Ligament Extracellular Matrix Research Articles

The anterior cruciate ligament in murine post-traumatic osteoarthritis: markers and mechanics

BackgroundKnee joint injuries, common in athletes, have a high risk of developing post-traumatic osteoarthritis (PTOA). Ligaments, matrix-rich connective tissues, play important mechanical functions stabilising the knee joint, and yet their role post-trauma is not understood. Recent studies have shown that ligament extracellular matrix structure is compromised in the early stages of spontaneous osteoarthritis (OA) and PTOA, but it remains unclear how ligament matrix pathology affects ligament mechanical function. In this study, we aim to investigate both structural and mechanical changes in the anterior cruciate ligament (ACL) in a mouse model of knee trauma.MethodsKnee joints were analysed following non-invasive mechanical loading in male C57BL/6 J mice (10-week-old). Knee joints were analysed for joint space mineralisation to evaluate OA progression, and the ACLs were assessed with histology and mechanical testing.ResultsJoints with PTOA had a 33–46% increase in joint space mineralisation, indicating OA progression. Post-trauma ACLs exhibited extracellular matrix modifications, including COL2 and proteoglycan deposition. Additional changes included cells expressing chondrogenic markers (SOX9 and RUNX2) expanding from the ACL tibial enthesis to the mid-substance. Viscoelastic and mechanical changes in the ACLs from post-trauma knee joints included a 20–21% decrease in tangent modulus at 2 MPa of stress, a decrease in strain rate sensitivity at higher strain rates and an increase in relaxation during stress-relaxation, but no changes to hysteresis and ultimate load to failure were observed.ConclusionsThese results demonstrate that ACL pathology and viscoelastic function are compromised in the post-trauma knee joint and reveal an important role of viscoelastic mechanical properties for ligament and potentially knee joint health.

The Role of the Non-Collagenous Extracellular Matrix in Tendon and Ligament Mechanical Behavior: A Review.

Tendon is a connective tissue that transmits loads from muscle to bone, while ligament is a similar tissue that stabilizes joint articulation by connecting bone to bone. The 70-90% of tendon and ligament's extracellular matrix (ECM) is composed of a hierarchical collagen structure that provides resistance to deformation primarily in the fiber direction, and the remaining fraction consists of a variety of non-collagenous proteins, proteoglycans, and glycosaminoglycans (GAGs) whose mechanical roles are not well characterized. ECM constituents such as elastin, the proteoglycans decorin, biglycan, lumican, fibromodulin, lubricin, and aggrecan and their associated GAGs, and cartilage oligomeric matrix protein (COMP) have been suggested to contribute to tendon and ligament's characteristic quasi-static and viscoelastic mechanical behavior in tension, shear, and compression. The purpose of this review is to summarize existing literature regarding the contribution of the non-collagenous ECM to tendon and ligament mechanics, and to highlight key gaps in knowledge that future studies may address. Using insights from theoretical mechanics and biology, we discuss the role of the non-collagenous ECM in quasi-static and viscoelastic tensile, compressive, and shear behavior in the fiber direction and orthogonal to the fiber direction. We also address the efficacy of tools that are commonly used to assess these relationships, including enzymatic degradation, mouse knockout models, and computational models. Further work in this field will foster a better understanding of tendon and ligament damage and healing as well as inform strategies for tissue repair and regeneration.

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.

Viscoelastic characteristics of the canine cranial cruciate ligament complex at slow strain rates.

