SOX9+ enthesis cells are associated with spinal ankylosis in ankylosing spondylitis.

OBJECTIVE: Although cartilage degeneration and invasion of the subchondral bone plate in entheseal lesion has been considered to consequently lead bony ankylosis in ankylosing spondylitis (AS), no evident mechanisms are known. DESIGN: To identify histopathological and physiological changes in enthesitis-related ankylosis in AS, we performed molecular characterization of transcription factors and surface markers, and transcriptome analysis with human tissues. Entheseal tissue containing subchondral bone was obtained from the facet joints of 9 patients with AS and 10 disease controls, and assessed by using differential staining techniques. Enthesis cells were isolated, characterized, stimulated with TNF and/or IL-17A, and analysed by cell-based experimental tools. RESULTS: We found diffusely distributed granular tissue and cartilage in the subchondral bone in AS. Co-expression of SOX9, a specific transcription factor in cartilage, and matrix metalloproteinase 13 (MMP13) was found in the granular tissues within the subchondral bone from AS patients. Intriguingly, SOX9 expression was significantly higher in AS enthesis cells than controls and correlated with TNFR1 and IL-17RA expressions, which is important for high reactivity to TNF and IL-17A cytokines. Co-stimulation by TNF and IL-17A resulted in accelerated mineralization/calcification features, and increased OCN expression in AS enthesis cells. Furthermore, SOX9 overexpression in enthesis leads to promoting mineralization feature by TNF and IL-17A stimuli. Finally, OCN expression is elevated in the destructive enthesis of advanced AS. CONCLUSION: These findings provide insight into the links between inflammation and the mineralization of entheseal tissue as the initiation of spinal ankylosis, emphasizing the importance of SOX9+ enthesis cells.

Collagen Organization in Facet Capsular Ligaments Varies With Spinal Region and With Ligament Deformation.

The spinal facet capsular ligament (FCL) is primarily comprised of heterogeneous arrangements of collagen fibers. This complex fibrous structure and its evolution under loading play a critical role in determining the mechanical behavior of the FCL. A lack of analytical tools to characterize the spatial anisotropy and heterogeneity of the FCL's microstructure has limited the current understanding of its structure-function relationships. Here, the collagen organization was characterized using spatial correlation analysis of the FCL's optically obtained fiber orientation field. FCLs from the cervical and lumbar spinal regions were characterized in terms of their structure, as was the reorganization of collagen in stretched cervical FCLs. Higher degrees of intra- and intersample heterogeneity were found in cervical FCLs than in lumbar specimens. In the cervical FCLs, heterogeneity was manifested in the form of curvy patterns formed by collections of collagen fibers or fiber bundles. Tensile stretch, a common injury mechanism for the cervical FCL, significantly increased the spatial correlation length in the stretch direction, indicating an elongation of the observed structural features. Finally, an affine estimation for the change of correlation length under loading was performed which gave predictions very similar to the actual values. These findings provide structural insights for multiscale mechanical analyses of the FCLs from various spinal regions and also suggest methods for quantitative characterization of complex tissue patterns.

In vivo evaluation of hybrid patches composed of PLA based copolymers and collagen/chondroitin sulfate for ligament tissue regeneration.

Biomaterials for soft tissues regeneration should exhibit sufficient mechanical strength, demonstrating a mechanical behavior similar to natural tissues and should also promote tissues ingrowth. This study was aimed at developing new hybrid patches for ligament tissue regeneration by synergistic incorporation of a knitted structure of degradable polymer fibers to provide mechanical strength and of a biomimetic matrix to help injured tissues regeneration. PLA- Pluronic® (PLA-P) and PLA-Tetronic® (PLA-T) new copolymers were shaped as knitted patches and were associated with collagen I (Coll) and collagen I/chondroitine-sulfate (Coll CS) 3-dimensional matrices. In vitro study using ligamentocytes showed the beneficial effects of CS on ligamentocytes proliferation. Hybrid patches were then subcutaneously implanted in rats for 4 and 12 weeks. Despite degradation, patches retained strength to answer the mechanical physiological needs. Tissue integration capacity was assessed with histological studies. We showed that copolymers, associated with collagen and chondroitin sulfate sponge, exhibited very good tissue integration and allowed neotissue synthesis after 12 weeks in vivo. To conclude, PLA-P/CollCS and PLA-T/CollCS hybrid patches in terms of structure and composition give good hopes for tendon and ligament regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1778-1788, 2017.

