BMP-12 treatment of adult mesenchymal stem cells in vitro augments tendon-like tissue formation and defect repair in vivo

We characterized the differentiation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) into tenocyte-like cells in response to bone morphogenetic protein-12 (BMP-12). BM-MSCs were prepared from Sprague-Dawley rats and cultured as monolayers. Recombinant BMP-12 treatment (10 ng/ml) of BM-MSCs for 12 hours in vitro markedly increased expression of the tenocyte lineage markers scleraxis (Scx) and tenomodulin (Tnmd) over 14 days. Treatment with BMP-12 for a further 12-hour period had no additional effect. Colony formation assays revealed that ~80% of treated cells and their progeny were Scx- and Tnmd-positive. BM-MSCs seeded in collagen scaffolds and similarly treated with a single dose of BMP-12 also expressed high levels of Scx and Tnmd, as well as type I collagen and tenascin-c. Furthermore, when the treated BM-MSC-seeded scaffolds were implanted into surgically created tendon defects in vivo, robust formation of tendon-like tissue was observed after 21 days as evidenced by increased cell number, elongation and alignment along the tensile axis, greater matrix deposition and the elevated expression of tendon markers. These results indicate that brief stimulation with BMP-12 in vitro is sufficient to induce BM-MSC differentiation into tenocytes, and that this phenotype is sustained in vivo. This strategy of pretreating BM-MSCs with BMP-12 prior to in vivo transplantation may be useful in MSC-based tendon reconstruction or tissue engineering.     

Scleraxis positively regulates the expression of tenomodulin, a differentiation marker of tenocytes

Tenomodulin (TeM) is a type II transmembrane glycoprotein containing a C-terminal anti-angiogenic domain and is predominantly expressed in tendons and ligaments. Here we report that TeM expression is closely associated with the appearance of tenocytes during chick development and is positively regulated by Scleraxis (Scx). At stage 23, when Scx expression in the syndetome has extended to the tail region, TeM was detectable in the anterior eight somites. At stage 25, TeM and Scx were both detectable in the regions adjacent to the myotome. Double positive domains for these genes were flanked by a dorsal TeM single positive and a ventral Scx single positive domain. At stage 28, the expression profile of TeM in the axial tendons displayed more distinct morphological features at different levels of the vertebrae. At stage 32 and later, Scx and TeM showed similar expression profiles in developing tendons. Retroviral expression of Scx resulted in the significant upregulation of TeM in cultured tenocytes, but not in chondrocytes. In addition, the misexpression of RCAS-cScx by electroporation into the hindlimb could not induce the generation of additional tendons, but did result in the upregulation of TeM expression in the tendons at stage 33 and later. These findings suggest that TeM is a late marker of tendon formation and that Scx positively regulates TeM expression in a tendon cell lineage-dependent manner.     

Mechanical force modulates scleraxis expression in bioartificial tendons

Following tendon injury, cartilage, bone and fat metaplasia are often observed, making the optimization of tenocyte differentiation an important clinical goal. In this study we examined the effect of static and cyclic mechanical loading on the expression of genes which play a role in tenocyte differentiation and function, namely scleraxis (Scx) and Type I collagen (Col1a1), and determined the effect of varying mechanical parameters including (1) static vs dynamic load, (2) increasing strain magnitude, (3) inclusion of 10 s rest periods, and (4) increasing cycle number. Cyclic loading resulted in a greater increase of tenocyte gene expression than static loading over 3 weeks in culture. Increasing strain levels potentiated the induction of tenocyte genes. The insertion of a 10 s rest periods further enhanced tenocyte gene expression, as did increasing repetition numbers. These results suggest that mechanical signaling exerts an important influence on the expression of genes which play a role in determining the tendon phenotype. Further work is required to confirm and extend these findings in primary cells such as resident tendon progenitor/stem cells, in order to provide an improved understanding of biology from which optimized rehabilitation programs can be developed.     

