Tendon Formation Research Articles

Isolation of human marrow-derived mesenchymal stem cells

Adult bone marrow is the site of intense hematopoietic activity that is housed in a unique connective tissue called bone marrow stroma. The stroma establishes a niche in which specific hematopoietic lineage pathways are managed in proximity to a rich vascular network. The bone and its osteoblasts, osteoclasts, and “resting cells” are also contributors to this microenvironment. Also present within this mix of cells are rare multipotent cells that can differentiate along several mesenchymal lineage pathways, culminating in the formation of bone, cartilage, muscle, marrow stroma, tendon and ligament, fat, and other connective tissues.

P64. Evaluation of Dysphagia Caused by Ossification of Anterior Longitudinal Ligament of Cervical Spine

BACKGROUND CONTEXT: Ossification of anterior longitudinal ligament (OALL) is associated with diffuse idiopathic skeletal hyperostosis (DISH), which is characterized by extensive spinal osteophyte formation and ossification of tendons, ligaments, and fasciae. Huge ossification of cervical anterior longitudinal ligament may cause dysphagia by compressing the cervical esophagus. Symptoms of dysphagia are subjective, making the determination of an operative indication difficult. There have been no useful evaluative methods of dysphagia caused by cervical osteophytes.

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.

The alchemy of tendon repair: a primer for the (S)mad scientist

During vertebrate development, mesenchymal progenitors capable of forming bone, cartilage, muscle, fat, or tendon arise from either neural crest or somitic mesoderm. Transcriptional programs that specify mesenchymal cell fates are initiated and modified by paracrine cues provided by TGF-beta superfamily members and mediated in part via the regulated assembly of Smad-containing multiprotein transcription factor complexes. In this issue of the JCI, Hoffmann and colleagues have identified that Smad8 activation drives tendon formation from C3H10T1/2 cells, a murine cell line that recapitulates many features of normal multipotent mesenchymal cells (see the related article beginning on page 940). Cells programmed to the tenocyte cell fate in vitro formed tenogenic grafts in vivo. These results add to the accumulating evidence that proliferating, multipotent mesenchymal progenitor cells can be programmed to yield multiple cell types--e.g., osteoblasts, myocytes, chondrocytes, and tenocytes--that may be useful in cell-based therapeutic approaches to musculoskeletal diseases.

455. Genetically Engineered Adult Stem Cells and Hybrid Scaffolds as a Platform for Intervertebral Disc Regeneration

Current therapeutic solutions for intervertebral disc (IVD) degeneration include prosthetic implants, which fail in the long term. Adult mesenchymal stem cells (MSCs), progenitor cells that give rise to chondroblasts, tenocytes, and osteoblasts, are excellent candidates for tissue engineering of skeletal tissues. We have previously shown that MSCs overexpressing the Brachyury or the Smad8/BMP-2 genes induced cartilage and tendon formation respectively. Therefore, we hypothesized that genetically engineered MSCs combined with a hybrid of polymeric scaffolds could be used to replace a degenerative IVD. A hybrid of fibrin gel and a filamentous Poly Lactide-Glycolide acid (PLGA) scaffold were molded into the shape of a rat IVD (1a). 1X106 MSCs over expressing the Brachyury transcription factor were suspended in the fibrin gel, forming the center part of the construct. 3X106 MSCs expressing the Smad8/BMP-2 genes were seeded on the PLGA, forming the outer ring of the construct. Grafts were implanted subcutaneously in C3H/HeN mice or used to replace caudal discs of Nude rats. The cells were also labeled with Luciferase or GFP gene, which enabled non- invasive monitoring of cell survival using the Roper Chemiluminescence1s Imaging System, or minimally invasively using the Fibered confocal microscopy system (CellVisio). MRI was utilized to compare the engineered disc water content to a native one. Grafts were harvested after three weeks, fixed in 4% formalin and paraffin/plastic embedded. Histological sections were subjected to immunohistology against Collagen I, II, and VI. Our results indicated that an IVD-like tissue was formed subcutaneously and in the IVD space. Smad8/BMP-2 expressing MSCs formed an annulus fibrosus (AF)-like tissue in the outer portion of the graft (1b). MSCs expressing the Brachyury gene gave raise to cartilaginous nucleus pulposus (NP)-like tissue in the center of the construct, positively stained against Collagen II and VI (1c). The cells survived at least two weeks post implantation as was demonstrated by the bioluminescence and the high-resolution fluorescence imaging systems (1d-e). MRI analysis indicated that similar water content was present in the engineered IVD compared to adjacent intact controls. This is the first report demonstrating the use of genetically engineered MSCs for IVD tissue engineering. We conclude, that engineered biological disc composed of genetically engineered MSCs seeded on an IVD-shaped hybrid biodegradable scaffolds can serve as a platform for IVD regeneration. (See Figure 1).

