Cruciate Ligament Cell Sheets Can Be Rapidly Produced on Thermoresponsive poly(glycidyl ether) Coating and Successfully Used for Colonization of Embroidered Scaffolds.

Anterior cruciate ligament (ACL) cell sheets combined with biomechanically competent scaffolds might facilitate ACL tissue engineering. Since thermoresponsive polymers allow a rapid enzyme-free detachment of cell sheets, we evaluated the applicability of a thermoresponsive poly(glycidyl ether) (PGE) coating for cruciate ligamentocyte sheet formation and its influence on ligamentocyte phenotype during sheet-mediated colonization of embroidered scaffolds. Ligamentocytes were seeded on surfaces either coated with PGE or without coating. Detached ligamentocyte sheets were cultured separately or wrapped around an embroidered scaffold made of polylactide acid (PLA) and poly(lactic-co-ε-caprolactone) (P(LA-CL)) threads functionalized by gas-phase fluorination and with collagen foam. Ligamentocyte viability, protein and gene expression were determined in sheets detached from surfaces with or without PGE coating, scaffolds seeded with sheets from PGE-coated plates and the respective monolayers. Stable and vital ligamentocyte sheets could be produced within 24 h with both surfaces, but more rapidly with PGE coating. PGE did not affect ligamentocyte phenotype. Scaffolds could be colonized with sheets associated with high cell survival, stable gene expression of ligament-related type I collagen, decorin, tenascin C and Mohawk after 14 d and extracellular matrix (ECM) deposition. PGE coating facilitates ligamentocyte sheet formation, and sheets colonizing the scaffolds displayed a ligament-related phenotype.

Mutations in COMP cause familial carpal tunnel syndrome.

Carpal tunnel syndrome (CTS) is the most common peripheral nerve entrapment syndrome, affecting a large proportion of the general population. Genetic susceptibility has been implicated in CTS, but the causative genes remain elusive. Here, we report the identification of two mutations in cartilage oligomeric matrix protein (COMP) that segregate with CTS in two large families with or without multiple epiphyseal dysplasia (MED). Both mutations impair the secretion of COMP by tenocytes, but the mutation associated with MED also perturbs its secretion in chondrocytes. Further functional characterization of the CTS-specific mutation reveals similar histological and molecular changes of tendons/ligaments in patients' biopsies and the mouse models. The mutant COMP fails to oligomerize properly and is trapped in the ER, resulting in ER stress-induced unfolded protein response and cell death, leading to inflammation, progressive fibrosis and cell composition change in tendons/ligaments. The extracellular matrix (ECM) organization is also altered. Our studies uncover a previously unrecognized mechanism in CTS pathogenesis.

Recruitment of monocytes and mature macrophages in mouse pubic symphysis relaxation during pregnancy and postpartum recovery†.

Appropriate remodeling of the female lower reproductive tract and pelvic floor is essential during normal mammalian pregnancy, labor, and postpartum recovery. During mouse pregnancy, in addition to reproductive tract modifications, the pubic symphysis (PS) is remodeled into a soft interpubic ligament (IpL) to provide safe delivery of the offspring and fast postpartum recovery. Although temporal changes in the phenotypes of myeloid cells, such as mononuclear phagocytes, are crucial to remodeling the lower reproductive tract organs in preparation for a safe delivery, little is known about the involvement of recruited monocytes or macrophages in mouse PS remodeling. We used combined light microscopy, electron microscopy, and qPCR analysis to investigate the profile of recruited monocytes and macrophage polarization markers in C57Bl6 mouse interpubic tissues during pregnancy (D12, D18, and D19) and early days postpartum (1 dpp and 3 dpp) to better identify their presence in proper remodeling of the mouse PS. Our morphological data show that the number of recruited monocytes is increased in interpubic tissues and that recruited monocytes differentiate into proinflammatory or anti-inflammatory macrophage phenotypes from D18 to 3 dpp, which may contribute to dynamic changes in the gene expression of specific inflammatory mediators involved in interpubic tissue remodeling at these time points. Therefore, our morphological and quantitative gene expression data suggest that both differentiated macrophages from recruited monocytes and polarized macrophages may collaborate for IpL relaxation at labor and the appropriate repair of the PS after the first pregnancy.

