Minispheroids as a Tool for Ligament Tissue Engineering: Do the Self-Assembly Techniques and Spheroid Dimensions Influence the Cruciate Ligamentocyte Phenotype?

Spheroid culture might stabilize the ligamentocyte phenotype. Therefore, the phenotype of lapine cruciate ligamentocyte (L-CLs) minispheroids prepared either by hanging drop (HD) method or by using a novel spheroid plate (SP) and the option of methyl cellulose (MC) for tuning spheroid formation was tested. A total of 250 and 1000 L-CLs per spheroid were seeded as HDs or on an SP before performing cell viability assay, morphometry, gene expression (qRT-PCR) and protein immunolocalization after 7 (HD/SP) and 14 (SP) days. Stable and viable spheroids of both sizes could be produced with both methods, but more rapidly with SP. MC accelerated the formation of round spheroids (HD). Their circular areas decreased significantly during culturing. After 7 days, the diameters of HD-derived spheroids were significantly larger compared to those harvested from the SP, with a tendency of lower circularity suggesting an ellipsoid shape. Gene expression of decorin increased significantly after 7 days (HD, similar trend in SP), tenascin C tended to increase after 7 (HD/SP) and 14 days (SP), whereas collagen type 1 decreased (HD/SP) compared to the monolayer control. The cruciate ligament extracellular matrix components could be localized in all mini-spheroids, confirming their conserved expression profile and their suitability for ligament tissue engineering.

Maintenance of Ligament Homeostasis of Spheroid-Colonized Embroidered and Functionalized Scaffolds after 3D Stretch.

Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.

Cyclically stretched ACL fibroblasts emigrating from spheroids adapt their cytoskeleton and ligament-related expression profile.

Mechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell-cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.

Mechanoreceptors observed in a ligamentous structure between the posterior horn of the lateral meniscus and the anterior cruciate ligament.

PURPOSE: A histological study of a structure between the posterior horn of the lateral meniscus and the anterior cruciate ligament. METHODS: Bilateral fresh-frozen cadaveric knees of two male donors (age 71 and 76 years) with no history of prior knee injury were examined. All dissections were performed by one experienced orthopaedic surgeon. Haematoxylin and Eosin staining was used to reveal tissue morphology. Goldner trichrome staining was used to evaluate the connective tissue. S100 and PGP 9.5 labelling were used for immunohistochemical analysis. RESULTS: In all cadaveric knees, a structure between the posterior horn of the lateral meniscus and the anterior cruciate ligament was identified. Histological analysis confirmed the ligamentous nature of this structure. Furthermore, Golgi tendon organs were observed within the ligamentous structure. CONCLUSION: This is the first study showing the presence of mechanoreceptors within the ligamentous structure between the posterior horn of the lateral meniscus and the anterior cruciate ligament. The ligamentous structure could contribute to stability of the knee by providing proprioceptive input, while preservation of the ligamentous structure might ensure a better functional outcome after surgery.

Mechanical injury and IL-1ß regulated LOXs and MMP-1, 2, 3 expression in ACL fibroblasts co-cultured with synoviocytes.

OBJECTIVE: Interleukin (IL)-1ß in the joint cavity increases to promote healing after anterior cruciate ligament (ACL) injury. Synovial tissue is a major joint microenvironmental regulator after ACL injury. The purpose of this study was to investigate the effects of synovial cells (SCs) on lysyl oxidase (LOX) and matrix metalloproteinase (MMP) production by ACL fibroblasts (ACLfs) in the presence of IL-1ß. RESULTS: This study sheds light on the regulation of LOX and MMP-1, -2, -3 expression by ACLfs co-cultured with SCs and treated with IL-1ß. LOX and MMP-1, 2, 3 gene/protein expression in IL-1ß/stretch-stimulated ACLfs co-cultured with SCs were measured by real-time quantitative PCR and Western blot. Meanwhile, MMP-2 activity was analyzed by zymogram. The results showed that co-culture with SCs increased LOX and MMP-1, -2, -3 gene and protein expression in the presence of IL-1ß. Next, ACLfs were subjected to 12% mechanical stretch to simulate pathological injury. Under these conditions, SCs inhibited IL-1ß-mediated upregulation of LOXs. However, IL-1ß enhanced the expression of MMP-1, -2, -3 in injured ACLfs. CONCLUSIONS: SCs can either inhibit or increase LOX production in the presence of IL-1ß, while promoting the accumulation of MMP in injured ACLfs. These results may provide crucial insights into the mechanisms underlying ACL poor healing capacity after injury.