Isolation and Characterization of Human Mesenchymal Stem Cells From Facet Joints and Interspinous Ligaments.

STUDY DESIGN: A descriptive in vitro study on isolation and differentiation of human mesenchymal stem cells (MSCs) derived from the facet joints and interspinous ligaments. OBJECTIVE: To isolate cells from the facet joints and interspinous ligaments and investigate their surface marker profile and differentiation potentials. SUMMARY OF BACKGROUND DATA: Lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament are progressive conditions characterized by the hypertrophy and ossification of ligaments and joints within the spinal canal. MSCs are believed to play a role in the advancement of these diseases and the existence of MSCs has been demonstrated within the ligamentum flavum and posterior longitudinal ligament. The aim of this study was to investigate whether these cells could also be found within facet joints and interspinous ligaments. METHODS: Samples were harvested from 10 patients undergoing spinal surgery. The MSCs from facet joints and interspinous ligaments were isolated using direct tissue explant technique. Cell surface antigen profilings were performed via flow cytometry. Their lineage differentiation potentials were analyzed. RESULTS: The facet joints and interspinous ligaments-derived MSCs have the tri-lineage potential to be differentiated into osteogenic, adipogenic, and chondrogenic cells under appropriate inductions. Flow cytometry analysis revealed both cell lines expressed MSCs markers. Both facet joints and interspinous ligaments-derived MSCs expressed marker genes for osteoblasts, adipocytes, and chondrocytes. CONCLUSION: The facet joints and interspinous ligaments may provide alternative sources of MSCs for tissue engineering applications. The facet joints and interspinous ligaments-derived MSCs are part of the microenvironment of the human ligaments of the spinal column and might play a crucial role in the development and progression of degenerative spine conditions.

Are myofibroblasts activated in idiopathic carpal tunnel syndrome? an immunohistochemical study.

OBJECTIVES: This study aims to investigate whether myofibroblasts participate in the fibrotic process of idiopathic carpal tunnel syndrome (CTS). PATIENTS AND METHODS: Forty patients (12 males, 28 females; median age 50.85 ± 11.2 years; range 30 to 71 years) who were operated in our clinic between March 2010 and August 2010 were included in the study. Twenty-five idiopathic CTS patients were assigned to the study group, and 15 trauma patients were assigned to the control group. Samples were taken from both transverse carpal ligament and subsynovial connective tissue (SSCT) of participants and they were analyzed by immunohistochemical method. Four immunohistochemical markers were used to analyze myofibroblast existence and vascular wall thickness (alpha smooth muscle actin [α-SMA]), collagen type IV antibodies, and T (CD3) and B (CD20) lymphocytes. RESULTS: The existence of myofibroblasts (α-SMA) in SSCT of patients who were in early phase of idiopathic CTS was shown through the positive reaction of their antibodies with fibroblasts. A significantly increased reaction of α-SMA and collagen antibodies in vascular structure of SSCT demonstrated increased vascular wall thickness and vascularity in the study group (p<0.01). No significant difference was detected between the two groups in terms of T and B lymphocyte antibody reaction (inflammation). CONCLUSION: The findings of this study indicate a potential for myofibroblasts to be activated during the early phase of the disease and contribute to the onset of disease. Further studies with larger sample sizes would be of great assistance in determining the role of myofibroblasts in idiopathic CTS.

An algorithm for the calculation of three-dimensional collagen fiber orientation in ligaments using angle-sensitive MRI.

