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CypA activates Wnt/β-catenin signaling

Clinicopathological characteristics

Purpose: Glioma-initiating cells (GIC) are glioma stem–like cells that contribute to glioblastoma (GBM) development, recurrence, and resistance to chemotherapy and radiotherapy. They have recently become the focus of novel treatment strategies. Cyclophilin A (CypA) is a cytosolic protein that belongs to the peptidyl–prolyl isomerase (PPIase) family and the major intracellular target of the immunosuppressive drug cyclosporin A (CsA). In this study, we investigate the functions of CypA and its mechanism of action in GICs' development.Experimental Design: We analyzed differences in CypA expression between primary tumors and neurospheres from the GDS database, both before and after GIC differentiation. A series of experiments was conducted to investigate the role of CypA in GIC stemness, self-renewal, proliferation, radiotherapy resistance, and mechanism. We then designed glutathione S-transferase (GST) pulldown and coimmunoprecipitation assays to detect signaling activity.Results: In this study, we demonstrated that CypA promotes GIC stemness, self-renewal, proliferation, and radiotherapy resistance. Mechanistically, we found that CypA binds β-catenin and is recruited to Wnt target gene promoters. By increasing the interaction between β-catenin and TCF4, CypA enhances transcriptional activity.Conclusions: Our results demonstrate that CypA enhances GIC stemness, self-renewal, and radioresistance through Wnt/β-catenin signaling. Due to its promotive effects on GICs, CypA is a potential target for future glioma therapy. Clin Cancer Res; 23(21); 6640–9. ©2017 AACR.

Supramolecular hydrogels are widely used as 3D scaffolds and delivery platforms in tissue engineering applications. However, hydrophobic therapeutic agents exhibit weak compatibility in hydrogel scaffolds along with aggregation and precipitation. Herein, simvastatin drugs used as BMP-2 stimulators are encapsulated into the layer space of LAPONITE® via electrostatic interactions and ion exchange efficiently, and supramolecular hydrogels could be fabricated with a self-healing, injectable and sustained drug release nature. Hydrogels encapsulated with 10 μg mL−1 simvastatin drug show good osteogenic differentiation in vitro. Moreover, the loading of demineralized bone matrix particles could enhance the capacity for osteogenesis via improving the expression of BMP-2 synergistically. The integrated hydrogels could be implanted into cranial defect sites for bone regeneration in vivo. This work provides the first demonstration of molecular and supramolecular engineering of hydrogels to load osteoinductive agents hierarchically for bone regeneration, contributing to the development of a brand-new strategy for dealing with compatibility between scaffolds and osteogenic agents.

Administration of exosomes is considered an attractive cell-free approach to skeletal repair and pathological disease treatment. However, poor yield for the production technique and unexpected therapeutic efficacy of exosomes have been obstacles to their widespread use in clinical practices. Here, we report an alternative strategy to produce exosome-related vesicles with high yields and improved regenerative capability. An extrusion approach was employed to amass exosome mimetics (EMs) from human mesenchymal stem cells (hMSCs). The collected EMs had a significantly increased proportion of vesicles positive for the exosome-specific CD-63 marker compared with MSC-derived exosomes. EMs were further obtained from genetically modified hMSCs in which expression of noggin, a natural bone morphogenetic protein antagonist, was down-regulated to enhance osteogenic properties of EMs. Moreover, the administration of hMSC-EMs in conjunction with an injectable chitosan hydrogel into mouse nonhealing calvarial defects demonstrated robust bone regeneration. Importantly, mechanistic studies revealed that the enhanced osteogenesis by EMs in which noggin was suppressed was mediated via inhibition of miR-29a. These findings demonstrate the great promise of MSC-mediated EMs and modulation of small RNA signaling for skeletal regeneration and cell-free therapy.

Supramolecular hydrogels have great potential as biomaterials for tissue engineering applications or vehicles for delivering therapeutic agents. Herein, a self-healing and pro-osteogenic hydrogel system is developed based on the self-assembly of laponite nanosheets and guanidinylated chitosan, where laponite works as a physical crosslinker with osteoinductive properties to form a network structure with a cationic guanidine group on chitosan chains. The hydrogels can be prepared with varying ratios of chitosan to laponite and display self-healing and injectable properties because of supramolecular forces as well as osteoinductive activity due to nanoclay. They enhance cell adhesion and promote osteogenic differentiation of mesenchymal stem cells by activating the Wnt/β-catenin signaling pathway. In addition, the hydrogel is used as a malleable carrier for the demineralized bone matrix (DBM). The loading of the DBM does not affect the self-healing and injectable natures of hydrogels while enhancing the osteogenic capacity, indicating that advanced allograft bone formulations with carriers can facilitate handling and bone healing. This work provides the first demonstration of therapeutic supramolecular design for the treatment of bone defects.