Bone morphogenetic proteins: from structure to clinical use

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor ss superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.

Stem cells in craniofacial and dental tissue engineering

Mesenchymal stem cells (MSC) have been identified in a variety of adult tissues as a population of pluripotential self-renewing cells. Based on their adherence and colony forming properties, a small number of MSC can be isolated from most mesenchymal tissues as well as bone marrow. In the presence of one or more growth factors, these cells commit to lineages that lead to the formation of bone, cartilage, muscle, tendon and adipose tissue; recent studies indicate that stem cells for cementum, dentine and the periodontal ligament also exist. All of these cells can be expanded in vitro, and, embedded in a scaffold, inserted into defects to promote healing and tissue replacement. Increased understanding of the molecular mechanism directing lineage specification and morphogenesis is providing a rational approach for the regeneration of craniofacial tissues and oral structures.

Genetic analysis of interactions between the somitic muscle, cartilage and tendon cell lineages during mouse development

Proper formation of the musculoskeletal system requires the coordinated development of the muscle, cartilage and tendon lineages arising from the somitic mesoderm. During early somite development, muscle and cartilage emerge from two distinct compartments, the myotome and sclerotome, in response to signals secreted from surrounding tissues. As the somite matures, the tendon lineage is established within the dorsolateral sclerotome, adjacent to and beneath the myotome. We examine interactions between the three lineages by observing tendon development in mouse mutants with genetically disrupted muscle or cartilage development. Through analysis of embryos carrying null mutations in Myf5 and Myod1, hence lacking both muscle progenitors and differentiated muscle, we identify an essential role for the specified myotome in axial tendon development, and suggest that absence of tendon formation in Myf5/Myod1 mutants results from loss of the myotomal FGF proteins, which depend upon Myf5 and Myod1 for their expression, and are required, in turn, for induction of the tendon progenitor markers. Our analysis of Sox5/Sox6 double mutants, in which the chondroprogenitors are unable to differentiate into cartilage, reveals that the two cell fates arising from the sclerotome, axial tendon and cartilage are alternative lineages, and that cartilage differentiation is required to actively repress tendon development in the dorsolateral sclerotome.

Bone morphogenetic protein 13 stimulates cell proliferation and production of collagen in human patellar tendon fibroblasts

Background Recombinant human (rh) bone morphogenetic protein 13 (BMP13) has been shown to induce the formation of tendon and ligament tissues in animal experiments. The role of BMP13 in tissue regeneration in human tendons remains unexplored, however.Material and methods We collected healthy human patellar tendon samples for histological examination and tendon fibroblast culture. The cultured cells were incubated in the presence and absence of rhBMP13 and the effect of the protein on cell proliferation was measured using 5-bromo-2’-deoxyuridine uptake.Results BMP13 was detectable by immunohisto-chemical staining in healthy patellar tendon samples, and was located exclusively in active tenoblasts and perivascular mesenchymal cells but not in interstitial tenocytes. The expression of proliferating cell nuclear antigen (PCNA) and pro-collagen type I showed a similar distribution. In vitro studies showed that rhBMP13 can increase proliferation of tendon fibroblasts and increase the gene expression of pro-collagen type I in tendon fibroblast culture.Interpretation Our findings indicate that BMP13 may be involved in the matrix remodeling process in adult tendon, and that it may play a role in tissue regeneration in tendons.