Ligaments including the cruciate ligaments support and transfer loads between bones applied to the knee joint organ. The functions of these ligaments can get compromised due to changes to their viscoelastic material properties. Currently there are discrepancies in the literature on the viscoelastic characteristics of knee ligaments which are thought to be due to tissue variability and different testing protocols.The aim of this study was to characterise the viscoelastic properties of healthy cranial cruciate ligaments (CCLs), from the canine knee (stifle) joint, with a focus on the toe region of the stress-strain properties where any alterations in the extracellular matrix which would affect viscoelastic properties would be seen. Six paired CCLs, from skeletally mature and disease-free Staffordshire bull terrier stifle joints were retrieved as a femur-CCL-tibia complex and mechanically tested under uniaxial cyclic loading up to 10 N at three strain rates, namely 0.1%, 1% and 10%/min, to assess the viscoelastic property of strain rate dependency. The effect of strain history was also investigated by subjecting contralateral CCLs to an ascending (0.1%, 1% and 10%/min) or descending (10%, 1% and 0.1%/min) strain rate protocol. The differences between strain rates were not statistically significant. However, hysteresis and recovery of ligament lengths showed some dependency on strain rate. Only hysteresis was affected by the test protocol and lower strain rates resulted in higher hysteresis and lower recovery. These findings could be explained by the slow process of uncrimping of collagen fibres and the contribution of proteoglycans in the ligament extracellular matrix to intra-fibrillar gliding, which results in more tissue elongations and higher energy dissipation. This study further expands our understanding of canine CCL behaviour, providing data for material models of femur-CCL-tibia complexes, and demonstrating the challenges for engineering complex biomaterials such as knee joint ligaments.

Dual-energy CT collagen density mapping of wrist ligaments reveals tissue remodeling in CPPD patients: first results from a clinical cohort

ObjectivesTo evaluate differences in collagen density as detected by dual-energy computed tomography (DECT) of wrist ligaments between patients with calcium pyrophosphate-dihydrate deposition disease (CPPD) and a control group in order to gain insight into changes of the extracellular matrix in response to crystal deposition.Materials and methodsThis retrospective study included 28 patients (18 with CPPD, 10 controls) who underwent DECT of the wrist. Collagen density maps were reconstructed from the DECT datasets and used to measure densities in regions of interest (ROIs) placed in the scapholunate (SL) ligament (dorsal, palmar, proximal), lunotriquetral (LT) ligament, and extensor carpi radialis (ECR) tendon, (n = 260 measurements). The presence of calcifications on standard CT images in these regions was assessed by a blinded reader. Densities were compared with nonparametric tests, and linear regression analysis was performed to investigate the impact of age, sex, and CT- detected calcium deposition on collagen density.ResultsCollagen density in the SL ligament was significantly higher in CPPD patients than in controls (overall mean: 265.4 ± 32.1 HU vs. 196.3 ± 33.8 HU; p < 0.001). In the ECR tendon, collagen densities did not differ significantly (p = 0.672): 161.3 ± 20.1 HU in CPPD vs. 163.6 ± 12.0 HU in controls. Regression analysis showed that diagnosis, but not age or calcification, had a significant impact on collagen density.ConclusionCollagen density of the SL ligament is significantly higher in CPPD patients than in control patients. Further research is needed to understand these changes in the extracellular matrix of ligaments in CPPD.

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.

Cellular-Responsive Poly(ethylene glycol) Hydrogels as a Synthetic Periodontal Ligament Extracellular Matrix

Cellular-Responsive Poly(ethylene glycol) Hydrogels as a Synthetic Periodontal Ligament Extracellular Matrix

Decellularized Iliotibial Band Recolonized with Allogenic Homotopic Fibroblasts or Bone Marrow-Derived Mesenchymal Stromal Cells.

Decellularized scaffolds present promising biomimetic approaches in various fields of tissue engineering. Different tissues have been selected for decellularization, among them extracellular matrix (ECM)-rich tissues such as tendons, ligaments and cartilage. The dense ECM of ligaments is particularly challenging to achieve a completely non-immunogenic ECM void of any cells. Here, the methods for decellularization adapted to ligamentous tissue of the iliotibial band (ITB) are presented along with cell isolation and several recolonization techniques using allogenic ITB-derived fibroblasts or mesenchymal stromal cells (MSCs).

Specific collagen peptides benefit the biosynthesis of matrix molecules of tendons and ligaments

Background An intact composition of extracellular matrix (ECM) collagens, proteoglycans and elastic fibres are responsible for the constitutional strength of tendons and ligaments [1,2]. It is known that pathophysiological changes in the ECM could lead to reduced extension properties and diminished capacity of energy absorption of ligaments and tendons and could promote diseases like patellar tip syndrome, tendinopathy and rupture [3,4]. In a clinical study it could be demonstrated that the oral ingestion of specific collagen peptides improved extension properties of the finger joints [5]. Aim of the present study was to investigate the impact of a specific collagen peptide composition (FORTIGEL) on the extracellular matrix of ligaments and Achilles tendons. Previous experimental studies confirmed the stimulatory impact of these bioactive collagen peptides on the ECM biosynthesis of joint cartilage tissue [6-8].