Hydrogels have been widely used in bone tissue engineering due to their tunable characteristics that allow facile modifications with various biochemical properties to support cell growth and guide proper cell functions. Herein, we report a design of hydrogel-siRNA conjugate that facilitates osteogenesis via gene silencing and activation of bone morphogenetic protein (BMP) signaling. A sulfonate hydrogel is prepared by modifying chitosan with sulfoacetic acid to mimic a natural sulfated polysaccharide and to provide a hydrogel surface that enables BMP binding. Then, siRNA targeting noggin, an endogenous extracellular antagonist of BMP signaling, is covalently conjugated to the sulfonate hydrogel by visible blue light crosslinking. The sulfonate hydrogel-siRNA conjugate is efficient to bind BMPs and also successfully prolongs the release of siRNA for sustained noggin suppression, thereby resulting in significantly increased osteogenic differentiation. Lastly, demineralized bone matrix (DBM) is incorporated into the sulfonate hydrogel-siRNA conjugate, wherein the DBM incorporation induces noggin expression via a negative feedback mechanism that regulates BMP signaling in DBM. However, simultaneous delivery of siRNA downregulates noggin thus facilitating endogenous BMP activity and enhancing the osteogenic efficacy of DBM. These findings support a promising hydrogel RNA silencing platform for bone tissue engineering applications.

Demineralized bone matrix (DBM), a potential alternative to autologous bone-graft, has been increasingly used for clinical bone repair; however, its application in larger defects isn't successful partly due to the rapid dispersion of DBM particles and relatively lower osteoinductivity. Here, a novel strategy is created to complement the osteoinductivity of DBM by incorporating DBM in biopolymer hydrogel combined with the abrogation of BMP antagonism. Combined treatment of DBM + noggin-suppression displays increased osteogenic potency of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. Injectable chitosan (MeGC)-based hydrogel with heparinization (Hep-MeGC) is further developed to localize and stabilize DBM. Noggin-suppression reveals the significant increase in osteogenesis of hBMSCs in the photopolymerizable Hep-MeGC hydrogels with the encapsulation of DBM. Moreover, the combination of DBM + noggin-suppression in the injectable Hep-MeGC hydrogel displays a robust bone healing in mouse critical-sized calvarial defects in vivo. The mechanistic analysis demonstrates that noggin-suppression increased DBM osteoinductivity by stimulating endogenous BMP/Smad signals. These results have shown promise in DBM's ability as a prominent bone grafting material while being coupled with gene editing mechanism and a localizing three-dimensional scaffold. Together, this approach poses a significant increase in the efficiency of DBM-mediated craniofacial bone repair and dental osteointegration.

Bone repair is a complex process involving the sophisticated interplay of osteogenic stem cells, extracellular matrix, and osteoinductive factors, and it is affected by bacterial toxins and oxidative stress. Inspired by the nature of plant-derived phytochemicals and inorganic-organic analogues of the bone extracellular matrix, we report herein the facile design of a nanoclay-organic hydrogel bone sealant (NoBS) that integrates multiple physico-chemical cues for bone regeneration into a single system. Assembly of phytochemical-modified organic chitosan and silica-rich inorganic nanoclay serves as highly biocompatible and osteoconductive extracellular matrix mimics. The decorated phytochemical exerts inherent bactericidal and antioxidant activities, and acts as an intermolecular networking precursor for gelation with injectable and self-healing capabilities. Moreover, the NoBS exerts osteoinductive effects mediated by the nanoclay, which regulates the Wnt/β-catenin pathway, along with the addition of osteoinductive signals, resulting in bone regeneration in a non-healing cranial defect. Engineering of this integrated bone graft substitute with multifunctional properties inspired by natural materials may suggest a promising and effective approach for creating a favorable microenvironment for optimal bone healing.

Hydrogels with controlled degradation and sustained bactericidal activities are promising biomaterial substrates to repair or regenerate the injured tissue. In this work, we present a unique pair of lysozyme and chitosan as a hydrogel that can promote cell growth and proliferation while concomitantly preventing infection during the gradual process of hydrogel degradation and tissue ingrowth. Lysozyme and chitosan containing cell adhesion motifs are chemically modified with photoreactive methacrylate moieties to obtain a crosslinked hydrogel network by visible light irradiation. The resulting lysozyme-chitosan conjugate successfully modulates the degradation rate of hydrogels while promoting cell adhesion, proliferation, and matrix formation with no cytotoxicity. The hydrogel also exerts an intrinsic antibacterial effect by combining antimicrobial features of chitosan and lysozyme. This work demonstrates an advanced hydrogel platform with dual function of tunable degradation and infection control for tissue engineering and wound healing applications.