A method based on angle-sensitive magnetic resonance imaging for determining unknown orientations of collagen fibers in ligaments is presented. Collagen fibers were stepwise rotated around two independent axes within a 3T magnetic resonance tomograph (from 0° to 180°, step size=10°). Analyzing the magnetic resonance signal intensity of each voxel as a function of the rotation angle, directions were calculated by means of a computational algorithm. The accuracy of the algorithm was validated using 1000 random test directions, revealing an average deviation of 4.4° (median±standard deviation: 2.7°±9°). Subsequently, the presented method was applied to three specimens of the human iliotibial tract mounted in different directions in a rotatable plastic box. Polarized light microscopy was used to verify parallel alignment of the collagen fibers in the three specimens. The calculated directions were compared with the directions of the specimens. Analyzing each voxel separately, average deviations (median±standard deviation) in the three specimens were: 11.2° (3.6°±20.4°), 12° (5°±24.1°), and 20.4° (8.7°±27°). If the magnetic resonance signal intensity of each voxel was averaged with the intensity of immediately neighboring voxels, average deviations (median±standard deviation) were 8.5° (3.6°±17.4°), 6.2° (0°±18°), and 9.2° (5°±19.2°). The presented method has the potential to be applied in situ to anatomical structures like cartilage, ligaments, tendons and fascia.

Attachment of the anteroinferior glenohumeral ligament-labrum complex to the glenoid: an anatomic study.

PURPOSE: To determine the morphology of the attachment of the anteroinferior glenohumeral ligament-labrum complex (AIGHL-LC) to the anterior rim of the glenoid. METHODS: Sixty-six cadaveric shoulders with a mean age of 81 years were studied. The length of the AIGHL-LC attachment in the superoinferior direction and its depth in the mediolateral direction at the 2-, 3-, 4-, and 5-o'clock positions were measured. The radial histologic sections from the center of the glenoid at the 2- and 4-o'clock positions were used for histologic examinations. RESULTS: The mean length of the AIGHL-LC attachment was 11.7 mm. The mean depth was 4.7 mm (1.6 mm on the articular cartilage and 3.0 mm on the glenoid neck) at the 2-o'clock position, 6.7 mm (2.4 mm and 4.3 mm, respectively) at the 3-o'clock position, 8.4 mm (3.0 mm and 5.4 mm, respectively) at the 4-o'clock position, and 6.8 mm (2.5 mm and 4.3 mm, respectively) at the 5-o'clock position. The depth of the AIGHL-LC attachment was the greatest at the 4-o'clock position (P < .01) and the smallest at the 2-o'clock position (P < .05). Histologically, the AIGHL-LC attached to both the cartilage and bone in 52 shoulders (86.7%) at the 2-o'clock position and in 53 shoulders (88.3%) at the 4-o'clock position. CONCLUSIONS: The depth of the AIGHL-LC attachment was the greatest at the 4-o'clock position and the smallest at the 2-o'clock position. At the 4-o'clock position, the AIGHL-LC attaches to both the articular cartilage and bone in 88% of shoulders whereas it attaches only to bone in 12%. CLINICAL RELEVANCE: This study provides fundamental information on the AIGHL-LC attachment. Because healing of the AIGHL-LC to the articular cartilage cannot be expected, the same attachment area as to the bone and cartilage observed in normal shoulders needs to be created on the glenoid neck during Bankart repair to obtain the physiological strength of the AIGHL-LC.

Macroscopic and microscopic analysis of the thumb carpometacarpal ligaments: a cadaveric study of ligament anatomy and histology.