Potential use of silkworm gut fiber braids as scaffolds for tendon and ligament tissue engineering.

Tendon and ligament tissue engineering require scaffolds for the treatment of various conditions in the medical field. These must meet requirements such as high tensile strength, biocompatibility, fast and stable repair and a rate of degradation that allows the repair of the damaged tissue. In this work, we propose the use of silkworm gut fiber braids as materials to temporarily replace and repair this type of tissues. The mechanical characterization of the braids made with different number of silk gut fibers is provided, as well as a descriptive analysis of the proliferation and adhesion of cultures of adult human mesenchymal stem cells from bone marrow and fibroblasts (L929) on the braids. As expected, the breaking force increases linearly in the scaffold with the number of fibers, thus being a parameter adaptable to the specific requirements of the tissue to repair and the animal model of study. On the other hand, in all of the cases studied, the values obtained for the elastic modulus of the hydrated fibers were in the range of the ones reported for various human tendons and ligaments. Moreover, the scaffold demonstrated excellent biocompatibility in vitro, allowing the adhesion and proliferation, in the same culture conditions, of the two cell types studied, therefore posing as an ideal candidate to be employed in future in vivo studies that allow elucidating its behavior in the articular environment or extra-articular tendinous areas. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res B Part B: 2019. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2209-2215, 2019.

Ligamentous structures in human glans penis.

The corpus spongiosum reportedly occupies a larger proportion of the human glans penis than does the penile body, embedding the end of the corpus cavernosus (CC). However, anatomic descriptions about the fibrous structures of glans penis in the literature cause confusion during dissection and reconstructive surgery. Forty-five penises of formalin-embalmed cadavers were dissected sagittally along the course of the distal urethra and observed macroscopically. Dense connective tissues adjacent to the fossa navicularis and spongiosum parts of the glans were cropped, and underwent Masson's trichrome and Verhoeff-Van-Gieson staining. Most (55.5%) of the specimens had distinct fibrous bands toward the distal tips of the glans penis, which elongated from the tunica albuginea of the CC. They comprised longitudinal collagen bundles continuous to the outer longitudinal layer of the tunica albuginea covering the CC and were intermingled with sparse elastic fibres. This architecture either did not reach the distal end of the glans penis (35.5% of cases), or was obscure or dispersed in all directions (9.0% of cases). The structural dimorphism and the variations in the ratio of dense connective tissue components of the fibrous skeleton are considered to contribute to the varying degrees of flexibility, distensibility and rigidity of the human glans penis.

Mesenchymal stem cell interacted with PLCL braided scaffold coated with poly-l-lysine/hyaluronic acid for ligament tissue engineering.

The challenge of finding an adapted scaffold for ligament tissue engineering remains unsolved after years of researches. A technology to fabricate a multilayer braided scaffold with flexible and elastic poly (l-lactide-co-caprolactone) (PLCL 85/15) has been recently pioneered by our team. In this study, polyelectrolyte multilayer films (PEM) with poly-l-lysine (PLL)/ hyaluronic acid (HA) were deposited on this scaffold. After PEM modification, polygonal (PLL) and particle-like (HA) structures were present on the braided scaffold with no significant variation of fibers Young's modulus. Wharton's jelly mesenchymal stem cells (WJ-MSC) and bone marrow mesenchymal stem cells (BM-MSC) showed good metabolic activity on scaffolds. They presented a spindled shape along the fiber longitudinal direction, and crossed the fibers to form cell bridges. Collagen type I, collagen type III, and tenascin-C secreted by MSCs were detected on day 14. Moreover, one-layer modified scaffold presented increased chemotaxis. As a conclusion, our results indicate that this braided PLCL scaffold with one-layer PEM modification shows inspiring potential with satisfying mechanical properties and biocompatibility. It opens new perspectives to incorporate growth factors within PEM-modified braided PLCL scaffold for ligament tissue engineering and to recruit endogenous cells after implantation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3042-3052, 2018.

Ultrasound-Mediated Gene Delivery Enhances Tendon Allograft Integration in Mini-Pig Ligament Reconstruction.