Elastin content is high in the canine cruciate ligament and is associated with degeneration

Cruciate ligaments (CLs) are primary stabilisers of the knee joint and canine cranial cruciate ligament disease (CCLD) and rupture is a common injury. Elastin fibres, composed of an elastin core and fibrillin containing microfibrils, are traditionally considered minor components of the ligament extracellular matrix (ECM). However, their content and distribution in CLs is unknown. The purposes of this study were to determine the elastin content of canine CLs and to ascertain its relationship to other biochemical components and histological architecture.Macroscopically normal CLs were harvested from Greyhounds (n=11), a breed with a low risk of CCLD. Elastin, collagen and sulfated glycosaminoglycan content were measured and histological scoring systems were developed to quantify ECM changes using a modified Vasseur score (mVS) and oxytalan fibre (bundles of microfibrils) staining. Elastin contents were 9.86±3.97% dry weight in the cranial CL and 10.79±4.37% in the caudal CL, respectively, and did not alter with advancing histological degeneration. All CLs demonstrated mild degenerative changes, with an average mVS score of 11.9±3.3 (maximum 24). Increasing degeneration of the ligament ECM showed a positive correlation (r=0.690, P<0.001) with increased oxytalan fibre staining within the ECM.Elastin is an abundant protein in CLs forming a greater proportion of the ligament ECM than previously reported. The appearance of oxytalan fibres in degenerative CL ECM may reflect an adaptive or reparative response to normal or increased loads. This finding is important for future therapeutic or ligament replacement strategies associated with cranial CL injury.

The molecular composition of the extracellular matrix of the human iliolumbar ligament

Background contextThe human iliolumbar ligament connects the transverse process of L5 to the iliac crest and contributes to lumbosacral stability and has been associated with low back pain. However, different opinions exist regarding the functional relevance of the ligament. PurposeIn the present study, we analyze the regional molecular composition of the ligament extracellular matrix. Study designSpecial attention is given to the attachment sites, to determine whether the ligament is subjected to a certain mechanical environment. MethodsIliolumbar ligament samples, extending from one enthesis to the other, were removed from 11 cadavers and fixed in methanol. Cryosections were immunolabeled with a panel of antibodies directed against collagens, glycosaminoglycans, proteoglycans, matrix proteins, and neurofilament. ResultsThe mid-substance of the ligament labeled for all the molecules normally found in dense fibrous connective tissue including types I, III, and VI collagen, versican, dermatan -, chondroitin 4 -, and keratan sulfate. However, both entheses were fibrocartilaginous and labeled for type II collagen, aggrecan, and chondroitin 6- sulfate. A common feature was fat between the fiber bundles near the entheses. Occasionally this fat contained nerve fibers. ConclusionsThe existence of fibrocartilaginous entheses suggests that the insertion sites of the ligament are subject to both tensile and compressive loading—probably because of insertional angle changes between ligament and bone during loading. Our findings support the suggestion that the iliolumbar ligament might play an important role in the stabilization of the lumbosacral junction.

Transcription Factor Mohawk and the Pathogenesis of Human Anterior Cruciate Ligament Degradation