BACKGROUND: Stability and mobility represent the paradoxical demands of the human thumb carpometacarpal joint, yet the structural origin of each functional demand is poorly defined. As many as sixteen and as few as four ligaments have been described as primary stabilizers, but controversy exists as to which ligaments are most important. We hypothesized that a comparative macroscopic and microscopic analysis of the ligaments of the thumb carpometacarpal joint would further define their role in joint stability. METHODS: Thirty cadaveric hands (ten fresh-frozen and twenty embalmed) from nineteen cadavers (eight female and eleven male; average age at the time of death, seventy-six years) were dissected, and the supporting ligaments of the thumb carpometacarpal joint were identified. Ligament width, length, and thickness were recorded for morphometric analysis and were compared with use of the Student t test. The dorsal and volar ligaments were excised from the fresh-frozen specimens and were stained with use of a triple-staining immunofluorescent technique and underwent semiquantitative analysis of sensory innervation; half of these specimens were additionally analyzed for histomorphometric data. Mixed-effects linear regression was used to estimate differences between ligaments. RESULTS: Seven principal ligaments of the thumb carpometacarpal joint were identified: three dorsal deltoid-shaped ligaments (dorsal radial, dorsal central, posterior oblique), two volar ligaments (anterior oblique and ulnar collateral), and two ulnar ligaments (dorsal trapeziometacarpal and intermetacarpal). The dorsal ligaments were significantly thicker (p < 0.001) than the volar ligaments, with a significantly greater cellularity and greater sensory innervation compared with the anterior oblique ligament (p < 0.001). The anterior oblique ligament was consistently a thin structure with a histologic appearance of capsular tissue with low cellularity. CONCLUSIONS: The dorsal deltoid ligament complex is uniformly stout and robust; this ligament complex is the thickest morphometrically, has the highest cellularity histologically, and shows the greatest degree of sensory nerve endings. The hypocellular anterior oblique ligament is thin, is variable in its location, and is more structurally consistent with a capsular structure than a proper ligament.

The anterolateral ligament of the human knee: an anatomic and histologic study.

PURPOSE: The functional anatomy of the knee is frequently studied but remains incompletely understood. Numerous authors have described a structure in the lateral knee connecting the lateral femoral condyle with the lateral meniscus and tibial plateau. The goal of this study is to define the incidence, anatomy, and histology of this structure, the anterolateral ligament. METHODS: The incidence of the ligament was determined in 30 consecutive patients undergoing total knee arthroplasty (TKA) for medial compartment osteoarthritis. The anatomy and histology were evaluated using 10 cadaveric knees. RESULTS: The anterolateral ligament was noted to be present in all 40 knees. In all cases, it was noted to take origin near or on the popliteus tendon insertion and insert into the lateral meniscus and tibial plateau 5 mm distal to the articular surface and posterior to Gerdy's Tubercle. The average width of the relatively flat structure was 8.2 ± 1.5 mm, and the average length was 34.1 ± 3.4 mm. Histologic analysis revealed a discreet structure with a fibrous core surrounded by synovium. Fibers blended with the popliteus at its origin and with the lateral meniscus as it passed distally. CONCLUSIONS: The anterolateral ligament may play a role in preventing anterior tibial translation. The role, if any, of this structure in meniscal stability and the pathology of meniscal tears remain unclear. LEVEL OF EVIDENCE: Not applicable-Descriptive Anatomic Study.

High origin of the anterior band of the inferior glenohumeral ligament: MR arthrography with anatomic and histologic correlation in cadavers.

INTRODUCTION: The anterior band of the inferior glenohumeral ligament has been described to arise from the anteroinferior labrum, but we have observed that in some persons its origin is from the anterior or anterosuperior labrum, creating diagnostic difficulties. MATERIALS AND METHODS: Ten fresh unembalmed cadaveric shoulders underwent magnetic resonance arthrography (MRA) using a posterior approach with a 1.5 T GE magnet, with the following sequences: T1-weighted fast spin-echo in axial, coronal and sagittal planes, and T1 fat-suppressed spin-echo in the axial plane (TR/TE 600/20, section thickness 2.5 mm, 0.5 mm interslice space, number of signals acquired, two, field of view 12 × 12 cm, and matrix 512 × 256 pixels). Following imaging, the shoulders were frozen and later sectioned using a band saw into 3-mm sections corresponding to the axial imaging plane. Histological analysis was also performed to determine the origin of the anterior band. RESULTS: Four of the ten shoulders had an origin of the anterior band above or at the 3 o'clock position: one at the 1 o'clock position, two at the 2 o'clock position, and one at the 3 o'clock position. In another shoulder, the anterior band of the inferior glenohumeral ligament originated from the middle glenohumeral ligament, and in five other shoulders, the anterior band originated from the anteroinferior labrum as has been described in the literature. CONCLUSIONS: This finding is of clinical significance as a high origin of the anterior band of the inferior glenohumeral ligament leads to MR arthrographic finding that can simulate those of labral tears or detachments.