Ligament injuries occur frequently, substantially hindering routine daily activities and sports participation in patients. Surgical reconstruction using autogenous or allogeneic tissues is the gold standard treatment for ligament injuries. Although surgeons routinely perform ligament reconstructions, the integrity of these reconstructions largely depends on adequate biological healing of the interface between the ligament graft and the bone. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would lead to significantly improved ligament graft integration. To test this hypothesis, an anterior cruciate ligament reconstruction procedure was performed in Yucatan mini-pigs. A collagen scaffold was implanted in the reconstruction sites to facilitate recruitment of endogenous mesenchymal stem cells. Ultrasound-mediated reporter gene delivery successfully transfected 40% of cells recruited to the reconstruction sites. When BMP-6 encoding DNA was delivered, BMP-6 expression in the reconstruction sites was significantly enhanced. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to significantly enhanced osteointegration in all animals 8 weeks after surgery. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively improve ligament reconstruction in large animals, thereby addressing a major unmet orthopedic need and offering new possibilities for translation to the clinical setting.

Peroxisome proliferator-activated receptor-γ and its related pathway in bone marrow mesenchymal stem cell differentiation co-cultured with mechanically stretched ligament fibroblasts.

The occurrence of pelvic floor dysfunctional disease (PFD) is closely related with elasticity, toughness, and functional changes of the connective tissue of the pelvic support tissue. Bone marrow mesenchymal stem cells (BMSCs) have been confirmed to have the capacity to differentiate into a variety of cell types such as osteoblasts, chondroblasts, adipocytes and fibroblasts. Therefore, BMSCs have the potential to improve the clinical outcomes for PFD. Peroxisome proliferator-activated receptor-γ (PPAR-γ), a ligand activated transcription factor, has acquired a great deal of attention as it is involved in the fibrosis and cell differentiation. However, how it is regulated during the process of the differentiation of BMSCs into fibroblasts remains to be defined. The present study investigated the underlying mechanisms of PPAR-γ effect of mechanical stretch on the differentiation of BMSCs induced by pelvic ligament fibroblasts. PPAR-γ expression was decreased during the differentiation of BMSCs into fibroblasts by co-cultured stretched fibroblasts. Addition of transforming growth factor-ß1 (TGF-ß1) reduced PPAR-γ expression and promoted the differentiation of BMSCs. With the employment of endogenous ligand, activation of PPAR-γ suppressed the BMSC differentiation. Similar effects were also observed with overexpression of PPAR-γ gene. In addition, decrease of PPAR-γ by the use of shRNA targeting rat PPAR-γ significantly contributed to BMSC differentiation to fibroblasts. These results indicate that PPAR-γ negatively regulates the differentiation of BMSCs into fibroblasts.

The posterior epidural ligament in the thoracic region: a cadaveric and histological study.

The existence of posterior epidural ligaments (PEL) has been established in the lumbar region, but they have hitherto not been shown to exist in the thoracic vertebral column. Their identification is of clinical significance in respect to incidental durotomy and the circulation of cerebrospinal fluid (CSF). Fourteen thoracic spine sections were dissected by cutting through the intervertebral disc and separating the ligamentum flavum from the vertebra above. The dural sheath was gently retracted anteriorly to identify macroscopic connections between the ligamentum flavum and the dura. Macroscopic connections observed were dissected out, retaining some dural sheath and ligamentum flavum. Histological staining with haematoxylin and eosin and Miller's elastin stain was used to investigate cellular connections. Thoracic PELs were positively identified in 5 of the 14 cadavers (35.7%). Histology showed similarities between the thoracic and lumbar PELs. Fifteen separate PELs were identified within these five thoracic sections. The thoracic PEL has sufficient tensile strength to present a risk to the integrity of the dural sheath during surgery, and surgeons should be aware of these connections when operating on the thoracic spine. PELs may also contribute to the circulation of CSF in the spinal subarachnoid space.

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).

Combining electrospinning and cell sheet technology for the development of a multiscale tissue engineered ligament construct (TELC).