Variations in collagen fibril structure in tendons

Variation of collagen fibril structure in tendon was investigated by x-ray diffraction. Anatomically distinct tendons from single species, as well as tendons from different species, were examined to determine the variations that exist in both the axial and lateral structure of the collagen fibrils. The meridional diffraction is derived from the axial collagen fibril structure. Anatomically distinct tendons of a particular species give meridional patterns that are indistinguishable within experimental error. The meridional diffraction patterns from tendons of different mammals are similar but show small species-specific variations, most noticeably in the 14th–18th orders. Tendons of birds also give meridional patterns that are similar to each other, but the avian patterns differ considerably from the mammalian ones. Avian tendons give stronger odd and weaker even low orders, a feature consistent with a reduced gap:overlap ratio, and have a distinctive intensity pattern for the higher meridional orders. Interpretation of these differences has been approached using biochemical data, diffraction by reconsituted fibers of purified collagen, and Fourier transform analysis. From these methods, it appears that the variations observed in the lower orders (2nd–8th) and in the higher orders (29th–52nd) are probably related to differences in the primary structure of the Type I collagen found in the different species. The variations observed in the 14th–18th orders appear not to be related to features within the triple-helical domain of the molecule. Equatorial diffraction yields information on the lateral packing of collagen molecules in the fibrils, and considerable variation was seen in different tendons. Rat tail tendon gives sharp Bragg reflections, demonstrating the presence of a crystalline lateral arrangement of molecules in the fibril. For the first time, sharp lattice reflections similar to those in rat tail tendon have been observed in nontail tendons, including rat achilles tendon, rabbit leg tendon, and wing and leg tendons of quail. In the rabbit and quail tendons, one of the strong equatorial reflections characteristic of the rat tendon pattern, at 1.26 nm, was absent. The positions of the equatorial maxima, which are a measure of intermolecular spacing, varied considerably, being smallest in the specimens displaying crystalline packing. The intermolecular distance in chiken and turkey leg tendons is longer than that found in mammalian tendons, or in avian wing tendons, which supports the hypothesis that a larger intermolecular spacing is characteristic of tendons that calcify. Thus, x-ray diffraction indicates there are reproducible differences in both the axial and lateral structure of collagen fibrils among different tendons. This work on tendon, a tissue containing almost exclusively Type I collagen as its major component, should serve as a basis for analyzing the structure of other connective tissues, which contain different genetic types of collagen and larger amounts of noncollagenous components.     

Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells

The proepicardial organ is an important transient structure that contributes cells to various cardiac lineages. However, its contribution to the coronary endothelium has been disputed, with conflicting data arising in chick and mouse. Here we resolve this conflict by identifying two proepicardial markers, Scleraxis (Scx) and Semaphorin3D (Sema3D), that genetically delineate heretofore uncharacterized proepicardial subcompartments. In contrast to previously fate-mapped Tbx18/WT-1-expressing cells that give rise to vascular smooth muscle, Scx- and Sema3D-expressing proepicardial cells give rise to coronary vascular endothelium both in vivo and in vitro. Furthermore, Sema3D(+) and Scx(+) proepicardial cells contribute to the early sinus venosus and cardiac endocardium, respectively, two tissues linked to vascular endothelial formation at later stages. Taken together, our studies demonstrate that the proepicardial organ is a molecularly compartmentalized structure, reconciling prior chick and mouse data and providing a more complete understanding of the progenitor populations that establish the coronary vascular endothelium.     

Influence of decorin and biglycan on mechanical properties of multiple tendons in knockout mice

Evaluations of tendon mechanical behavior based on biochemical and structural arrangement have implications for designing tendon specific treatment modalities or replacement strategies. In addition to the well studied type I collagen, other important constituents of tendon are the small proteoglycans (PGs). PGs have been shown to vary in concentration within differently loaded areas of tendon, implicating them in specific tendon function. This study measured the mechanical properties of multiple tendon tissues from normal mice and from mice with knock-outs of the PGs decorin or biglycan. Tail tendon fascicles, patellar tendons (PT), and flexor digitorum longus tendons (FDL), three tissues representing different in vivo loading environments, were characterized from the three groups of mice. It was hypothesized that the absence of decorin or biglycan would have individual effects on each type of tendon tissue. Surprisingly, no change in mechanical properties was observed for the tail tendon fascicles due to the PG knockouts. The loss of decorin affected the PT causing an increase in modulus and stress relaxation, but had little effect on the FDL. Conversely, the loss of biglycan did not significantly affect the PT, but caused a reduction in both the maximum stress and modulus of the FDL. These results give mechanical support to previous biochemical data that tendons likely are uniquely tailored to their specific location and function. Variances such as those presented here need to be further characterized and taken into account when designing therapies or replacements for any one particular tendon.     