To investigate the expression and function of Mohawk (MKX) in human adult anterior cruciate ligament (ACL) tissue and ligament cells from normal and osteoarthritis (OA)-affected knees. Knee joints were obtained at autopsy (within 24-48 hours postmortem) from 13 donors with normal knees (mean ± SD age 36.9 ± 11.0 years), 16 donors with knee OA (age 79.7 ± 11.4 years), and 8 aging donors without knee OA (age 76.9 ± 12.9 years). All cartilage surfaces were graded macroscopically. MKX expression was analyzed by immunohistochemistry and quantitative polymerase chain reaction. ACL-derived cells were used to study regulation of MKX expression by interleukin-1β (IL-1β). MKX was knocked down with small interfering RNA (siRNA) to analyze the function of MKX in extracellular matrix (ECM) production and differentiation in ACL-derived cells. The expression of MKX was significantly decreased in ACL-derived cells from OA knees compared with normal knees. Consistent with this finding, immunohistochemistry analysis showed that MKX-positive cells were significantly reduced in ACL tissue from OA donors, in particular in cells located in disorientated fibers. In ACL-derived cells, IL-1β strongly suppressed MKX expression and reduced expression of the ligament ECM genes COL1A1 and TNXB. In contrast, SOX9, a chondrocyte master transcription factor, was up-regulated by IL-1β treatment. Importantly, knockdown of MKX expression with siRNA up-regulated SOX9 expression in ACL-derived cells, whereas the expression of COL1A1 and TNXB was reduced. Reduced expression of MKX is a feature of degenerated ACL in OA-affected joints, and this may be mediated in part by IL-1β. MKX appears necessary to maintain the tissue-specific cellular differentiation status and ECM production in adult human tendons and ligaments.

Update on the aetiopathogenesis of canine cranial cruciate ligament disease

Cranial cruciate ligament disease (CCLD) is the most common cause of hindlimb lameness in the dog, being associated with and eventually leading to stifle osteoarthritis. Canine cranial cruciate ligament disease is a gradual degeneration of the ligament extracellular matrix (ECM) leading to ligament rupture. The aetiopathogenesis of this condition is still poorly understood but several risk factors have been identified such as breed, bodyweight, gender and conformation. Recent developments in this area include the role of genetics, stifle joint conformation, ligament ECM metabolism, and inflammation associated with immune-mediated disease within the stifle joint. A genetic mode of inheritance has been demonstrated in the Newfoundland which is predisposed to CCLD. Increased cellular metabolism within the cranial cruciate ligament has been directly associated with increased craniocaudal stifle joint laxity in dog breeds at high risk of CCLD. Conformation abnormalities, such as a narrowed distal femoral intercondylar notch, in high risk breeds have been shown to be associated with alterations in cranial cruciate ligament ultrastructure. Increased production of inflammatory cytokines, such as cathepsins and interleukins, by the stifle synovial cells may occur secondary to or may be an inciting cause of ligament degeneration. Future research endeavours will focus on the association between immune-mediated response and fibrocartilaginous metaplasia and matrix degradation within the cranial cruciate ligament, and whether this can be altered in all susceptible dogs or only certain breeds.

Comparison of Potentials Between Stem Cells Isolated from Human Anterior Cruciate Ligament and Bone Marrow for Ligament Tissue Engineering

We have previously isolated and identified stem cells from human anterior cruciate ligament (ACL). The purpose of this study was to evaluate the differences in proliferation, differentiation, and extracellular matrix (ECM) formation abilities between bone marrow stem cells (BMSCs) and ACL-derived stem cells (LSCs) from the same donors when cultured with different growth factors, including basic fibroblast growth factor (bFGF), epidermal growth factor, and transforming growth factor-beta 1 (TGF-beta1). Ligament tissues and bone marrow aspirate were obtained from patients undergoing total knee arthroplasty and ACL reconstruction surgeries. Proliferation, colony formation, and population doubling capacity as well as multilineage differentiation potentials of LSCs and BMSCs were compared. Gene expression and ECM production for ligament engineering were also evaluated. It was found that BMSCs possessed better osteogenic differentiation potential than LSCs, while similar adipogenic and chondrogenic differentiation abilities were observed. Proliferation rates of both LSCs and BMSCs were enhanced by bFGF and TGF-beta1. TGF-beta1 treatment significantly increased the expression of type I collagen, type III collagen, fibronectin, and alpha-smooth muscle actin in LSCs, but TGF-beta1 only upregulated type I collagen and tenascin-c in BMSCs. Protein quantification further confirmed the results of differential gene expression and suggested that LSCs and BMSCs increase ECM production upon TGF-beta1 treatment. In summary, in comparison with BMSCs, LSCs proliferate faster and maintain an undifferentiated state with bFGF treatment, whereas under TGF-beta1 treatment, LSCs upregulate major tendinous gene expression and produce a robust amount of ligament ECM protein, making LSCs a potential cell source in future applications of ACL tissue engineering.

