. However, no calcium deposition was found in the cocultured BMSCs in the TGF-treated group. This may indicate TGF-β3 delayed the mineralization process of chondrocytes.

The fibrous tissues prevalent throughout the body possess an ordered structure that underlies their refined and robust mechanical properties. Engineered replacements will require recapitulation of this exquisite architecture in three dimensions. Aligned nanofibrous scaffolds can dictate cell and matrix organization; however, their widespread application has been hindered by poor cell infiltration due to the tight packing of fibers during fabrication. Here, we develop and validate an enabling technology in which tunable composite nanofibrous scaffolds are produced to provide instruction without impediment. Composites were formed containing two distinct fiber fractions: slow-degrading poly(ε-caprolactone) and water-soluble, sacrificial poly(ethylene oxide), which can be selectively removed to increase pore size. Increasing the initial fraction of sacrificial poly(ethylene oxide) fibers enhanced cell infiltration and improved matrix distribution. Despite the removal of >50% of the initial fibers, the remaining scaffold provided sufficient instruction to align cells and direct the formation of a highly organized ECM across multiple length scales, which in turn led to pronounced increases in the tensile properties of the engineered constructs (nearly matching native tissue). This approach transforms what is an interesting surface phenomenon (cells on top of nanofibrous mats) into a method by which functional, 3D tissues (>1 mm thick) can be formed, both in vitro and in vivo. As such, this work represents a marked advance in the engineering of load-bearing fibrous tissues, and will find widespread applications in regenerative medicine.

Introduction: The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs., Methods: Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR., Results: Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs., Conclusions: Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.

Magnesium has recently been explored as a potential biomaterial for degradable orthopedic implants but its effect on fibrocartilage remains unknown. The objective of this study was to assess the effect of high concentrations of magnesium ions on the matrix production of goat costal fibrochondrocytes in vitro. Cells were cultured using a scaffoldless approach with media containing magnesium chloride (MgCl(2)) or magnesium sulfate (MgSO(4)) at concentrations of 20, 50, and 100 mM in addition to the baseline magnesium concentration of 0.8 mM MgSO(4). At 4 weeks, there were no significant differences in compressive tangent modulus and total matrix production between constructs cultured in 20 mM Mg(2+) and the 0.8 mM Mg(2+) control (435 ± 47 kPa). There was a significant decrease in compressive tangent modulus compared to the 0.8 mM Mg(2+) constructs in the 50 mM MgCl(2) and MgSO(4) groups, while the 100 mM groups were not mechanically testable (p < 0.05). The collagen and glycosaminoglycan (GAG) content of the 50 and 100 mM MgCl(2) and MgSO(4) constructs was significantly lower than the control (6.9 ± 0.5% and 16.5 ± 1.3% per dry weight, respectively) (p < 0.05). The results show that goat costal fibrochondrocytes exhibit a high degree of resiliency to magnesium ion concentrations up to 20 mM in vitro.

Study Design: Human annulus fibrosus tissue and cells were analyzed for the presence of chemokine receptors and the migratory effect of selected chemokines., Objective: To investigate spontaneous repair mechanisms and underlying cell recruitment in response to annular tears and degenerative defects., Summary of Background Data: Resorption of herniated disc tissue and the attempt to close annulus tears with repair tissue occur spontaneously. Although chemokines are suggested to play a role in resorption of herniated disc tissue, the role of chemokines in annulus fibrosus homeostasis and repair remains unclear., Methods: Cells were isolated from annulus fibrosus tissue and expanded in the presence of human serum. Multiwell chemotaxis assays were used to analyze the migratory effect of human serum and 0 to 1000 nM concentrations of the chemokines CXCL7, CXCL10, CXCL12, CCL25, and XCL1 on annulus fibrosus cells (AFCs) (n = 9 per chemokine and dose). Presence of corresponding chemokine receptors in AFCs was determined by real-time polymerase chain reaction analysis and immunohistochemistry., Results: Serum (0.1%-10%) significantly (P < 0.01) stimulates the migration of AFCs. Compared with untreated cells, the migration of cells was significantly (P < 0.01) enhanced upon stimulation with 100 to 1000 nM CXCL10 and 1000 nM XCL1. Chemokine receptors showed low expression levels in expanded AFCs as assessed by polymerase chain reaction. Immunohistochemical staining of the CXCL10 receptor CXCR3 and the XCL1 receptor XCR1 showed that the presence of the particular receptors in AFCs expanded under conventional cell culture conditions. In native annulus fibrosus tissue, CXCR3 was evident, whereas XCR1 could not be detected., Conclusion: The findings suggest that chemokines, in particular CXCL10, effectively recruit isolated AFCs. This suggests that chemokines are involved in annulus fibrosus homeostasis and potentially in spontaneous annulus repair attempts. This might have important implications for biological annulus-sealing strategies.