Structure and function of tuna tail tendons

The caudal tendons in tunas and other scombrid fish link myotomal muscle directly to the caudal fin rays, and thus serve to transfer muscle power to the hydrofoil-like tail during swimming. These robust collagenous tendons have structural and mechanical similarity to tendons found in other vertebrates, notably the leg tendons of terrestrial mammals. Biochemical studies indicate that tuna tendon collagen is composed of the (alpha1)(2),alpha2 heterotrimer that is typical of vertebrate Type I collagen, while tuna skin collagen has the unusual alpha1,alpha2,alpha3 trimer previously described in the skin of some other teleost species. Tuna collagen, like that of other fish, has high solubility due to the presence of an acid-labile intermolecular cross-link. Unlike collagen in mammalian tendons, no differences related to cross-link maturation were detected among tendons in tuna ranging from 0.05 to 72 kg (approx. 0.25-6 years). Tendons excised post-mortem were subjected to load cycling to determine the modulus of elasticity and resilience (mean of 1.3 GPa and 90%, respectively). These material properties compare closely to those of leg tendons from adult mammals that can function as effective biological springs in terrestrial locomotion, but the breaking strength is substantially lower. Peak tendon forces recorded during steady swimming appear to impose strains of much less than 1% of tendon length, and no more than 1.5% during bursts. Thus, the caudal tendons in tunas do not appear to function as elastic storage elements, even at maximal swimming effort.     

Extracorporeal Shock Wave Treatment (ESWT) Improves In Vitro Functional Activities of Ruptured Human Tendon-Derived Tenocytes

In vitro models of human tenocytes derived from healthy as well as from ruptured tendons were established, characterized and used at very early passage (P1) to evaluate the effects of Extracorporeal Shock Wave Treatment (ESWT). The molecular analysis of traditional tenocytic markers, including Scleraxis (Scx), Tenomodulin (Tnm), Tenascin-C (Tn-C) and Type I and III Collagens (Col I and Col III), permitted us to detect in our samples the simultaneous expression of all these genes and allowed us to compare their levels of expression in relationship to the source of the cells and treatments. In untreated conditions, higher molecular levels of Scx and Col I in tenocytes from pathological compared to healthy samples have been detected, suggesting – in the cells from injured tendon – the natural trigger of an early differentiation and repairing program, which depends by Scx and requires an increase in collagen expression. When ESWT (at the dose of 0.14 mJ/mm²) was applied to cultured tenocytes explanted from injured source, Scx and Col I were significantly diminished compared to healthy counterpart, indicating that such natural trigger maybe delayed by the treatment, in order to promote cellular repair. Herein, we show for the first time that ESWT enhances in vitro functional activities of ruptured tendon-derived tenocytes, such as proliferation and migration, which could probably contributes to tendon healing in vivo.     

Collagen fibril diameter distribution does not reflect changes in the mechanical properties of in vitro stress-deprived tendons

The purpose of this study was to determine if an association exists between the tensile properties and the collagen fibril diameter distribution in in vitro stress-deprived rat tail tendons. Rat tail tendons were paired into two groups of 21 day stress-deprived and 0 time controls and compared using transmission electron microscopy (n = 6) to measure collagen fibril diameter distribution and density, and mechanical testing (n =6) to determine ultimate stress and tensile modulus. There was a statistically significant decrease in both ultimate tensile strength (control: 17.95+/-3.99 MPa, stress-deprived: 6.79+/-3.91 MPa) and tensile modulus (control: 312.8+/-89.5 MPa, stress-deprived: 176.0+/-52.7 MPa) in the in vitro stress-deprived tendons compared to controls. However, there was no significant difference between control and stress-deprived tendons in the number of fibrils per tendon counted, mean fibril diameter, mean fibril density, or fibril size distribution. The results of this study demonstrate that the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons is not correlated with the collagen fibril diameter distribution and, therefore, the collagen fibril diameter distribution does not, by itself, dictate the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons.     