A finite dissipative theory of temporary interfibrillar bridges in the extracellular matrix of ligaments and tendons

The structural integrity and the biomechanical characteristics of ligaments and tendons result from the interactions between collagenous and non-collagenous proteins (e.g. proteoglycans, PGs) in the extracellular matrix. In this paper, a dissipative theory of temporary interfibrillar bridges in the anisotropic network of collagen type I, embedded in a ground substance, is derived. The glycosaminoglycan chains of decorin are assumed to mediate interactions between fibrils, behaving as viscous structures that transmit deformations outside the collagen molecules. This approach takes into account the dissipative effects of the unfolding preceding fibrillar elongation, together with the slippage of entire fibrils and the strain-rate-dependent damage evolution of the interfibrillar bridges. Thermodynamic consistency is used to derive the constitutive equations, and the transition state theory is applied to model the rearranging properties of the interfibrillar bridges. The constitutive theory is applied to reproduce the hysteretic spectrum of the tissues, demonstrating how PGs determine damage evolution, softening and non-recoverable strains in their cyclic mechanical response. The theoretical predictions are compared with the experimental response of ligaments and tendons from referenced studies. The relevance of the proposed model in mechanobiology research is discussed, together with several applications from medical practice to bioengineering science.

Development of a Silk Cable-Reinforced Gelatin/Silk Fibroin Hybrid Scaffold for Ligament Tissue Engineering

The objective of this study was to develop a silk cable-reinforced gelatin/silk fibroin hybrid scaffold for ligament tissue engineering. The scaffold was fabricated by lyophilizing the cross-linked gelatin and silk fibroin mixture with braided silk cables. Scanning electronic microscopy (SEM) observation showed that microporous gelatin/silk fibroin sponges formed around silk cables mimicked the microstructures of ligament extracellular matrix (ECM). The silk cables significantly increased the tensile strength of the scaffold to meet the mechanical requirements for ligament tissue engineering. The scaffold possessed good cell adhesion property, and when mesenchymal stem cells (MSCs) were seeded on it, cells proliferated profusely. After 2 weeks of culture, seeded MSCs were distributed uniformly throughout the scaffold and were highly viable. Occurrence of cell death during culture was not significant. Deposition of collagen on the scaffold was found to increase with time. Differentiation of MSCs into ligament fibroblasts was verified by expressions of ligament ECM specific genes including collagen type I, collagen type III, and tenascin-C in mRNA and protein level. Immunohistochemistry stains also confirmed the production of key ligament ECM components on the scaffold. The results demonstrate that silk cable-reinforced gelatin/silk fibroin scaffold possesses the appropriate mechanical properties and has enlarged surface area. It is also capable of supporting cell proliferation and differentiation for ligament tissue engineering.

In vivo study of anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold

Although most in vitro studies indicate that silk is a suitable biomaterial for ligament tissue engineering, in vivo studies of implanted silk scaffolds for ligament reconstruction are still lacking. The objective of this study is to investigate anterior cruciate ligament (ACL) regeneration using mesenchymal stem cells (MSCs) and silk scaffold. The scaffold was fabricated by incorporating microporous silk sponges into knitted silk mesh, which mimicked the structures of ligament extracellular matrix (ECM). In vitro culture demonstrated that MSCs on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of ligament-specific genes also increased with time. Then MSCs/scaffold was implanted to regenerate ACL in vivo. After 24 weeks, histology observation showed that MSCs were distributed throughout the regenerated ligament and exhibited fibroblast morphology. The key ligament ECM components including collagen I, collagen III, and tenascin-C were produced prominently. Furthermore, direct ligament–bone insertion with typical four zones (bone, mineralized fibrocartilage, fibrocartilage, ligament) was reconstructed, which resembled the native structure of ACL–bone insertion. The tensile strength of regenerated ligament also met the mechanical requirements. Moreover, its histological grading score was significantly higher than that of control. In conclusion, the results imply that silk scaffold has great potentials in future clinical applications.