Microscopic analysis of the iliofemoral and ischiofemoral ligaments in the hip joint: collagen fiber direction and crimp distribution.

Since no previous studies have described the functional significance of the iliofemoral and ischiofemoral ligaments on the basis of microscopic analyses, we examined the direction of collagen fiber alignment and crimp distribution of the collagen fibers in sections cut in different directions. Polarized microscopic images of sections along the longitudinal (L) and transverse (T) planes of each ligament were obtained from 20 cadavers (8 males and 12 females, age at death 81.7 ± 9.4 years old). Results showed that the microscopic direction of collagen fibers in the iliofemoral ligament was parallel to the macroscopic direction, suggesting that this ligament may play a part in restricting extension of the hip joint. In contrast, the microscopic direction of collagen fibers in the ischiofemoral ligament was not parallel to the macroscopic direction, suggesting that this ligament may contribute not only to the restriction of medial rotation but also retstriction of flexion of the hip joint. From the low density of the crimp distribution in the L plane, the iliofemoral ligament may contribute to stability of the hip joint in the standing position in the living body. In conclusion, the microscopic observations of the direction of collagen fibers as well as the crimp distribution shown in the present study provide a better understanding of the functional significance of the iliofemoral and ischiofemoral ligaments.

Mesenchymal stem cells.

Stem cells have two features: the ability to differentiate along different lineages and the ability of self-renewal. Two major types of stem cells have been described, namely, embryonic stem cells and adult stem cells. Embryonic stem cells (ESC) are obtained from the inner cell mass of the blastocyst and are associated with tumorigenesis, and the use of human ESCs involves ethical and legal considerations. The use of adult mesenchymal stem cells is less problematic with regard to these issues. Mesenchymal stem cells (MSCs) are stromal cells that have the ability to self-renew and also exhibit multilineage differentiation. MSCs can be isolated from a variety of tissues, such as umbilical cord, endometrial polyps, menses blood, bone marrow, adipose tissue, etc. This is because the ease of harvest and quantity obtained make these sources most practical for experimental and possible clinical applications. Recently, MSCs have been found in new sources, such as menstrual blood and endometrium. There are likely more sources of MSCs waiting to be discovered, and MSCs may be a good candidate for future experimental or clinical applications. One of the major challenges is to elucidate the mechanisms of differentiation, mobilization, and homing of MSCs, which are highly complex. The multipotent properties of MSCs make them an attractive choice for possible development of clinical applications. Future studies should explore the role of MSCs in differentiation, transplantation, and immune response in various diseases.

Annular ligament of the elbow: MR arthrography appearance with anatomic and histologic correlation.

OBJECTIVE: As part of the lateral ligament complex of the elbow, the annular ligament has an adjunctive role in stabilizing the proximal radioulnar joint. Knowledge of the anatomy of this ligament may enhance our understanding of its functional role in maintaining integrity of the elbow joint in fracture and dislocation. The purpose of this study is to provide a detailed analysis of the anatomy of the annular ligament using MR arthrography with anatomic and histologic correlation in cadavers. MATERIALS AND METHODS: MR arthrography of six fresh cadaveric elbows using coronal, axial, and sagittal planes was performed after injection of intraarticular contrast material. The MR arthrography appearance of the annular ligament was correlated with anatomic sectioning and histologic analysis. RESULTS: On MR arthrography, the axial and sagittal planes provided the best evaluation of the annular ligament. Although the anterior attachment of annular ligament was a single band, the posterior attachment was fenestrated in two of six specimens. Anatomic inspection confirmed the MR observations. On histology, the annular ligament appeared to be part of a complex supporting structure continuous with the elbow joint capsule, adjacent ligaments, and muscles. CONCLUSION: The annular ligament is a complex structure formed from the capsule, lateral collateral ligamentous complex, and supinator muscle that act in unison to stabilize the proximal radioulnar joint. Its posterior attachment to the ulna can be fenestrated normally. A detailed analysis of anatomy of this ligament allows us to better understand its functional role in fractures and dislocations of the elbow joint.