Six1 is not involved in limb tendon development, but is expressed in limb connective tissue under Shh regulation

Mice deficient for the homeobox gene Six1 display defects in limb muscles consistent with the Six1 expression in myogenic cells. In addition to its myogenic expression domain, Six1 has been described as being located in digit tendons and as being associated with connective tissue patterning in mouse limbs. With the aim of determining a possible involvement of Six1 in tendon development, we have carefully characterised the non-myogenic expression domain of the Six1 gene in mouse and chick limbs. In contrast to previous reports, we found that this non-myogenic domain is distinct from tendon primordia and from tendons defined by scleraxis expression. The non-myogenic domain of Six1 expression establishes normally in the absence of muscle, in Pax3 −/− mutant limbs. Moreover, the expression of scleraxis is not affected in early Six1 −/− mutant limbs. We conclude that the expression of the Six1 gene is not related to tendons and that Six1, at least on its own, is not involved in limb tendon formation in vertebrates. Finally, we found that the posterior domain of Six1 in connective tissue is adjacent to that of the secreted factor Sonic hedgehog and that Sonic hedgehog is necessary and sufficient for Six1 expression in posterior limb regions.

Coordinated development of muscles and tendons of theDrosophilaleg

Since Miller's morphological description, the Drosophila leg musculature and its formation has not been revisited. Here, using a set of GFP markers and confocal microscopy, we analyse Drosophila leg muscle development, and describe all the muscles and tendons present in the adult leg. Importantly, we provide for the first time evidence for tendons located internally within leg segments. By visualising muscle and tendon precursors, we demonstrate that leg muscle development is closely associated with the formation of internal tendons. In the third instars discs, in the vicinity of tendon progenitors, some Twist-positive myoblasts start to express the muscle founder cell marker dumbfounded (duf). Slightly later, in the early pupa, epithelial tendon precursors invaginate inside the developing leg segments, giving rise to the internal string-like tendons. The tendon-associated duf-lacZ-expressing muscle founders are distributed along the invaginating tendon precursors and then fuse with surrounding myoblasts to form syncytial myotubes. At mid-pupation, these myotubes grow towards their epithelial insertion sites, apodemes, and form links between internally located tendons and the leg epithelium. This leads to a stereotyped pattern of multifibre muscles that ensures movement of the adult leg.

Effect of recombinant human bone morphogenetic protein-12 (rhBMP-12) on regeneration of periodontal attachment following tooth replantation in dogs.

Subcutaneous and intramuscular implants of bone morphogenetic protein-12 (BMP-12) have been shown to induce formation of tendon and ligament tissue. BMP-12 induced a new attachment with a distinct fibrocartilaginous zone at the tendon-bone interface in the rat tendon-bone attachment model. Surgical controls showed poor healing and failure to reform the appropriate tendon-bone attachment morphologically. Application of recombinant human BMP-12 (rhBMP-12) to periodontal defects suggests that rhBMP-12 has the potential to support regeneration of the periodontal ligament (PDL). The objective of this pilot study was to evaluate this effect of rhBMP-12 in a tooth replantation model. Six, young adult, male Hound Labrador mongrel dogs were used. Maxillary and/or mandibular incisor and premolar teeth were extracted and the PDL was either left "intact" or removed by root planing. rhBMP-12 (1.0 mg/ml) or a buffer control was topically applied to teeth with "intact" PDL in contralateral jaw quadrants in each of 3 animals. The teeth were immersed in 1.0 ml of the rhBMP-12 or the buffer solution for 10 min and then replanted. The remaining three animals received rhBMP-12 (1.0 mg/ml) and the buffer control in a similar fashion applied to teeth instrumented to remove the PDL and cementum, and surface demineralized with citric acid. The animals were euthanized at 8 weeks postsurgery and block sections were collected and processed for histopathologic analysis. No dramatic differences were found between teeth receiving topical rhBMP-12 and the buffer control. Application of rhBMP-12 did not have an apparent effect on new cementum and PDL formation in the tooth replantation model. Moreover, application of rhBMP-12 did not increase nor did it decrease the apparent presence and extent of ankylosis along the root surface compared to the control. The observations from this study do not support the use of topical rhBMP-12 to support the reestablishment of the PDL including regeneration of cementum and functionally oriented fibers, and to prevent ankylosis and root resorption following replantation of teeth.