Various mechanical stresses in vivo induce disc cell apoptosis and intervertebral disc (IVD) degeneration, but the underlying molecular mechanism is not fully known. The aim of this study was to investigate the role of endoplasmic reticulum stress in cyclic stretch-induced apoptosis of rat annulus fibrosus (AF) cells. Flexercell Tension Plus system was used to apply cyclic stretch to rat annulus fibrosus cells at a frequency of 0.5 Hz with 20% elongation for 12, 24, 36, or 48 h. Apoptosis was detected by flow cytometry, and nuclei morphologic changes were visualized by Hoechst 33258 staining and caspase-8, 9 activity assays. The expression of the markers of endoplasmic reticulum stress including CHOP, GRP78, and caspase-12 were determined by RT-PCR and Western blot. Mitochondrial membrane potential change was observed by JC-1 staining in situ. In addition, the levels of the nitric oxide (NO) were determined with the Griess reaction and fluorescence staining. The results indicated that cyclic stretch at a frequency of 0.5 Hz with 20% elongation-induced apoptosis in rat AF cells. Prolonged exposure of the unphysiologically cyclic stretch to AF cells caused NO overproduction, up-regulation of endoplasmic reticulum stress markers including CHOP, GRP78, and caspase-12, depolarization of mitochondria and activation of caspase-9. However, cyclic stretch at this level had no effect on caspase-8 activity. In addition, specific inhibitor of caspase-12 (Z-ATAD-FMK) and caspase-9 (Z-LEHD-FMK) partly suppressed cyclic stretch-induced AF cell apoptosis and the anti-apoptotic effects of the caspase inhibitors were additive. Our data suggest that endoplasmic reticulum stress, likely mediated by NO, contributes to the AF cell apoptosis induced by cyclic stretch in addition to the mitochondrial pathway. These findings could be helpful to understand the mechanism of disc cell apoptosis, the root cause of IVD degeneration.

Fibrocartilaginous tissues such as the meniscus serve critical load-bearing roles, relying on arrays of collagen fibers to resist tensile loads experienced with normal activity. As these structures are frequently injured and possess limited healing capacity, there exists great demand for tissue-engineered replacements. Toward recreating the structural features of these anisotropic tissues in vitro, we employ scaffolds composed of co-aligned nanofibers that direct mesenchymal stem cell (MSC) orientation and the formation of organized extracellular matrix (ECM). Concomitant with ECM synthesis, the mechanical properties of constructs increase with free-swelling culture, but ultimately failed to achieve equivalence with meniscal fibrocartilage. As mechanical forces are essential to the development and maintenance of musculoskeletal tissues, this work examined the effect of cyclic tensile loading on MSC-laden nanofibrous constructs. We hypothesized that loading would modulate the transcriptional behavior of MSCs, spur the deposition of ECM, and lead to enhancements in construct mechanical properties compared to free-swelling controls. Fiber-aligned scaffolds were seeded with MSCs and dynamically loaded daily in tension or maintained as nonloaded controls for 4 weeks. With mechanical stimulation, fibrous gene expression increased, collagen deposition increased, and the tensile modulus increased by 16% relative to controls. These results show that dynamic tensile loading enhances the maturation of MSC-laden aligned nanofibrous constructs, suggesting that recapitulation of the structural and mechanical environment of load-bearing tissues results in increases in functional properties that can be exploited for tissue engineering applications.