Mechano growth factor pretreatment yield mechanical stimuli induced cell stress responses in ligament fibroblasts of osteoarthritis via activating ATF-2.

OBJECTIVE: The purpose of this study is to investigate whether mechanical growth factor (MGF) promotes mechanical response to ligament fibroblasts in osteoarthritis knee cavity via activating transcription factor 2 (ATF-2). RESULTS: Osteoarthritis ligament fibroblasts (OA-LFs) were suffered from 12% static mechanical stretch to mimic mechanical force mediated ligament injury. Meanwhile, OA-LFs were treated with MGF before and during mechanical stretch. We observed that OA delayed LFs response to mechanical injury, while MGF pretreatment promoted cells timely feedback the mechanically stimuli by inducing cellular stress. Additionally, MGF accelerated the ligament injury repair by promoting cell migration, decreasing the MMP-2 activity, and remitting the cell deformation via ATF-2 activating in cells. CONCLUSIONS: Our study shows that MGF pretreatment of OA-LFs can respond quickly to mechanical damage and repair ligament tissue by activating ATF-2. Therefore, MGF has potential as a therapeutic for OA patients.

Anterior cruciate ligament remnant cells have different potentials for cell differentiation based on their location.

Histological and cytological observations of the human anterior cruciate ligament (ACL) had been described, but the differentiation potency based on their location is still unknown. To determine and compare proliferation and differentiation potential of cells derived from distal and middle thirds of the ACL remnant, ACL remnant was initially marked at the distal third (within 10 mm from the tibial insertion) and middle third (between 10-20 mm from the tibial insertion) and then dissected. Both the middle and distal third regions of ACL remnant were analyzed using CD34+ cell counting. Cell proliferation rate did not differ in both middle and distal third regions of ACL remnant, but they showed different characteristics in cell differentiation depending on their location. The distal third region of the ACL remnant had a tendency for chondrogenic differentiation with higher expression of CD34+ cells. On the other hand, the middle third region of ACL remnant had a strong tendency for osteogenic and ligamentous differentiation. Characteristics of the ACL remnant tissues should be considered when performing remnant-preserving or harvesting ACL remnants for tissue engineering.

Enhanced Growth of Lapine Anterior Cruciate Ligament-Derived Fibroblasts on Scaffolds Embroidered from Poly(l-lactide-co-ε-caprolactone) and Polylactic Acid Threads Functionalized by Fluorination and Hexamethylene Diisocyanate Cross-Linked Collagen Foams.

Reconstruction of ruptured anterior cruciate ligaments (ACLs) is limited by the availability and donor site morbidity of autografts. Hence, a tissue engineered graft could present an alternative in the future. This study was undertaken to determine the performance of lapine (L) ACL-derived fibroblasts on embroidered poly(l-lactide-co-ε-caprolactone) (P(LA-CL)) and polylactic acid (PLA) scaffolds in regard to a tissue engineering approach for ACL reconstruction. Surface modifications of P(LA-CL)/PLA by gas-phase fluorination and cross-linking of a collagen foam using either ethylcarbodiimide (EDC) or hexamethylene diisocyanate (HMDI) were tested regarding their influence on cell adhesion, growth and gene expression. The experiments were performed using embroidered P(LA-CL)/PLA scaffolds that were seeded dynamically or statically with LACL-derived fibroblasts. Scaffold cytocompatibility, cell survival, numbers, metabolic activity, ultrastructure and sulfated glycosaminoglycan (sGAG) synthesis were evaluated. Quantitative real-time polymerase chain reaction (QPCR) revealed gene expression of collagen type I (COL1A1), decorin (DCN), tenascin C (TNC), Mohawk (MKX) and tenomodulin (TNMD). All tested scaffolds were highly cytocompatible. A significantly higher cellularity and larger scaffold surface areas colonized by cells were detected in HMDI cross-linked and fluorinated scaffolds compared to those cross-linked with EDC or without any functionalization. By contrast, sGAG synthesis was higher in controls. Despite the fact that the significance level was not reached, gene expressions of ligament extracellular matrix components and differentiation markers were generally higher in fluorinated scaffolds with cross-linked collagen foams. LACL-derived fibroblasts maintained their differentiated phenotype on fluorinated scaffolds supplemented with a HMDI cross-linked collagen foam, making them a promising tool for ACL tissue engineering.