Enhanced differentiation of mesenchymal stem cells co-cultured with ligament fibroblasts on gelatin/silk fibroin hybrid scaffold

The differentiation of mesenchymal stem cells (MSCs) towards fibroblasts is a crucial issue in ligament tissue engineering. This study aims to investigate the feasibility of using co-culture system to induce the differentiation of MSCs for constructing the tissue-engineered ligament in vitro. A kind of silk cable-reinforced gelatin/silk fibroin hybrid scaffold was used to provide three-dimensional (3-D) culture environments for MSCs. The 3-D co-culture system was set up by culturing MSCs/scaffold and ligament fibroblasts in the transwell insert and lower chamber, respectively. The regulatory effects of fibroblasts on MSCs were determined. After 2 weeks of co-culture the MSCs showed faster proliferation and higher DNA content compared with MSCs non-co-cultured. The MSCs were distributed uniformly throughout the scaffold and showed good viability. The collagen production also increased significantly with culture time. The MSCs in co-culture system were proved to differentiate into ligament fibroblasts by expressing ligament extra-cellular matrix (ECM)-specific genes including collagen I, collagen III, and tenascin-C in mRNA and protein level. The immunohistochemistry staining also confirmed the synthesis of key ligament ECM components. This study reveals that specific regulatory signals released from fibroblasts in 3-D co-culture system can enhance the differentiation of MSCs for ligament tissue engineering.

The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system

Differentiation of mesenchymal stem cells (MSCs) into anterior cruciate ligament (ACL) cells is regulated by many factors. Mechanical stress affects the healing and remodeling process of ACL after surgery in important ways. Besides, co-culture system had also showed the promise to induce MSCs toward different kinds of cells on current research. The purpose of this study was to investigate the gene expression of ACL cells’ major extracellular matrix (ECM) component molecules of MSCs under three induction groups. In addition, to follow our previous study, cell electrophoresis technique and mRNA level gene expression of MSC protein were also used to analyze the differentiation of MSCs. The results reveal that specific regulatory signals which released from ACL cells appear to be responsible for supporting the selective differentiation toward ligament cells in co-culture system and mechanical stress promotes the secretion of key ligament ECM components. Therefore, the combined regulation could assist the development of healing and remolding of ACL tissue engineering. Furthermore, this study also verifies that cell electrophoresis could be used in investigation of cell differentiation. Importantly, analysis of the data suggests the feasibility of utilizing MSCs in clinical applications for repairing or regenerating ACL tissue.

CONSTITUENT PROTEOGLYCANS OF THE HUMAN YELLOW LIGAMENT EXTRACELLULAR MATRIX

Three types of proteoglycans (PG-I, II and III) present in the human yellow ligament extracellular matrix were separated using a combination of CsCl isopycnic density gradient centrifugation, DEAE-Sephacel ion-exchange chromatography, gel-filtration on Sepharose CL-4B and CL-2B, and Octyl-Sepharose CL-4B chromatography. Proteoglycan-I was a high-molecular-weight proteoglycan, which was excluded by Sepharose CL-4B, and its molecular weight, estimated using sodium dedecvl sulfate-polyacrylamide gel electrophoresis, was over 350,000. The glycosaminoglycan chain of PG-I was composed of chondroitin 6-sulfate, and PG-I appeared to be an aggrecan-type proteoglycan. Proteoglycan-Il and III were low-molecular-weight proteoglycans with molecular weights of 190,000 and 105,000, respectively. The core protein sizes of PG-II and III were similar, 44,000, but anti-human decorin antibody reacted with PG--III, not with PG-II. The glycosaminoglycan chain of PG-II contained dermatan sulfate and chondroitin 6-sulfate, whereas that of PG-III contained the former only. In the light of these results, PG-II and III were suggested to be bigIycan- and decorin-type proteoglycans, respectively. The major proteoglvcan in the human yellow ligament extracellular matrix was PG-IIL Hirosaki Med. 1. 57: 49-58, 2006