Periodontal repair in dogs: effect of recombinant human bone morphogenetic protein-12 (rhBMP-12) on regeneration of alveolar bone and periodontal attachment.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been shown to stimulate alveolar bone and cementum formation in periodontal defects but not a functionally oriented periodontal ligament (PDL). Subcutaneous and intramuscular implants of BMP-12 have been shown to induce tendon formation and ligament-like tissue. The objective of this study was to evaluate rhBMP-12 for periodontal regeneration, in particular PDL formation. Six young adult Hound Labrador mongrel dogs were used. Routine supraalveolar periodontal defects were created around the mandibular premolar teeth. Three animals received rhBMP-12(0.04 mg/ml) in an absorbable collagen sponge (ACS) carrier vs. rhBMP-12(0.2 mg/mL)/ACS in contralateral defects. Three animals received rhBMP-12(1.0 mg/ml)/ACS vs. rhBMP-2(0.2 mg/ml)/ACS (total implant volume/defect approximately 1 ml). The animals were euthanized 8 weeks postsurgery and block biopsies were processed for histometric analysis. Bone regeneration appeared increased in sites receiving rhBMP-2/ACS compared to sites receiving rhBMP-12/ACS. Cementum regeneration was similar comparing sites implanted with rhBMP-2/ACS to sites implanted with rhBMP-12/ACS. In contrast, sites receiving rhBMP-12/ACS exhibited a functionally oriented PDL bridging the gap between newly formed bone and cementum whereas this was a rare observation in sites receiving rhBMP-2/ACS. Ankylosis appeared increased in sites receiving rhBMP-2/ACS compared to those receiving rhBMP-12/ACS. The outcomes of this study suggest that rhBMP-12 may have significant effects on regeneration of the PDL. Additional preclinical evaluation is needed to confirm these initial observations prior to clinical application.

384. Stem Cells Expressing SMAD 8: A Platform for Tendon Regeneration

Current reconstructive techniques to repair torn tendons consist of autografts, allografts or synthetic prosthesis. Unfortunately, none of these surgical alternatives provide a long-term adequate solution. In this study, we defined a novel mechanism that mediates the differentiation of MSCs into tendon based on the SMAD8 signaling molecule, which could be used as a regenerative platform. A variant of SMAD8, deficient of the MH1 domain, was transfected to MSCs that express bone morphogenetic protein 2 (BMP2). The in vitro phenotype of the engineered MSCs was evaluated using RT-PCR, Histochemistry and morphology. In addition, the cells were injected into subcutaneous tissue and into the kidney capsule of female C3H/HeN mice in order to assess the tissue formation in vivo. Engineered MSCs, also expressing Lacz or Luciferase marker genes, were implanted on a Collagen sponge into a 3 mm partial defect of nude rats Achilles tendon. 5 weeks post implantation, the area of the injection or implantation was isolated and analyzed by light and electron microscopy, histochemical staining, RT-PCR and double quantum filtered (DQF) MRI. Cell survival in the implantation site was non-invasively and quantitatively demonstrated in vivo by the detection of Luciferase bioluminescence, using a CCCD system. Our results indicated a tenocytic phenotype of the engineered MSCs, based on microscopy and the expression of the tendon characteristic genes, Six 1and Scleraxis. Formation of tendon tissue in vivo was confirmed by the analysis of injected implants, which revealed the dense connective tissue with parallel-organized fibers and spindle shaped cells (A). Electron microscopic analysis showed tightly packed collagen fibers adjacent to fibroblast-shaped cells with active Endoplasmic Reticulum (B). Moreover, we were also able to repair Achilles tendon defects with the engineered MSCs. CCCD imaging quantitatively monitored cells survival in the operated tendon, and imunohistochmical staining detected the labeled implanted cells in site (C &F). DQF MRI analysis proved that highly ordered Collagen fibers were formed in the site of implantation as opposed to the control group (D, E). Since the expression of BMP2 is known to drive MSCs to osteogenic differentiation, we propose that the expression of SMAD8 in MSCs induces an inhibition of the BMP2 signaling pathway, resulting in the differentiation of MSCs into tendon cells. This is the first study showing that a particular SMAD signaling cascade is involved in tendon formation. Moreover, this is the first time a regenerative effect is shown to be exerted by the combination of a secreted factor and a signaling molecule. These findings may have considerable importance for therapeutic avenue in tendon in which SMAD 8 signaling plays a pivotal role Figure 1.