SV40 Transfected Human Anterior Cruciate Ligament Derived Ligamentocytes-Suitable as a Human in Vitro Model for Ligament Reconstruction?

Cultured human primary cells have a limited lifespan undergoing dedifferentiation or senescence. Anterior cruciate ligaments (ACL) are hypocellular but tissue engineering (TE) requires high cell numbers. Simian virus (SV) 40 tumor (T) antigen expression could extend the lifespan of cells. This study aimed to identify cellular changes induced by SV40 expression in human ACL ligamentocytes by comparing them with non-transfected ligamentocytes and tissue of the same donor to assess their applicability as TE model. Human ACL ligamentocytes (40-year-old female donor after ACL rupture) were either transfected with a SV40 plasmid or remained non-transfected (control) before monitored for SV40 expression, survival, and DNA content. Protein expression of cultured ligamentocytes was compared with the donor tissue. Ligamentocyte spheroids were seeded on scaffolds embroidered either from polylactic acid (PLA) threads solely or combined PLA and poly (L-lactide-co-ε-caprolactone) (P(LA-CL)) threads. These scaffolds were further functionalized with fluorination and fibrillated collagen foam. Cell distribution and survival were monitored for up to five weeks. The transfected cells expressed the SV40 antigen throughout the entire observation time, but often exhibited random and incomplete cell divisions with significantly more dying cells, significantly more DNA and more numerous nucleoli than controls. The expression profile of non-transfected and SV40-positive ligamentocytes was similar. In contrast to controls, SV40-positive cells formed larger spheroids, produced less vimentin and focal adhesions and died on the scaffolds after 21 d. Functionalized scaffolds supported human ligamentocyte growth. SV40 antigen expressing ligamentocytes share many properties with their non-transfected counterparts suggesting them as a model, however, applicability for TE is limited.

Ultrastructural characterization of cells in the tibial stump of ruptured human anterior cruciate ligament, their changes and significance with duration of injury.

Fibroblasts and myofibroblasts have been known to be present in both ruptured and intact human anterior cruciate ligament (ACL), and although their relevant histology and immunochemistry have been studied in the past, ultrastructural features of these cells are largely lacking. Therefore, we aim to characterise the ultrastructural details of these cells with the help of transmission electron microscopy (TEM) and to study the changes and their significance with duration of injury. Samples from 60 ruptured human ACL undergoing surgery were obtained and categorised according to duration of injury and observed under TEM with main focus on the following ultrastructural features: cellular morphology, presence of rough endoplasmic reticulum, Golgi apparatus, lamina, myofilaments, and presence of myofibroblasts. These features were further correlated with the duration of injury and association, if any, determined using appropriate statistical analysis. A total of 54 male and 6 female patients with mean duration of the injury of 23.01 ± 26.09 weeks (2-108 weeks) were included in the study and categorised into five groups based on duration of injury as follows: I (< 6 weeks), II (7-12 weeks), III (13-20 weeks), IV (21-50 weeks) and V (> 50 weeks). There was a significant association between the above-mentioned ultrastructural features and the duration of injury (p < 0.05) except for the presence of ovoid fibroblast cells (p = 0.53). Furthermore, number of myofibroblasts and cells with Golgi apparatus and rough endoplasmic reticulum was seen to peak at 13-20 weeks following injury. We describe ultrastructural features of fibroblast of different morphology along with myofibroblasts in the ligaments following injury, the changes in which might have a potential bearing on ligament healing.

Generation of Human-Induced Pluripotent Stem Cells From Anterior Cruciate Ligament.

Human-induced pluripotent stem cells (hiPSCs) are reprogrammed somatic cells and are an excellent cell source for tissue engineering applications, disease modeling, and for understanding human development. HiPSC lines have now been generated from a diverse range of somatic cell types and have been reported to retain an epigenetic memory of their somatic origin. To date, the reprogramming of a true ligament has not been reported. The aim of this study is to generate iPSCs from human anterior cruciate ligament (ACL) cells. ACL cells from three above-knee amputation donors, with donor matched dermal fibroblasts (DFs) were tested for reprogramming using an existing DF reprogramming protocol. ACL cells were, however, more sensitive than donor matched DF to transforming growth factor-ß (TGF-ß); displaying marked contraction, increased proliferation and increased TNC and COMP expression in vitro, which hindered reprogramming to iPSCs. Modification of the protocol by scoring the cell monolayer or by removal of TGF-ß during ACL reprogramming resulted in emerging colonies being easier to identify and extract, increasing reprogramming efficiency. Following 30 passages in culture, the generated ACL derived iPSCs displayed pluripotency markers, normal karyotype and can successfully differentiate to cells of the three embryonic germ layers. This study illustrates it is possible to generate hiPSCs from ligament and identifies optimized ligament reprogramming conditions. ACL derived iPSCs may provide a promising cell source for ligament and related tissue engineering applications. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:92-104, 2020.