Although much is known about the effects of uniaxial mechanical loading on fibrocartilage development, the stress fields to which fibrocartilaginous regions are subjected to during development are mutiaxial. That fibrocartilage develops at tendon-to-bone attachments and in compressive regions of tendons is well established. However, the three-dimensional (3D) nature of the stresses needed for the development of fibrocartilage is not known. Here, we developed and applied an in vitro system to determine whether fibrocartilage can develop under a state of periodic hydrostatic tension in which only a single principal component of stress is compressive. This question is vital to efforts to mechanically guide morphogenesis and matrix expression in engineered tissue replacements. Mesenchymal stromal cells in a 3D culture were exposed to compressive and tensile stresses as a result of an external tensile hydrostatic stress field. The stress field was characterized through mechanical modeling. Tensile cyclic stresses promoted spindle-shaped cells, upregulation of scleraxis and type one collagen, and cell alignment with the direction of tension. Cells experiencing a single compressive stress component exhibited rounded cell morphology and random cell orientation. No difference in mRNA expression of the genes Sox9 and aggrecan was observed when comparing tensile and compressive regions unless the medium was supplemented with the chondrogenic factor transforming growth factor beta3. In that case, Sox9 was upregulated under static loading conditions and aggrecan was upregulated under cyclic loading conditions. In conclusion, the fibrous component of fibrocartilage could be generated using only mechanical cues, but generation of the cartilaginous component of fibrocartilage required biologic factors in addition to mechanical cues. These studies support the hypothesis that the 3D stress environment influences cell activity and gene expression in fibrocartilage development.

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of tissues to replace diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Mesenchymal progenitors such as bone marrow stromal cells (BMSCs) are an attractive cell source for fibrocartilage tissue engineering, but the types or combinations of signals required to promote fibrochondrocyte-specific differentiation remain unclear. The present study investigated the influences of cyclic tensile loading on the chondrogenesis of BMSCs and the development of engineered fibrocartilage. Cyclic tensile displacements (10%, 1 Hz) were applied to BMSC-seeded fibrin constructs for short (24 h) or extended (1-2 weeks) periods using a custom loading system. At early stages of chondrogenesis, 24 h of cyclic tension stimulated both protein and proteoglycan synthesis, but at later stages, tension increased protein synthesis only. One week of intermittent cyclic tension significantly increased the total sulfated glycosaminoglycan and collagen contents in the constructs, but these differences were lost after 2 weeks of loading. Constraining the gels during the extended culture periods prevented contraction of the fibrin matrix, induced collagen fiber alignment, and increased sulfated glycosaminoglycan release to the media. Cyclic tension specifically stimulated collagen I mRNA expression and protein synthesis, but had no effect on collagen II, aggrecan, or osteocalcin mRNA levels. Overall, these studies suggest that the combination of chondrogenic stimuli and tensile loading promotes fibrochondrocyte-like differentiation of BMSCs and has the potential to direct fibrocartilage development in vitro.

Study Design: To develop a construction algorithm in which electrospun nanofibrous scaffolds are coupled with a biocompatible hydrogel to engineer a mesenchymal stem cell (MSC)-based disc replacement., Objective: To engineer a disc-like angle-ply structure (DAPS) that replicates the multiscale architecture of the intervertebral disc., Summary of Background Data: Successful engineering of a replacement for the intervertebral disc requires replication of its mechanical function and anatomic form. Despite many attempts to engineer a replacement for ailing and degenerated discs, no prior study has replicated the multiscale hierarchical architecture of the native disc, and very few have assessed the mechanical function of formed neo-tissues., Methods: A new algorithm for the construction of a disc analogue was developed, using agarose to form a central nucleus pulposus (NP) and oriented electrospun nanofibrous scaffolds to form the anulus fibrosus region (AF). Bovine MSCs were seeded into both regions and biochemical, histologic, and mechanical maturation were evaluated with in vitro culture., Results: We show that mechanical testing in compression and torsion, loading methods commonly used to assess disc mechanics, reveal equilibrium and time-dependent behaviors that are qualitatively similar to native tissue, although lesser in magnitude. Further, we demonstrate that cells seeded into both AF and NP regions adopt distinct morphologies that mirror those seen in native tissue, and that, in the AF region, this ordered community of cells deposit matrix that is organized in an angle-ply configuration. Finally, constructs demonstrate functional development with long-term in vitro culture., Conclusion: These findings provide a new approach for disc tissue engineering that replicates multi-scale form and function of the intervertebral disc, providing a foundation from which to build a multi-scale, biologic, anatomically and hierarchically relevant composite disc analogue for eventual disc replacement.