Ligament tissue rupture is a common sport injury. Although current treatment modalities can achieve appropriate reconstruction of the damaged ligament, they present significant drawbacks, mostly related to reduced tissue availability and pain associated with tissue harvesting. Stem cell based tissue regeneration combined with electrospun scaffolds represents a novel treatment method for torn ligaments. In this study, a low fiber density polycaprolactone (PCL) electrospun mesh and sheep mesenchymal stem cells (sMSCs) were used to develop tissue engineered ligament construct (TELC) in vitro. The assembly of the TELC was based on the spontaneous capacity of the cells to organize themselves into a cell sheet once seeded onto the electrospun mesh. The cell sheet matured over 4 weeks and strongly integrated with the low fiber density electrospun mesh which was subsequently processed into a ligament-like bundle and braided with two other bundles to develop the final construct. Live/dead assay revealed that the handling of the construct through the various phases of assembly did not cause significant difference in viability compared to the control. Mechanical evaluation demonstrated that the incorporation of the cell sheet into the braided construct resulted in significantly modifying the mechanical behavior. A stress/displacement J-curve was observed for the TELC that was similar to native ligament, whereas this particular feature was not observed in the non-cellularized specimens. The regenerative potential of the TELC was evaluated ectopically in immunocompromized rats, compared to non cellularized electrospun fiber mesh and this demonstrated that the TELC was well colonized by host cells and that a significant remodelling of the implanted construct was observed. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 399-409, 2018.

Modulating microfibrillar alignment and growth factor stimulation to regulate mesenchymal stem cell differentiation.

The ideal tissue engineering (TE) strategy for ligament regeneration should recapitulate the bone - calcified cartilage - fibrocartilage - soft tissue interface. Aligned electrospun-fibers have been shown to guide the deposition of a highly organized extracellular matrix (ECM) necessary for ligament TE. However, recapitulating the different tissues observed in the bone-ligament interface using such constructs remains a challenge. This study aimed to explore how fiber alignment and growth factor stimulation interact to regulate the chondrogenic and ligamentous differentiation of mesenchymal stem cells (MSCs). To this end aligned and randomly-aligned electrospun microfibrillar scaffolds were seeded with bone marrow derived MSCs and stimulated with transforming growth factor ß3 (TGFß3) or connective tissue growth factor (CTGF), either individually or sequentially. Without growth factor stimulation, MSCs on aligned-microfibers showed higher levels of tenomodulin (TNMD) and aggrecan gene expression compared to MSCs on randomly-oriented fibers. MSCs on aligned-microfibers stimulated with TGFß3 formed cellular aggregates and underwent robust chondrogenesis, evidenced by increased type II collagen expression and sulphated glycosaminoglycans (sGAG) synthesis compared to MSCs on randomly-oriented scaffolds. Bone morphogenetic protein 2 (BMP2) and type I collagen gene expression were higher on randomly-oriented scaffolds stimulated with TGFß3, suggesting this substrate was more supportive of an endochondral phenotype. In the presence of CTGF, MSCs underwent ligamentous differentiation, with increased TNMD expression on aligned compared to randomly aligned scaffolds. Upon sequential growth factor stimulation, MSCs expressed types I and II collagen and deposited higher overall levels of collagen compared to scaffolds stimulated with either growth factor in isolation. These findings demonstrate that modulating the alignment of microfibrillar scaffolds can be used to promote either an endochondral, chondrogenic, fibrochondrogenic or ligamentous MSC phenotype upon presentation of appropriate biochemical cues. STATEMENT OF SIGNIFICANCE: Polymeric electrospun fibers can be tuned to match the fibrillar size and anisotropy of collagen fibers in ligaments, and can be mechanically competent. Therefore, their use is attractive when attempting to tissue engineer the bone-ligament interface. A central challenge in this field is recapitulating the cellular phenotypes observed across the bone-ligament interface. Here we demonstrated that it is possible to direct MSCs seeded onto aligned electrospun fibres towards either a ligamentogenic, chondrogenic or fibrochondrogenic phenotype upon presentation of appropriate biochemical cues. This opens the possibility of using aligned microfibrillar scaffolds that are spatially functionalized with specific growth factors to direct MSC differentiation for engineering the bone-ligament interface.

The role of GPX1 in the pathogenesis of female pelvic organ prolapse.