Analysis of early cellular responses of anterior cruciate ligament fibroblasts seeded on different molecular weight polycaprolactone films functionalized by a bioactive poly(sodium styrene sulfonate) polymer.

With the growing number of anterior cruciate ligament (ACL) ruptures and the increased interest for regenerative medicine procedures, many studies are now concentrated on developing bioactive and biodegradable synthetic ligaments. For this application, the choice of raw materials with appropriate physicochemical characteristics and long-term degradation features is essential. Polycaprolactone (PCL) has the advantage of slow degradation that depends on its molecular weight. This study evaluates two PCL materials: a technical grade (PC60: 60 kDa) versus a medical grade (PC12: 80 kDa), both before and after functionalization with poly(sodium styrene sulfonate) (pNaSS). After determining the grafting process had little to no effect on the PCL physicochemical properties, sheep ACL fibroblast responses were investigated. The PC12 films induced a significantly lower expression of the tumor necrosis factor alpha inflammatory gene compared to the PC60 films. Both film types induced an overproduction of fibroblast growth factor-2 and transforming growth factor beta compared to the controls on day 5 and demonstrated collagen gene expression profiles similar to the controls on day 7. Upon protein adsorption, pNaSS grafting caused a rapid cell adhesion in the first 30 min and an increased adhesion strength (1.5-fold higher). Moreover, after 7 days, an increase in cell density and actin network development were noted on the grafted films.

Enhancement of in vitro proliferation and bioactivity of human anterior cruciate ligament fibroblasts using an in situ tissue isolation method and basic fibroblast growth factor culture conditions: A pilot analysis.

BACKGROUND: Previous studies have reported poor proliferation and bioactivity of human anterior cruciate ligament fibroblasts (hACLFs) after injury. As hACLFs are one of the most significant and indispensable source of seed cells in constructing tissue-engineered ligament, enhancing hACLF proliferation would offer favorable cellular-biological ability and induce the extracellular matrix secretion of hACLFs after loading on multiple types of scaffolds. Enhancing the bioactivity of hACLFs would improve tissue repair and functional recovery after tissue-engineered ligament transplantation. This study compared cells prepared by collagenase digestion and the in situ culture of tissue pieces and investigated the effect of basic fibroblast growth factor (bFGF) on hACLFs. METHODS: Six adult patients participated in this study. Of these patients, tissues from three were compared after culture establishment through collagenase digestion or in situ tissue isolation. hACLF phenotypic characteristics were assessed, and the effect of bFGF on hACLF cultures was observed. hACLFs cultured with and without bFGF served as the experimental and control groups, respectively. Cell Counting Kit-8 was used to detect proliferation. The expression of ligament-related genes and proteins was evaluated by immunofluorescence staining, real-time polymerase chain reaction (PCR) assays, and Western blot assays. RESULTS: The morphology of hACLFs isolated using the two methods differed after the 2nd passage. The proliferation of cells obtained by in situ culture was higher than that of cells obtained by collagenase digestion. hACLFs cultured with bFGF after the 3rd passage exhibited a higher proliferation rate than the controls. Immunofluorescence staining, real-time PCR, and Western blot analysis showed a significant increase in ligament-related gene and protein expression in the hACLFs cultured with bFGF. CONCLUSIONS: The in situ isolation of tissue pieces enhanced hACLF proliferation in vitro, and the hACLFs exhibited phenotypic characteristics of fibroblasts. hACLFs cultured with bFGF exhibited increased hACLF bioactivity.

Comparative histomorphometric analysis of cellular phenotype in canine stifle ligaments and tendon.