The attachment of dissimilar materials is a major challenge because of the high levels of stress that develop at such interfaces. An effective solution to this problem develops at the attachment of tendon (a compliant "soft tissue") to bone (a stiff "hard tissue"). This tissue, the "enthesis", transitions from tendon to bone through gradations in structure, composition, and mechanical properties. These gradations are not regenerated during tendon-to-bone healing, leading to a high incidence of failure after surgical repair. Understanding the development of the enthesis may allow scientists to develop treatments that regenerate the natural tendon-to-bone insertion. Recent work has demonstrated that both biologic and mechanical factors drive the development and morphogenesis of the enthesis. A cascade of biologic signals similar to those seen in the growth plate promotes mineralization of cartilage on the bony end of the enthesis and the formation of fibrocartilage on the tendon end of the enthesis. Mechanical loading is also necessary for the development of the enthesis. Removal of muscle load impairs the formation of bone, fibrocartilage, and tendon at the developing enthesis. This paper reviews recent work on the development of the enthesis, with an emphasis on the roles of biologic and mechanical factors.

Background: Entheses are fibrocartilaginous organs that bridge ligament with bone at their interface and add significant insertional strength. To replace a severely damaged ligament, a tissue-engineered graft preinstalled with interfacial fibrocartilage, which is being regenerated from stem cells, appears to be more promising than ligament-alone graft. Such a concept can be realized by a biomimetic approach of establishing a dynamic communication of stem cells with bone cells and/or ligament fibroblasts in vitro., Aim: The current study has two objectives. The first objective is to demonstrate functional coculture of bone marrow-derived stem cells (BMSCs) with mature bone cells/ligament fibroblasts as evidenced by gap-junctional communication in vitro. The second objective is to investigate the role of BMSCs in the regeneration of fibrocartilage within the coculture., Materials & Methods: Rabbit bone/ligament fibroblasts were dual-stained with DiI-Red and calcein (gap-junction permeable dye), and cocultured with unlabeled BMSCs at fixed ratio (1:10). The functional gap junction was demonstrated by the transfer of calcein from donor to recipient cells that was confirmed and quantified by flow cytometry. Type 2 collagen (cartilage extracellular matrix-specific protein) expressed by the mixed cell lines in the cocultures were estimated by real-time reverse transcription PCR and compared with that of the ligament-bone coculture (control)., Results: Significant transfer of calcein into BMSCs was observed and flow cytometry analyses showed a gradual increase in the percentage of BMSCs acquiring calcein with time. Cocultures that included BMSCs expressed significantly more type 2 collagen compared with the control., Conclusion: The current study, for the first time, reported the expression of gap-junctional communication of BMSCs with two adherent cell lines of musculoskeletal system in vitro and also confirmed that incorporation of stem cells augments fibrocartilage regeneration. The results open up a path to envisage a composite graft preinstalled with enthesial fibrocartilage using a stem cell-based coculture system.

The immature disc nucleus pulposus (NP) consists of notochordal cells (NCs). With maturation NCs disappear in humans, to be replaced by chondrocyte-like mature NP cells (MNPCs); this change in cell phenotype coincidences with early signs of disc degeneration. The reasons for NC disappearance are important to understand disc degeneration, but remain unknown, yet. This study investigated, whether loading induced a change from a notochordal nucleus phenotype to a chondrocyte-like one. An in vivo disc compression model with fixateur externe was used in 36 mature rabbits. Discs were compressed for different time periods (1, 28, 56 days), and compared with uncompressed control discs (56 days without treatment), and discs with sham compression (28 days). Nucleus cell phenotype was determined by histology and immunohistochemistry. NCs, but not MNPCs highly expressed bone-morphogenetic-protein 2 and cytokeratin 8, thus NC and MNPC numbers could be determined. A histologic score was used to detect structural endplate changes after compression (28 days). Control and sham compressed discs contained around 70% NCs and 30% MNPCs, to be decreased to <10% NCs after 28-56 days of loading. NC density fell sharply by >50% after 28-56 days of compression (P < 0.05 vs. controls). Signs of decreased endplate cellularity and increased endplate sclerosis and fibrosis were found after loading. These experiments show that NCs were less resistant to mechanical stress than MNPCs suggesting that increased intradiscal pressures after loading, and limited nutrition through structurally altered endplates could instigate the disappearance of NCs.