Hybrid scaffold with smoothened agonist sterosomes is engineered as a useful bone graft device for more efficacious bone healing., Biomaterial delivery of bioactive agents and manipulation of stem cell fate are an attractive approach to promote tissue regeneration. Here, smoothened agonist sterosome is developed using small-molecule activators [20S-hydroxycholesterol (OHC) and purmorphamine (PUR)] of the smoothened protein in the hedgehog pathway as carrier and cargo. Sterosome presents inherent osteoinductive property even without drug loading. Sterosome is covalently immobilized onto three-dimensional scaffolds via a bioinspired polydopamine intermediate to fabricate a hybrid scaffold for bone regeneration. Sterosome-immobilized hybrid scaffold not only provides a favorable substrate for cell adhesion and proliferation but also delivers bioactive agents in a sustained and spatially targeted manner. Furthermore, this scaffold significantly improves osteogenic differentiation of bone marrow stem cells through OHC/PUR-mediated synergistic activation of the hedgehog pathway and also enhances bone repair in a mouse calvarial defect model. This system serves as a versatile biomaterial platform for many applications, including therapeutic delivery and endogenous regenerative medicine.

The use of small molecular drugs with gene manipulation offers synergistic therapeutic efficacy by targeting multiple signaling pathways for combined treatment. Stimulation of mesenchymal stem cells (MSCs) with osteoinductive small molecule phenamil combined with suppression of noggin is a promising therapeutic strategy that increases bone morphogenetic protein (BMP) signaling and bone repair. Our cationic Sterosome formulated with stearylamine (SA) and cholesterol (Chol) is an attractive co-delivery system that not only forms stable complexes with small interfering RNA (siRNA) molecules but also solubilizes hydrophobic small molecules in a single vehicle, for directing stem cell differentiation. Herein, we demonstrate the ability of SA/Chol Sterosomes to simultaneously deliver hydrophobic small molecule phenamil and noggin-directed siRNA to enhance osteogenic differentiation of MSCs both in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. These results suggest a novel liposomal platform to simultaneously deliver therapeutic genes and small molecules for combined therapy.Statement of significanceApplication of phenamil, a small molecular bone morphogenetic protein (BMP) stimulator, combined with suppression of natural BMP antagonists such as noggin is a promising therapeutic strategy to enhance bone regeneration. Here, we present a novel strategy to co-deliver hydrophobic small molecule phenamil and noggin-targeted siRNA via cationic Sterosomes formed with stearylamine (SA) and high content of cholesterol (Chol) to enhance osteogenesis and bone repair. SA/Chol Sterosomes demonstrated high phenamil encapsulation efficiency, supported sustained release of encapsulated drugs, and significantly reduced drug dose requirements to induce osteogenic differentiation of mesenchymal stem cells (MSCs). Simultaneous deliver of phenamil and noggin siRNA in a single vehicle synergistically enhanced MSC osteogenesis and calvarial bone repair. This study suggests a new non-phospholipid liposomal formulation to simultaneously deliver small molecules and therapeutic genes for combined treatment.

Background Demineralized bone matrix (DBM), an allograft bone processed to better expose osteoinductive factors such as bone morphogenetic proteins (BMPs), is increasingly used for clinical bone repair. However, more extensive use of DBM is limited by its unpredictable osteoinductivity and low bone formation capacity. Commercial DBM products often employ polymeric carriers to enhance handling properties but such carriers generally do not possess bioactive functions. Heparin is a highly sulfated polysaccharide and is shown to form a stable complex with growth factors to enhance their bioactivities. In this study, a new heparinized synthetic carrier for DBM is developed based on photocrosslinking of methacrylated glycol chitosan and heparin conjugation. Results Heparinized chitosan exerts protective effects on BMP bioactivity against physiological stressors related to bone fracture healing. It also enhances the potency of BMPs by inhibiting the activity of BMP antagonist, noggin. Moreover, heparinized chitosan is effective to deliver bone marrow stromal cells and DBM for enhanced osteogenesis by sequestering and localizing the cell-produced or DBM-released BMPs. Conclusions This research suggests an essential approach of developing a new hydrogel carrier to stabilize the bioactivity of BMPs and improve the clinical efficacy of current bone graft therapeutics for accelerated bone repair.