Transforming growth factor-beta1 stimulation enhances Dupuytren's fibroblast contraction in response to uniaxial mechanical load within a 3-dimensional collagen gel.

PURPOSE: A function of fibroblasts is the generation of cytomechanical force within their surrounding extracellular matrix. Abnormalities in force generation may be the cause of many pathologic conditions including scarring, and some fibroproliferative disorders such as Dupuytren's disease, which is the focus of this report. METHODS: This work investigated the cytomechanical responses of Dupuytren's-derived fibroblasts to externally applied mechanical force using a culture force monitor model, with and without stimulation with the fibrosis-linked cytokine, transforming growth factor-beta1 (TGF-beta1). We compared these responses with cytomechanical responses of fibroblasts derived from the transverse carpal ligament. RESULTS: Dupuytren's fibroblasts display a significantly greater ability to contract a collagen matrix compared with control fibroblasts, with a maximum generated force of 131 dynes (p < .001). These cells did not exhibit a characteristic plateau phase in the contraction, which indicates a delay in achieving tensional homeostasis from Dupuytren's-derived cells. After being subjected to uniaxial overload and underload, Dupuytren's fibroblasts responded by increased force generation, whereas control fibroblasts responded by a reduction in force in response to an overload, and contraction in response to an underload. These changes were exacerbated by the addition of the profibrotic factor TGF-beta1, with a significant increase in generated force for all cell types, in particular during the early phase of fibroblast attachment and contraction, and a positive contraction gradient in response to overloading forces. CONCLUSIONS: These data suggest that cells derived from this fibrotic disease display characteristic abnormalities in force generation profiles. Their default response to loading or underloading is contraction, or increased force generation. This work highlights the role of TGF-beta1 as a mechano-transduction cytokine, which has an influence on the early phase cell of force generation, as well as a role in mechanical responses of cells to external mechanical stimuli. This, in turn, may influence the progression of Dupuytren's disease and the high rates of recurrence seen postoperatively.

An anatomic and histologic study of the coracohumeral ligament.

Although the anatomy and histology of the coracohumeral ligament (CHL) play an important role in the diagnosis and treatment of frozen shoulder, they remain unclear. Our objective was to study the anatomic features of the CHL and analyze its histology. Twenty-six fresh-frozen, normal cadaveric shoulders were used to examine the position and morphology of the CHL and their relationship with the superior glenohumeral ligament and to determine the CHL's histologic features in comparison with the joint capsule and coracoacromial ligament. The CHLs were all located in the rotator interval, with an irregular trapezoidal structure. The subacromial bursa was above the CHL, and the subcoracoid bursa was below the it. The CHLs in all shoulders originated from the lateral aspect of the base of the coracoid process. In 11 shoulders, it inserted into the supraspinatus tendon, whereas in 11 other shoulders, it inserted into the rotator interval. In 3 shoulders, the CHLs were split and inserted into both the supraspinatus and subscapularis tendons, respectively. Finally, the CHL in 1 shoulder only inserted into the subscapularis tendon. We also observed that the pectoralis minor tendons in 4 shoulders passed over the coracoid process top and inserted into the CHLs. In 11 shoulders, a complex of the CHL and the superior glenohumeral ligament was formed. Histologically, the CHL was found to be similar to the joint capsule without any ligament features. The position, morphology, and origin of the CHL did not change much, but its insertion varied greatly. In addition, the CHL had the histologic feature of a capsule, not a ligament.

Morphology and adhesion of human fibroblast cells cultured on bioactive polymer grafted ligament prosthesis.