Study Design: A novel degenerative disc disease model and sustained delivery method for corticosteroid in male Sprague-Dawley albino rats., Objectives: To develop a model of degenerative disc disease and to determine the effect of continuous sustained release of corticosteroid on the process of degeneration within the traumatized disc., Summary of Background Data: The current modalities of treating symptomatic degenerative disc disease are either conservative or surgical. However, there is no cure for the degenerative process and prevention, therefore, is the ideal treatment. An understanding of the mechanisms involved in disc degeneration is crucial to develop new methods for prevention and treatment, including appropriate delivery systems and dosages of repair factors., Methods: The L5-L6 intervertebral disc was pierced with a 23-gauge needle in 18 rats. The animals received either sham or corticosterone-charged tricalcium phosphate ceramic capsules. The rats were euthanized at 4 weeks. Chondrocytes in the transition zone areas were counted and compared statistically., Results: The surgical technique induced degeneration of the nucleus without evidence of inflammation at adjacent levels when compared with nontraumatized controls. The number of chondrocytes per area was significantly less in the sham group than in the control group. Corticosteroid treatment showed chondrocyte numbers similar to control in 4 of 5 different views of the disc. The anterior region of the disc had 50% less chondrocytes per area than the control; however, the chondrocyte numbers were 50% greater than in the same site from discs of sham animals., Conclusions: The results show the development of a degenerative disc animal model that can be used to test the effects of growth enhancing factors in disc repair. Administration of continuous sustained release of corticosterone can slow the process of degeneration within the traumatized disc in the rat model.

This study established serum-free, chemically defined conditions to generate fibrocartilage with human embryonic stem cells (hESCs). Three sequential experimental phases were performed to eliminate serum because of its variability and antigenic potential and characterize the performance of hESCs in serum-free and serum-based conditions. Each phase used a two-stage modular experiment: chondrogenic differentiation followed by scaffold-less tissue engineering, called self-assembly. Phase I studied serum effects, and showed that a 1% serum chondrogenic medium (CM) during differentiation resulted in uniform constructs, whereas a 20% serum CM did not. Furthermore, a no-serum CM during self-assembly led to a collagen content 50% to 200% greater than a 1% serum CM. Thus, a "serum standard" of 1% serum during differentiation and no serum during self-assembly was carried forward. Phase II compared this with serum-free formulations, using 5% knock-out serum replacer or 1-ng/mL transforming growth factor beta 1 (TGF-beta1). The TGF-beta1 group was chosen as a "serum-free standard" because it performed similarly to the serum standard in terms of morphological, biochemical, and biomechanical properties. In Phase III, the serum-free standard had significantly more collagen (100%) and greater tensile ( approximately 150%) and compressive properties ( approximately 80%) than the serum standard with TGF-beta1 treatment during self-assembly. These advances are important to the understanding of mechanisms of chondrogenesis and creating clinically relevant stem cell therapies.

The current brief review focuses on certain issues relating to form-function relationships that are evident at tendon or ligament attachment sites (entheses). It evaluates the development of entheses (both fibrocartilaginous and fibrous) and highlights again an issue largely ignored for decades - i.e. how entheses attached to the metaphyses of long bones manage to keep the same relative position as the bones grow in length. Attention is drawn to the manner in which enthesis fibrocartilage prevents direct cell-cell communication between osteocytes and tendon/ligament cells and how (in a healthy enthesis) it presents a physical barrier separating the blood supply of bone from that of tendon/ligament. The possibility that the thoracolumbar fascia, with its multitude of muscular associations and numerous sites of ligamentous attachment could increase stress concentration at entheses is raised, the structure and development of enthesophytes (bony spurs) is reviewed as is the concept of a synovio-entheseal complex (SEC). How these functional anatomical units (SECs) could trigger pain and inflammation in athletes is briefly discussed.

OBJECTIVES - Relaxin induces the matrix metalloproteinase MMP-1 (collagenase-1) in TMJ fibrocartilaginous cells, and this response is potentiated by beta-estradiol. We identified the MMP-1 promoter sites and transcription factors that are induced by relaxin with or without beta-estradiol in fibrocartilaginous cells. MATERIAL AND METHODS - Early passage cells were transiently transfected with the pBLCAT2 plasmid containing specific segments of the human MMP-1 promoter regulating the chloramphenicol acyl transferase (CAT) gene and co-transfected with a plasmid containing the beta-galactosidase gene. The cells were cultured in serum-free medium alone or medium containing 0.1 ng/ml relaxin, or 20 ng/ml beta-estradiol or both hormones, and lysates assayed for CAT and beta-galactosidase activity. RESULTS - Cells transfected with the -1200/-42 or -139/-42 bp MMP-1 promoter-reporter constructs showed 1.5-fold and 3-fold induction of CAT by relaxin in the absence or presence of beta-estradiol, respectively. Relaxin failed to induce CAT in the absence of the -137/-69 region of the MMP-1 promoter, which contains the AP-1-and PEA3-binding sites. Using wild type or mutated minimal AP-1 and PEA-3 promoters we found that both these promoter sites are essential for the induction of MMP-1 by relaxin. The mRNAs for transcription factors c-fos and c-jun, which together form the AP-1 heterodimer, and Ets-1 that modulates the PEA-3 site, were upregulated by relaxin or beta-estradiol plus relaxin. CONCLUSION - These studies show that both the AP-1 and PEA-3 promoter sites are necessary for the induction of MMP-1 by relaxin in fibrocartilaginous cells.