OBJECTIVE: To measure the density of cellular phenotypes in canine caudal cruciate ligament (CaCL), cranial cruciate ligament (CrCL), medial collateral ligament (MCL), and long digital extensor tendon (LDET). STUDY DESIGN: Ex-vivo study. METHODS: Ten CaCL, CrCL, MCL, and LDET obtained from 1 stifle of 10 dogs with no gross pathology were analyzed histologically. The density of cells with 3 nuclear phenotypes (fusiform, ovoid, and spheroid) was determined within the core region of each specimen. RESULTS: Cells with fusiform nuclei were most dense in the MCL (median [range], 319 [118-538] cells/mm2 ) and LDET (331 [61-463]), whereas cells with ovoid nuclei were most dense in the CaCL (276 [123-368]) and CrCL (212 [165-420]). The spheroid nuclear phenotype had the lowest density in all structures (31 [5-61] in CaCL, 54 [5-90] in CrCL, 2 [0-14] in MCL, and 5 [0-80] in LDET); however, the CrCL contained a denser population of spheroid cells compared with MCL and LDET (P < .05). Total cell densities did not differ among the 4 structures (P > .05). CONCLUSION: Phenotype density varied within the ligaments and tendon tested here. The cell population of CaCL and CrCL differed from that of dense collagenous tissues such as MCL and LDET. CLINICAL SIGNIFICANCE: The relatively higher density of spheroid phenotype in CrCL may reflect a distinctive native cellular population or a cellular transformation secondary to unique mechanical environment or hypoxia. This intrinsic cellular population may explain altered tissue properties prone to pathological rupture or poor healing potential of the canine CrCL.

Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells.

Periostin (POSTN), a secretory matricellular matrix protein, plays a multitude of biologic functions. Various splice variants of POSTN have been described; however, their expression pattern and functional implications are not completely understood. This study was undertaken to decipher the differential expression pattern of POSTN and its splice variants in various tissues and cell types. We show that POSTN was more highly expressed in anterior cruciate ligament (ACL) remnants compared with articular cartilage at the cellular and tissue level. Isoforms 1 and 8 were highly expressed only in articular chondrocytes, suggesting their splice-specific regulation in chondrocytes. To discern the role of total POSTN and full-length human POSTN isoform 1 (hPOSTN-001), we stably transfected human chondrosarcoma 1 (hCh-1) cell line with hPOSTN-001 using a pcDNA3.1-hPOSTN-001 construct. RNA-sequencing analysis of hCh-1 cells identified differentially expressed genes with a known role in chondrocyte function and osteoarthritis. Similar expression of a subset of candidate genes was revealed in ACL progenitor cells and chondrocytes as well as in ACL progenitor cells in which POSTN activity was altered by overexpression and by small interfering RNA gene knockdown. Cells expressing total POSTN, not isoform 1, exhibited increased cell adhesion potential. These findings suggest an important role for POSTN in the knee.-Cai, L., Brophy, R. H., Tycksen, E. D., Duan, X., Nunley, R. M., Rai, M. F. Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells.

Gene expression, protein profiling, and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint.

The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.

Identification and Characterization of Canine Ligament Progenitor Cells and Their Extracellular Matrix Niche.

Ligaments are prone to injury and degeneration in humans and animals, however the healing potential of ligament is poor and current treatment options ineffective. Stem cell-based therapies hold potential for treatment of ligament injuries. This study aimed to characterize a ligament progenitor cell (LPC) population and to identify specific niche components which could promote the survival and function of LPCs. LPCs were isolated from canine cranial cruciate ligament and characterized for clonogenicity, multipotency and marker expression. The extracellular matrix (ECM) composition was characterized by the novel application of a metabolic labeling and mass spectrometry technique. LPCs demonstrated clonogenicity, multipotency, and stem cell marker expression. A number of different collagens, glycoproteins, and proteoglycans were identified in the LPC niche using proteomics. Metabolic labeling of cells demonstrated unique turnover profiles for distinct ECM protein groups, indicating the importance of certain niche components for LPC survival and function. The newly synthesized niche components identified in this study could be exploited to aid identification of LPCs and to promote their survival and function for potential ligament repair strategies.

Expression of LOXs and MMP-1, 2, 3 by ACL Fibroblasts and Synoviocytes Impact of Coculture and TNF-α.