The anterior cruciate ligament (ACL) is the most important ligament for the knee stabilization. Unfortunately, it is also the most commonly injured. Synthetic polymers such as polyethylene terephthalate (PET) are widely used to fabricate ligament prostheses. In this study, we reported how to graft poly(sodium styrene sulfonate) (pNaSS) onto PET fabrics used to prepare ligament at a rate of about 4.5 x 10(-6) mol/g. In this study, we analyzed the morphology of human fibroblast MacCoy adhering onto the pNaSS grafted fabrics. Cell adhesion strength onto grafted and non grafted fabrics previously adsorbed with serum proteins was also evaluated after the application of shear stresses. Results showed that human fibroblast MacCoy adhered more strongly on the pNaSS grafted fabric compared to the non grafted one. The cell spreading is well on the grafted fiber even after the shear stress application: about 65% of cells remained adhered on the pNaSS grafted fabric as compared to 32% on the non grafted one. We concluded that Mac Coy human fibroblast cells strongly adhered onto the pNaSS functionalized PET prosthesis surface and showed a better spread cell morphology as well as a more homogeneous distribution than on the non grafted sample surfaces.

[Expressions of type I and type III collagens and tenascin-C in rat bone mesenchymal stem cells co-cultured with ligament fibroblasts].

Bone marrow mesenchymal stem cells (BMSCs) can be directed to differentiate into a variety of cell types depending on their micro-environment. In this study, rat BMSCs were co-cultured with rat ligament fibroblasts during different time courses. The mRNA expressions of type I, type III collagens and tenascin-C were measured by real time RT-PCR, and the corresponding protein levels of type I and type III collagens by radioimmunoassay. Results show that the mRNA expressions of type I and type III collagens in the BMSCs were 2 times up-regulated after a 6-day co-culture, and the relative mRNA expressions of type I and type III collagens were 3.9 +/- 0.2 and 1.9 +/- 0.2, while they were 1.9 +/- 0.3 and 0.8 +/- 0.1 in the control groups, respectively. The protein syntheses of these two collagens were also increased after a 12-day co-culture; the type I and type III collagens synthesis were 13.6 +/- 1.3 ng/microg and 5.9 +/- 0.5 ng/microg in co-culture groups and 12.4 +/- 0.8 ng/microg and 5.0 +/- 0.4 ng/microg in their control groups, respectively. Likewise, there was a 2 times enhancement in tenascin-C mRNA expression after the 12-day co-culture (0.07 +/- 0.02 by control group and 0.14 +/- 0.02 by co-culture group, P < 0. 05). These data suggest that the presence of the ligament fibroblast promotes the syntheses of type I and the III collagens and tenascin-C in the rat BMSC.

Mapping lubricin in canine musculoskeletal tissues.

Lubricin, also known as superficial zone protein or PRG4, has many distinct biological functions, including lubrication, antiadhesion, and as a regulator of cell growth. This study investigated lubricin in canine musculoskeletal tissues using RT-PCR and immunohistochemistry. One or more variants were noted in canine flexor digitorum profundus (FDP) tendon, Achilles tendon, patellar tendon, A2 pulley, anterior cruciate ligament (ACL), knee lateral collateral ligament (LCL), articular cartilage, meniscus, muscle, and skin. We found 6 N-terminal lubricin splicing variants. The variants with larger sizes were identified in FDP tendon, ACL, LCL, A2 pulley, and cartilage. Lubricin was distributed both on the tissue surfaces and at the interface of fiber bundles within tissues, but this distribution varied by tissue type. We conclude that lubricin is present in many tissues; variations in splicing and physical distribution suggest that the variants of lubricin may play different roles in different locations.

Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament.