Lmx1b expression during joint and tendon formation: localization and evaluation of potential downstream targets

The tetrapod limb exhibits distinct dorsoventral joint, tendon, and muscle asymmetry. The LIM-homeodomain transcription factor, Lmx1b, is required to achieve the dorsal character of these structures, but the mechanism by which Lmx1b orchestrates this asymmetrical development is unknown. To identify target tissues and genes regulated by Lmx1b, we examined Lmx1b expression during joint, tendon and muscle formation (9.5–16.5 dpc) and the expression of several genes spatially restricted to developing joints and associated tissues in normal and Lmx1b knockout (KO) mice including: Gdf-5, sFrp2, sFrp3, Six1 and Six2. Lmx1b was diffusely expressed in the undifferentiated dorsal mesoderm of the emerging limb bud (E9.5–E11.5). With progressive proximal to distal differentiation, Lmx1b expression localized to dorsal joint-forming regions, to developing tendons and ligaments, but not to migrating myocytes (E13.5–15.5). By E16.5, mature tendon and ligament associations were evident and Lmx1b expression had regressed. The expression patterns of Gdf-5 and sFrp3 at E15.5 were symmetrical along the dorsoventral axis in normal and Lmx1b KO mice. sFrp2, Six1 and Six2 exhibited asymmetrical dorsoventral expression and in Lmx1b KO mice, this asymmetry is lost; however, none were solely restricted to or excluded from dorsal Lmx1b expressing tissues.

Signals regulating tendon formation during chick embryonic development.

Tendons are collagen-rich structures that link muscle to cartilage. By using quail-chick chimeras, it has been shown that tendon and cartilage cells originate from the same mesodermic compartment, which is distinct from that giving rise to muscle cells. Axial tendons originate from the sclerotomal compartment, and limb tendons originate from the lateral plate, whereas axial and limb muscles derive from dermomyotomes. Despite these different embryologic origins, muscle and tendon morphogenesis occurs in close spatial and temporal association. Facilitated by the distinct embryologic origin of myogenic and tendon cells, surgical studies in the avian embryo have highlighted interactions between tendons and muscles, during embryonic development. However, these interactions seem to differ between axial and limb levels. The molecular mechanisms underlying muscle and tendon interactions have been shown recently to involve different members of the fibroblast growth factor family. This review covers the available data on the early steps of tendon formation in the limb and along the primary axis. The relationship with muscle morphogenesis will be highlighted.

Adhesion formation after flexor tendon repair: a histologic and biomechanical comparison of 2- and 4-strand repairs in a chicken model

Purpose Both increased handling and increased bulk at the repair site have been hypothesized as affecting adhesion formation and gliding after tendon repair. Tendons repaired with 2- and 4-strand techniques were compared using both biomechanical and histopathologic measurements to determine the influence of increasing strand number on adhesion formation and gliding. Methods The flexor digitorum profundus tendon of the right middle toe of 80 broiler chickens was cut and then repaired with either a single (2-strand) or double (4-strand) modified Kessler core suture, followed by a running epitendinous suture. The limb was immobilized after surgery. Birds were killed at either 3 days or 4 weeks after tendon repair and adhesion formation measured using either biomechanical testing or quantitative and qualitative histology. For biomechanical testing, the tendon was pulled free of the sheath and a force versus displacement curve was generated. Comparisons of peak force and work to peak were made. Histologic specimens were examined by a pathologist blinded to the treatment group who scored the length and density of adhesions and made qualitative observations. Results Both biomechanical and histologic data showed expected differences in adhesion formation for early (3 days) and late (4 weeks) healing but no significant differences between 2- and 4-strand repairs. Biomechanical testing of 4-week specimens showed a nonsignificant tendency toward greater work required to break adhesions in 4-strand repairs. Conclusions Adhesion formation and gliding resistance of tendons after 2- or 4-strand modified Kessler core suture were not significantly different, which suggests that simply increasing the number of strands crossing a repair does not necessarily result in more adhesions or resistance in this model.