Objective: To compare temporomandibular joint (TMJ) condylar cartilage cells in vitro to hyaline cartilage cells cultured in a three-dimensional (3D) environment for tissue engineering of mandibular condylar cartilage., Design: Mandibular condylar cartilage and hyaline cartilage cells were harvested from pigs and cultured for 6 weeks in polyglycolic acid (PGA) scaffolds. Both types of cells were treated with glucosamine sulfate (0.4 mM), insulin-like growth factor-I (IGF-I) (100 ng/ml) and their combination. At weeks 0 and 6, cell number, glycosaminoglycan (GAG) and collagen content were determined, types I and II collagen were visualized by immunohistochemistry and GAGs were visualized by histology., Results: Hyaline cartilage cells produced from half an order to a full order of magnitude more GAGs and collagen than mandibular condylar cartilage cells in 3D culture. IGF-I was a highly effective signal for biosynthesis with hyaline cartilage cells, while glucosamine sulfate decreased cell proliferation and biosynthesis with both types of cells. In vitro culture of TMJ condylar cartilage cells produced a fibrous tissue with predominantly type I collagen, while hyaline cartilage cells formed a fibrocartilage-like tissue with types I and II collagen. The combination of IGF and glucosamine had a synergistic effect on maintaining the phenotype of TMJ condylar cells to generate both types I and II collagen., Conclusion: Given the superior biosynthetic activity by hyaline cartilage cells and the practical surgical limitations of harvesting cells from the TMJ of a patient requiring TMJ reconstruction, cartilage cells from elsewhere in the body may be a potentially better alternative to cells harvested from the TMJ for TMJ tissue engineering. This finding may also apply to other fibrocartilages such as the intervertebral disc and knee meniscus in applications where a mature cartilage cell source is desired.

Study Design: Experimental and descriptive study of a xenotransplantation model in minipigs., Objective: To study survival and function of human mesenchymal stem cells (hMSCs) after transplantation into injured porcine spinal discs, as a model for cell therapy., Summary of Background Data: Biologic treatment options of the intervertebral disc are suggested for patients with chronic low back pain caused by disc degeneration., Methods: Three lumbar discs in each of 9 minipigs were injured by aspiration of the nucleus pulposus (NP), 2 weeks later hMSCs were injected in F12 media suspension (cell/med) or with a hydrogel carrier (Puramatrix) (cell/gel). The animals were sacrificed after 1, 3, or 6 months. Disc appearance was visualized by magnetic resonance imaging. Immunohistochemistry methods were used to detect hMSCs by antihuman nuclear antibody staining, and further performed for Collagen II, Aggrecan, and Collagen I. SOX 9, Aggrecan, Versican, Collagen IA, and Collagen IIA and Collagen IIB human mRNA expression was analyzed by real-time PCR., Results: At magnetic resonance imaging all injured discs demonstrated degenerative signs. Cell/gel discs showed fewer changes compared with cell/med discs and only injured discs at later time points. hMSCs were detected in 9 of 10 of the cell/gel discs and in 8 of 9 of the cell/med discs. Immunostaining for Aggrecan and Collagen type II expression were observed in NP after 3 and 6 months in gel/cell discs and colocalized with the antihuman nuclear antibody. mRNA expression of Collagen IIA, Collagen IIB, Versican, Collagen 1A, Aggrecan, and SOX9 were detected in both cell/med and cell/gel discs at the time points 3 and 6 months by real-time PCR., Conclusion: hMSCs survive in the porcine disc for at least 6 months and express typical chondrocyte markers suggesting differentiation toward disc-like cells. As in autologous animal models the combination with a three-dimensional-hydrogel carrier seems to facilitate differentiation and survival of MSCs in the disc. Xenotransplantation seems to be valuable in evaluating the possibility for human cell therapy treatment for intervertebral discs.