This study investigated four different connective tissue cell types to determine which cell type should be the source for seeding a tissue-engineered anterior cruciate ligament (ACL) replacement. Cells derived from the ACL, medial collateral ligament (MCL), achilles tendon (AT), and patellar tendon (PT) of New Zealand White rabbits were isolated and cultured. Each cell type was cultured in vitro after seeding on three-dimensional (3-D) braided polymer scaffolds and on tissue culture polystyrene that served as a control. Samples were evaluated and compared for their morphology, proliferation, and gene expression of fibronectin, type I and type III collagen. Scanning electron microscopy (SEM) photomicrographs verified cell attachment of all four types of connective tissue fibroblasts to the scaffolds. Preliminary results comparing proliferation indicate that cells obtained from the PT and AT have the fastest proliferation. Whereas gene expression of the phenotypic markers measured using real-time reverse transcription polymerase chain reaction (RT-PCR) indicates ACL cells have the highest gene expression for the matrix markers. This leads to the question of which cell type should be the cell source for tissue-engineering of ligament, the highly proliferating cells or the differentiated matrix producing cells. This study would suggest that ACL differentiated matrix producing cells are the most suitable cells for further study and development of a tissue-engineered ligament.

Molecular parameters indicating adaptation to mechanical stress in fibrous connective tissue.

The present study pursues the hypothesis that local compressive force and the occurrence of cartilage-specific transformation processes within the extracellular matrix of tendons and ligaments are directly correlated. We compare the pattern of certain marker molecules typical of (fibro)cartilage in select examples. Investigations are carried out of the extensor tendons of toes and fingers, the transverse ligament of the atlas, the transverse ligament of the acetabulum, and of the tendon of the superior oblique muscle and its trochlea. The marker molecules are detected with standardized immunohistochemical methods. The results show that certain molecules only occur under conditions of (relatively high) compressive stress, others being the result of tensile stress. The molecular spectrum of the molecules of the ECM allows qualifying conclusions as to the mechanical situation of a given part of the tissue. A quantifying statement about the intensity of compressive stress is not possible to make thus far, but the extension of the restructuring areas corresponds to the area of compressive stress. Depending on the intensity and duration of the local compressive strain, the molecules involved may be ordered chronologically according to their occurrence in the ECM. The glycosaminoglycans react at lower stress levels than the proteoglycans, which in turn react earlier than the collagens, especially with regard to the vanishing of type I collagen and the first occurrence of type II collagen. Of the glycosaminoglycans, dermatan sulfate and keratan sulfate occur first. These are detectable in virtually all cases. They are followed by chondroitin 4 sulfate. The last glycosaminoglycan, which occurs in already significantly fibrocartilaginous tissue, is chondroitin 6 sulfate. Under chronologically intensifying compressive stress in the increasingly fibrocartilaginous tissues, the proteoglycans versican and, to a lesser extent, tenascin--characteristic markers of fibrous tissue--can still be detected. However, their spatial expansion steadily decreases until they finally vanish. Contrastingly, aggrecan and link protein expression becomes increasingly prominent in such tissues. The spatial expansion of the adaptation zones in tendons and ligaments roughly corresponds with the zones subjected to compressive force; tensile stress alone does not result in a production of fibrocartilage. The questions asked at the beginning may thus be answered as follows: The molecular composition of the various fibrous connective tissues, such as tendons and ligaments, can be directly correlated with the respective tissue's mechanical function. As an expression of this regular interrelation, a ranking of certain ECM molecules may be set up that corresponds to the type, intensity, and duration of the mechanical stress. Grounded on this, it seems possible to prognosticate the occurrence of certain components in the ECM depending on the nature of the mechanical stress. The occurrence of certain molecules within the fibrocartilaginous tissue is of clinical importance in connection with various forms of rheumatoid arthritis and perhaps other diseases with an autoimmune-related etiology. Since a considerable part of the inflammatory destructions involved may at least indirectly result from autoimmune processes directed against the cartilage-type components of the ECM, every fibrocartilage constitutes a potential target to a certain degree. This applies particularly to those fibrocartilages whose ECM has a molecular composition closely resembling that of hyaline articular cartilage. Therefore, knowledge of the regional molecular composition allows a prediction of sites where clinical symptoms may occur in the course of rheumatoid arthritis.