Gestation and delivery can increase intra-abdominal pressure, which are well-known risk factors for Pelvic Organ Prolapse (POP). But the pathogenesis mechanism of POP remains unclear. Our previous research showed that the expression of glutathione peroxidase type 1 (GPX1) decreased in pelvic floor ligaments from POP patients, implying that oxidative stress (OS) may be related to POP. The aim of this study was to figure out the role of GPx1 regulation in the pathogenesis of POP. Women (>45 years) who received hysterectomy surgery were enrolled in this research, identified by screening. We applied mechanical strain of 0µ, 5333 µ to GPX1-overexpressing human uterosacral ligament fibroblasts (hUSLFs) isolated from menopausal women without POP respectively for 4 hours, in order to investigate the changes of cell apoptosis, oxidative status and ECM metabolism when cytomechanics model loaded on GPX1-overexpressing hUSLFs. Comparing with the non-transfection and mock-vehicle groups, we found that GPX1 not only protects hUSLFs from cell apoptosis, oxidative damage, but also improves the remodeling of ECM induced by mechanical stimulation. These results suggested that mechanical strain caused abnormalities of ECM metabolism via OS pathway in hUSLFs, which was involved in the pathogenesis of POP, and that GPx1 played a significant role in regulating mechanical strain induced POP.

Biofabrication of soft tissue templates for engineering the bone-ligament interface.

Regenerating damaged tissue interfaces remains a significant clinical challenge, requiring recapitulation of the structure, composition, and function of the native enthesis. In the ligament-to-bone interface, this region transitions from ligament to fibrocartilage, to calcified cartilage and then to bone. This gradation in tissue types facilitates the transfer of load between soft and hard structures while minimizing stress concentrations at the interface. Previous attempts to engineer the ligament-bone interface have utilized various scaffold materials with an array of various cell types and/or biological cues. The primary goal of this study was to engineer a multiphased construct mimicking the ligament-bone interface by driving differentiation of a single population of mesenchymal stem cells (MSCs), seeded within blended fibrin-alginate hydrogels, down an endochondral, fibrocartilaginous, or ligamentous pathway through spatial presentation of growth factors along the length of the construct within a custom-developed, dual-chamber culture system. MSCs within these engineered constructs demonstrated spatially distinct regions of differentiation, adopting either a cartilaginous or ligamentous phenotype depending on their local environment. Furthermore, there was also evidence of spatially defined progression toward an endochondral phenotype when chondrogenically primed MSCs within this construct were additionally exposed to hypertrophic cues. The study demonstrates the feasibility of engineering spatially complex soft tissues within a single MSC laden hydrogel through the defined presentation of biochemical cues. This novel approach represents a new strategy for engineering the ligament-bone interface. Biotechnol. Bioeng. 2017;114: 2400-2411. © 2017 Wiley Periodicals, Inc.

Extracellular matrix metabolism disorder induced by mechanical strain on human parametrial ligament fibroblasts.

Pelvic organ prolapse (POP) is a global health problem that may seriously impact the quality of life of the sufferer. The present study aimed to investigate the potential mechanisms underlying alterations in extracellular matrix (ECM) metabolism in the pathogenesis of POP, by investigating the expression of ECM components in human parametrial ligament fibroblasts (hPLFs) subject to various mechanical strain loads. Fibroblasts derived from parametrial ligaments were cultured from patients with POP and without malignant tumors, who underwent vaginal hysterectomy surgery. Fibroblasts at generations 3­6 of exponential phase cells were selected, and a four­point bending device was used for 0, 1,333 or 5,333 µ mechanical loading of cells at 0.5 Hz for 4 h. mRNA and protein expression levels of collagen type I α 1 chain (COL1A1), collagen type III α 1 chain (COL3A1), elastin, matrix metalloproteinase (MMP) ­2 and ­9, and transforming growth factor (TGF)­ß1 were detected by reverse transcription­quantitative polymerase chain reaction and western blotting, respectively. Under increased mechanical strain (5,333 µ), mRNA and protein expression levels of COL1A1, COL3A1 elastin and TGF­ß1 decreased, particularly COL1A1; however, mRNA and protein expression levels of MMP­2 and ­9 were significantly increased, compared with the control group (0 µ strain). Following 1,333 µ mechanical strain, mRNA and protein expression levels of COL1A1, COL3A1 elastin and MMP­2 increased, and MMP­9 decreased, whereas no significant differences were observed in TGF­ß1 mRNA and protein expression levels. In conclusion, ECM alterations may be involved in pathogenesis of POP, with decreased synthesis and increased degradation of collagen and elastin. Furthermore, the TGF­ß1 signaling pathway may serve an important role in this process and thus may supply a new target and strategy for understanding the etiology and therapy of POP.