Paper #156 The effects of bone morphogenetic proteins 13 on human patellar tendon fibroblasts

Bone morphogenetic proteins (BMPs) are a family of proteins that can induce bone and cartilage formation during embryogenesis and tissue repair. Human recombinant (hr) BMP13 has been recently cloned and proved to promote the formation of tendon and ligament tissues in in vivo experiments. But the roles of BMP13 on tissue regeneration in tendons remain unexamined. In the present study, human patellar tendon samples were collected for histological examination and preparation of tendon fibroblast culture. Immunohistochemical staining showed that BMP12 was detected on healthy patellar tendon samples, chiefly located on active tenoblasts and perivascular mesenchymal cells, with a similar distribution as proliferation cell nuclear antigens (PCNA), pro-collagen I and decorin. In vitro studies on tendon fibroblast culture showed that hrBMP13 could increase cell proliferation and the gene expression of pro-collagen I and biglycan, but the gene expression of decorin was not affected. Our findings suggest that BMP13 may play a role in tendon healing. Bone morphogenetic proteins (BMPs) are a family of proteins that can induce bone and cartilage formation during embryogenesis and tissue repair. Human recombinant (hr) BMP13 has been recently cloned and proved to promote the formation of tendon and ligament tissues in in vivo experiments. But the roles of BMP13 on tissue regeneration in tendons remain unexamined. In the present study, human patellar tendon samples were collected for histological examination and preparation of tendon fibroblast culture. Immunohistochemical staining showed that BMP12 was detected on healthy patellar tendon samples, chiefly located on active tenoblasts and perivascular mesenchymal cells, with a similar distribution as proliferation cell nuclear antigens (PCNA), pro-collagen I and decorin. In vitro studies on tendon fibroblast culture showed that hrBMP13 could increase cell proliferation and the gene expression of pro-collagen I and biglycan, but the gene expression of decorin was not affected. Our findings suggest that BMP13 may play a role in tendon healing.

The roles of bone morphogenetic protein (BMP) 12 in stimulating the proliferation and matrix production of human patellar tendon fibroblasts

Recombinant human (rh) bone morphogenetic protein 12 (BMP12) is proved to induce the formation of tendon and ligament tissues in animal experiments. But the roles of BMP12 on tissue regeneration in human tendons remain unexplored. In the present study, healthy human patellar tendon samples were collected for histological examination and preparation of tendon fibroblast culture. Immunohistochemical staining showed that BMP12 was detected on healthy patellar tendon samples, only located on active tenoblasts and perivascular mesenchymal cells but not in interstitial tenocytes. The expression of PCNA and procollagen type I also exhibited a similar distribution. It indicates that BMP12 may be involved in matrix remodeling process in adult tissues. In vitro studies showed that rhBMP12 could increase proliferation of tendon fibroblasts and increase the gene expression of procollagen type I and type III, but decrease the gene expression of decorin in tendon fibroblasts culture. Our findings suggest that BMP12 may play a role in early phases of tissue regeneration in tendons.

Fgf4 Positively Regulates scleraxis and Tenascin Expression in Chick Limb Tendons

In vertebrates, tendons connect muscles to skeletal elements. Surgical experiments in the chick have underlined developmental interactions between tendons and muscles. Initial formation of tendons occurs autonomously with respect to muscle. However, further tendon development requires the presence of muscle. The molecular signals involved in these interactions remain unknown. In the chick limb, Fgf4 transcripts are located at the extremities of muscles, where the future tendons will attach. In this paper, we analyse the putative role of muscle-Fgf4 on tendon development. We have used three general tendon markers, scleraxis, tenascin, and Fgf8 to analyse the regulation of these tendon-associated molecules by Fgf4 under different experimental conditions. In the absence of Fgf4, in muscleless and aneural limbs, the expression of the three tendon-associated molecules, scleraxis, tenascin, and Fgf8, is down-regulated. Exogenous implantation of Fgf4 in normal, aneural, and muscleless limbs induces scleraxis and tenascin expression but not that of Fgf8. These results indicate that Fgf4 expressed in muscle is required for the maintenance of scleraxis and tenascin but not Fgf8 expression in tendons.