Force transmission via axial tendons in undulating fish: a dynamic analysis

Sonomicrometrics of in vivo axial strain of muscle has shown that the swimming fish body bends like a homogenous, continuous beam in all species except tuna. This simple beam-like behavior is surprising because the underlying tendon structure, muscle structure and behavior are complex. Given this incongruence, our goal was to understand the mechanical role of various myoseptal tendons. We modeled a pumpkinseed sunfish, Lepomis gibbosus, using experimentally-derived physical and mechanical attributes, swimming from rest with steady muscle activity. Axially oriented muscle-tendons, transverse and axial myoseptal tendons, as suggested by current morphological knowledge, interacted to replicate the force and moment distribution. Dynamic stiffness and damping associated with muscle activation, realistic muscle force generation, and force distribution following tendon geometry were incorporated. The vertebral column consisted of 11 rigid vertebrae connected by joints that restricted bending to the lateral plane and endowed the body with its passive viscoelasticity. In reaction to the acceleration of the body in an inviscid fluid and its internal transmission of moment via the vertebral column, the model predicted the kinematic response. Varying only tendon geometry and stiffness, four different simulations were run. Simulations with only intrasegmental tendons produced unstable axial and lateral tail forces and body motions. Only the simulation that included both intra- and intersegmental tendons, muscle-enhanced segment stiffness, and a stiffened caudal joint produced stable and large lateral and axial forces at the tail. Thus this model predicts that axial tendons function within a myomere to (1) convert axial force to moment (moment transduction), (2) transmit axial forces between adjacent myosepta (segment coupling), and, intersegmentally, to (3) distribute axial forces (force entrainment), and (4) stiffen joints in bending (flexural stiffening). The fact that all four functions are needed to produce the most realistic swimming motions suggests that axial tendons are essential to the simple beam-like behavior of fish.     

Identification, content, and distribution of type VI collagen in bovine tendons

Tendon composition changes according to differentiation, mechanical load, and aging. In this study, we attempted to identify, localize, and quantify type VI collagen in bovine tendons. Type VI collagen was identified by the electrophoretic behavior of the alpha chains and Western blotting, and by rotary shadowing. Type VI collagen was extracted from powdered tendon with three sequential 24-h extractions with 4 M guanidine-HCl. The amount of type VI collagen was determined by enzyme-linked immunosorbent assay for purely tensional areas and for the compressive fibrocartilage regions of the deep flexor tendon of the digits, for the corresponding fetal and calf tendons, and for the extensor digital tendon. The distal fibrocartilaginous region of the adult tendon was richer in type VI collagen than the tensional area, reaching as much as 3.3 mg/g (0.33%) of the wet weight. Calf tendons showed an accumulation of type VI at the fibrocartilage site. Immunocytochemistry demonstrated that type VI collagen was evenly distributed in the tensional areas of tendons but was highly concentrated around the fibrochondrocytes in the fibrocartilages. The results demonstrate that tendons are variable with regard to the presence and distribution of type VI collagen. The early accumulation of type VI collagen in the region of calf tendon that will become fibrocartilage in the adult suggests that it is a good marker of fibrocartilage differentiation. Furthermore, the distribution of type VI collagen in tendon fibrocartilage indicates that it organizes the pericellular environment and may represent a survival factor for these cells.     

Indirect co-culture with tendons or tenocytes can program amniotic epithelial cells towards stepwise tenogenic differentiation

Background

Amniotic epithelial cells (AEC) have potential applications in cell-based therapy. Thus far their ability to differentiate into tenocytes has not been investigated although a cell source providing a large supply of tenocytes remains a priority target of regenerative medicine in order to respond to the poor self-repair capability of adult tendons. Starting from this premise, the present research has been designed firstly to verify whether the co-culture with adult primary tenocytes could be exploited in order to induce tenogenic differentiation in AEC, as previously demonstrated in mesenchymal stem cells. Since the co-culture systems inducing cell differentiation takes advantage of specific soluble paracrine factors released by tenocytes, the research has been then addressed to study whether the co-culture could be improved by making use of the different cell populations present within tendon explants or of the high regenerative properties of fetal derived cell/tissue.

Methodology/Principal Findings

Freshly isolated AEC, obtained from ovine fetuses at mid-gestation, were co-incubated with explanted tendons or primary tenocytes obtained from fetal or adult calcaneal tendons. The morphological and functional analysis indicated that AEC possessed tenogenic differentiation potential. However, only AEC exposed to fetal-derived cell/tissues developed in vitro tendon-like three dimensional structures with an expression profile of matrix (COL1 and THSB4) and mesenchymal/tendon related genes (TNM, OCN and SCXB) similar to that recorded in native ovine tendons. The tendon-like structures displayed high levels of organization as documented by the cell morphology, the newly deposited matrix enriched in COL1 and widespread expression of gap junction proteins (Connexin 32 and 43).

Conclusions/Significance

The co-culture system improves its efficiency in promoting AEC differentiation by exploiting the inductive tenogenic soluble factors released by fetal tendon cells or explants. The co-cultural system can be proposed as a low cost and easy technique to engineer tendon for biological study and cell therapy approach.