Mechanical stress influences the viability and morphology of human parametrial ligament fibroblasts.

The present study aimed to investigate damage to human parametrial ligament fibroblasts by detecting cell proliferation, cytoskeletal structure, cellular alterations and senescence. Uterosacral and cardinal ligaments were obtained from 10 patients with cervical intraepithelial neoplasia grade II­III, who had received total vaginal hysterectomies, and fibroblasts were derived from this tissue. Fibroblasts were stretched using a four­point bending system with a force of 0 (control), 1,333 µ strain (1 mm) or 5,333 µ strain (4 mm) for 4 h. The present study revealed that mechanical force significantly reduced cell proliferation and increased cell senescence. As mechanical force increased, the mitochondria of fibroblasts began to exhibit vacuolization, and the cell cytoskeleton began to depolymerize and rearrange. In conclusion, the present study demonstrated that mechanical forces within a certain range may induce cell damage via mitochondrial injury, cytoskeletal alterations and increased cell senescence, resulting in decreased cell viability of pelvic fibroblasts.

The Development of a Xenograft-Derived Scaffold for Tendon and Ligament Reconstruction Using a Decellularization and Oxidation Protocol.

PURPOSE: To evaluate the biological, immunological, and biomechanical properties of a scaffold derived by architectural modification of a fresh-frozen porcine patella tendon using a decellularization protocol that combines physical, chemical, and enzymatic modalities. METHODS: Porcine patellar tendons were processed using a decellularization and oxidation protocol that combines physical, chemical, and enzymatic modalities. Scaffolds (n = 88) were compared with native tendons (n = 70) using histologic, structural (scanning electron microscopy, porosimetry, and tensile testing), biochemical (mass spectrometry, peracetic acid reduction, DNA quantification, alpha-galactosidase [α-gal] content), as well as in vitro immunologic (cytocompatibility, cytokine induction) and in vivo immunologic nonhuman primate analyses. RESULTS: A decrease in cellularity based on histology and a significant decrease in DNA content were observed in the scaffolds compared with the native tendon (P < .001). Porosity and pore size were increased significantly (P < .001). Scaffolds were cytocompatible in vitro. There was no difference between native tendons and scaffolds when comparing ultimate tensile load, stiffness, and elastic modulus. The α-gal xenoantigen level was significantly lower in the decellularized scaffold group compared with fresh-frozen, nondecellularized tissue (P < .001). The in vivo immunological response to implanted scaffolds measured by tumor necrosis factor-α and interleukin-6 levels was significantly (P < .001) reduced compared with untreated controls in vitro. These results were confirmed by an attenuated response to scaffolds in vivo after implantation in a nonhuman primate model. CONCLUSIONS: Porcine tendon was processed via a method of decellularization and oxidation to produce a scaffold that possessed significantly less inflammatory potential than a native tendon, was biocompatible in vitro, of increased porosity, and with significantly reduced amounts of α-gal epitope while retaining tensile properties. CLINICAL RELEVANCE: Porcine-derived scaffolds may provide a readily available source of material for musculoskeletal reconstruction and repair while eliminating concerns regarding disease transmission and the morbidity of autologous harvest.

Early cellular responses of BMSCs genetically modified with bFGF/BMP2 co-cultured with ligament fibroblasts in a three-dimensional model in vitro.