This study aims to confirm the effects of synoviocytes (SCs) on regulating lysyl oxidases (LOXs) and matrix metalloproteinase (MMP)-1, 2, 3 in the normal and injured anterior cruciate ligament (ACL) fibroblasts response to tumor necrosis factor-α(TNF-α). The gene and protein expression levels of LOXs and MMP-1, 2, 3 in SCs cocultured ACL fibroblasts (ACLfs) induced by TNF-α and mechanical injury were analyzed by real-time polymerase chain reaction (PCR) and western bolting; the MMP-2 activity were analyzed by zymography. The results exhibited that TNF-α alone slightly downregulated the expressions of LOXs and upregulated the expression of MMP-1, 2, 3 in both normal and injured ACL fibroblasts. The decrease of LOXs and increase of MMP-1, 2, 3 in ACLfs response to TNF-α were further promoted by coculture. Taken together, these results show for the first time that the crosstalk between ACLfs and SCs could modulate the LOXs and MMP-1, 2, 3 synthesis in ACLfs in the presence of TNF-α. Accumulation of MMPs in the isolated fluid-containing space not only disrupts the balance of ACL healing, but also increases cartilage degradation and accelerates osteoarthritis (OA) in injured joint. Based on this mechanism, targeting inhibition of MMPs could provide a promising therapeutic strategy for acute ligament injury.

MGF E peptide improves anterior cruciate ligament repair by inhibiting hypoxia-induced cell apoptosis and accelerating angiogenesis.

Severe hypoxic microenvironment endangers cell survival of anterior cruciate ligament (ACL) fibroblasts and is harmful to ACL repair and regeneration. In the current study, we explored the effects of mechanogrowth factor (MGF) E peptide on the hypoxia-induced apoptosis of ACL fibroblasts and relevant mechanisms. It demonstrated that severe hypoxia promoted hypoxia-inducible factor-1α (HIF-1α) expression and caused cell apoptosis of ACL fibroblasts through increasing caspase 3/7/9 messenger RNA (mRNA), cleaved caspase 3 and proapoptotic proteins expression levels but decreasing antiapoptotic proteins expression levels. Fortunately, MGF E peptide effectively protected ACL fibroblasts against hypoxia-induced apoptosis through regulating caspase 3/7/9 mRNA, cleaved caspase 3 and apoptosis-relevant proteins expression levels. Simultaneously, mitochondrial, @@@MEK-ERK1/2 (extracellular-signal-regulated kinase 1/2), and phosphoinositide-3-kinase-protein kinase B (PI3K-Akt) pathways were involved in MGF E peptide regulating hypoxia-induced apoptosis of ACL fibroblasts. In rabbit ACL rupture model, MGF E peptide also decreased HIF-1α expression levels, cell apoptosis, and facilitated cell proliferation. In addition, MGF could accelerate angiogenesis after ACL injury probably owing to its recruitment of proangiogenesis cells by stromal cell-derived factor 1α/CXCR4 axis and stimulation of vascular endothelial growth factor α expression level. In conclusion, our findings suggested that MGF E peptide could be utilized for ACL repair and regeneration and supplied experimental support for its application in clinical ACL treatment as a potential strategy.

Different expression profiles of the lysyl oxidases and matrix metalloproteinases in human ACL fibroblasts after co-culture with synovial cells.

Purposes The anterior cruciate ligament (ACL) has poor functional healing response. The synovial tissue surrounding ACL ligament might be a major regulator of the microenvironment in the joint cavity after ACL injury, thus affecting the repair process. Using transwell co-culture, this study explored the direct influence of human synovial cells (HSCs) on ACL fibroblasts (ACLfs) by characterizing the differential expression of the lysyl oxidase family (LOXs) and matrix metalloproteinases (MMP-1, -2, -3), which facilitate extracellular matrix (ECM) repair and degradation, respectively. Methods The mRNA expression levels of LOXs and MMP-1, -2, -3 were analyzed by semi-quantitative PCR and quantitative real-time PCR. The protein expression levels of LOXs and MMP-1, -2, -3 were detected by western blot. Results We found that co-culture resulted in an increase in the mRNAs of LOXs in normal ACLfs and differentially regulated the expression of MMPs. Then we applied 12% mechanical stretch on ACLfs to induce injury and found the mRNA expression levels of LOXs in injured ACLfs were decreased in the co-culture group relative to the mono-culture group. Conversely, the mRNA expression levels of MMPs in injured ACLfs were promoted in the co-culture group compared with the mono-culture group. At translational level, we found that LOXs were lower while MMPs were highly expressed in the co-culture group compared to the mono-culture group. Conclusions The co-culture of ACLfs and HSCs, which mimicked the cell-to-cell contact in a micro-environment, could contribute to protein modulators for wound healing, inferring the potential reason for the poor self-healing of injured ACL.