Currently, a number of strategies including the implantation of bone marrow-derived mesenchymal stem cells (BMSCs) and growth factors have been developed to regenerate the tendon-to-bone interface after performing anterior cruciate ligament reconstruction. However, the mechanisms behind the interactions of the implanted BMSCs and tendon cells remain to be elucidated. The aim of this study was to evaluate the early cellular responses of BMSCs genetically modified with basic growth factor growth factor (bFGF)/bone morphogenic protein 2 (BMP2) and ligament fibroblasts in a three-dimensional co-culture model. BMSCs and ligament fibroblasts were both isolated from male Wistar rats. The BMSCs were then transfected with an adenoviral vector carrying bFGF or BMP2. The transfected BMSCs and ligament fibroblasts both encapsulated in alginate beads were co-cultured for 6 days in three-dimensional model. On days 0, 3 and 6, cell proliferation was assayed. On day 6, the expression of several tendon-bone related markers was evaluated. In the co-culture system, bFGF and BMP2 were highly expressed at the mRNA and protein level. During the process, bFGF significantly promoted cell proliferation, as well as the expression of scleraxis (SCX) and collagen (COL) type â…  (COL1) in the BMSCs; however, it markedly decreased the expression of phenotype markers in the ligament fibroblasts, including COL1 and COL3. BMP2 markedly increased the expression of alkaline phosphatase and osteocalcin in the BMSCs and ligament fibroblasts, whereas it had no obvious effect on cell proliferation and collagen synthesis in the ligament fibroblasts. The combination of bFGF and BMP2 resulted in the similarly enhanced proliferation of BMSCs and ligament fibroblasts as observed with bFGF alone; however, this combination more potently promoted osteogenic differentiation than did BMP2 alone. The findings of our study demonstrate the superiority of the combined use of growth factors in inducing osteogenic differentiation and provide a theoretical foundation for the regeneration of the tendon-to-bone interface.

Functional regeneration of ligament-bone interface using a triphasic silk-based graft.

The biodegradable silk-based scaffold with unique mechanical property and biocompatibility represents a favorable ligamentous graft for tissue-engineering anterior cruciate ligament (ACL) reconstruction. However, the low efficiency of ligament-bone interface restoration barriers the isotropic silk graft to common ACL therapeutics. To enhance the regeneration of the silk-mediated interface, we developed a specialized stratification approach implementing a sequential modification on isotropic silk to constitute a triphasic silk-based graft in which three regions respectively referring to ligament, cartilage and bone layers of interface were divided, followed by respective biomaterial coating. Furthermore, three types of cells including bone marrow mesenchymal stem cells (BMSCs), chondrocytes and osteoblasts were respectively seeded on the ligament, cartilage and bone region of the triphasic silk graft, and the cell/scaffold complex was rolled up as a multilayered graft mimicking the stratified structure of native ligament-bone interface. In vitro, the trilineage cells loaded on the triphasic silk scaffold revealed a high proliferative capacity as well as enhanced differentiation ability into their corresponding cell lineage. 24 weeks postoperatively after the construct was implanted to repair the ACL defect in rabbit model, the silk-based ligamentous graft exhibited the enhancement of osseointegration detected by a robust pullout force and formation of three-layered structure along with conspicuously corresponding matrix deposition via micro-CT and histological analysis. These findings potentially broaden the application of silk-based ligamentous graft for ACL reconstruction and further large animal study.

Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse.

Mechanical loading on pelvic supports contributes to pelvic organ prolapse (POP). However, the underlying mechanisms remain to be elucidated. Our previous study identified that mechanical strain induced oxidative stress (OS) and promoted apoptosis and senescence in pelvic support fibroblasts. The aim of the present study is to investigate the molecular signaling pathway linking mechanical force with POP. Using a four­point bending device, human uterosacral ligament fibroblasts (hUSLF) were exposed to mechanical tensile strain at a frequency of 0.3 Hz and intensity of 5333 µÎµ, in the presence or absence of LY294002. The applied mechanical strain on hUSLF resulted in apoptosis and senescence, and decreased expression of procollagen type I α1. Mechanical strain activated phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling and resulted in downregulated expression of glutathione peroxidase 1 and Mn­superoxide dismutase, and accumulation of intracellular reactive oxygen species. These effects were blocked by administration of LY294002. Furthermore, it was demonstrated that PI3K/Akt was activated in the uterosacral ligaments of POP patients, and that OS was increased and collagen type I production reduced. The results from the present study suggest that mechanical strain promotes apoptosis and senescence, and reduces collagen type I production via activation of PI3K/Akt-mediated OS signaling pathway in hUSLF. This process may be involved in the pathogenesis of POP as it results in relaxation and dysfunction of pelvic supports.