Additional file 4: Figure S4. Half-life extension of BMP-2 induced by Hep-MeGC. BMSCs were cultured with 100 ng mL− 1 of BMP-2 with MeGC (blue) and Hep-MeGC (orange) for prolonged condition and with 200 ng mL− 1 MMP-9 for proteolysis condition. The concentration of BMP-2 near cell layer were quantified by BMP-2 enzyme-linked immunosorbent assay.

Additional file 3: Figure S3. H&E staining images of hydrogel-DBM composites. The “H” indicates the location of hydrogel and “D” indicates the location of DBM with the proliferated cells. Scale bar is 50 μm.

Additional file 1: Figure S1. Relative ALP expression of BMSCs cultured with graded dosages of noggin, BMP-2, and either MeGC or Hep-MeGC (corresponding to Fig. 3a). *p

A silk scaffold exhibits high potential for the human anterior cruciate ligament (ACL) reconstruction due to its exceptional mechanics as well as biocompatibility. Inefficient ACL interface restoration is thought to be a major hurdle for the common implementation of a silk-based ligament graft. By integrating a stratified approach and gene immobilization, here we developed a gene-immobilized triphasic silk scaffold to enhance ACL osseointegration. Isotropic silk was divided into three regions (respectively corresponding to a ligament, fibrocartilage and the bone region of the native ACL interface) using a custom-made divider, and the lentiviral vector-encoded transforming growth factor beta-3 (TGF-β3) and bone morphogenetic protein-2 (BMP2) was further, respectively, immobilized to phosphatidylserine (PS)-coated fibrocartilage and the bone region of the triphasic silk scaffold. The in vitro assessments displayed that this gene-immobilized triphasic silk scaffold significantly promotes bone marrow mesenchymal stem cell (BMSC) proliferation and differentiation into corresponding cell lineage. Moreover, the gene-modified triphasic silk scaffold combined with BMSCs alone, which was rolled into a compact shaft to be implanted onto rabbit ACL-defect models, revealed roughly complete osseointegration restoration as a result of apparent three-layered tissue formation and robust mechanical ability as early as 12 weeks postoperatively. These outcomes demonstrated that employing the stratified approach and gene immobilization efficiently expedites silk-mediated ACL interface formation, expanding the therapeutic potential of the silk-based ligament graft for ACL reconstruction.

Vascular regeneration is thought to be crucial in the repair of damaged vessels as well as nonvascular tissues. A healthy endothelial layer provides homeostasis and prevents thrombosis in blood vessels. The variety of cells such as hematopoietic stem cells (HSCs), endothelial progenitor cells (EPCs), and mature endothelial cells (ECs), are revealed to play an important role in forming an endothelial layer. There are a number of biomolecules that have been identified to be capable of attacting these cells to participate in vascular repair. In terms of these findings, alternative strategies through the biomolecule modification of scaffold have been recently established to enhance in situ endothelialization for vascular regeneration. This article mainly reviews current and developing biomolecules that can be immobilized onto biomaterial surfaces to accelerate in situ endothelialization for vascular repair, providing potentials in further discovering novel tissue engineering therapeutics for the treatment of human vascular diseases.

Aberrant lineage commitment of mesenchymal stem cells (MSCs) in marrow contributes to abnormal bone formation due to reduced osteogenic and increased adipogenic potency. While several major transcriptional factors associated with lineage differentiation have been found during the last few decades, the molecular switch for MSC fate determination and its role in skeletal regeneration remains largely unknown, limiting creation of effective therapeutic approaches. Tribbles homolog 3 (Trb3), a member of tribbles family pseudokinases, is known to exert diverse roles in cellular differentiation. Here, we investigated the reciprocal role of Trb3 in the regulation of osteogenic and adipogenic differentiation of MSCs in the context of bone formation, and examined the mechanisms by which Trb3 controls the adipo-osteogenic balance. Trb3 promoted osteoblastic commitment of MSCs at the expense of adipocyte differentiation. Mechanistically, Trb3 regulated cell-fate choice of MSCs through BMP/Smad and Wnt/β-catenin signals. Importantly, in vivo local delivery of Trb3 using a novel gelatin-conjugated caffeic acid-coated apatite/PLGA (GelCA-PLGA) scaffold stimulated robust bone regeneration and inhibited fat-filled cyst formation in rodent non-healing mandibular defect models. These findings demonstrate Trb3-based therapeutic strategies that favor osteoblastogenesis over adipogenesis for improved skeletal regeneration and future treatment of bone-loss disease. The distinctive approach implementing a scaffold-mediated local gene transfer may further broaden the translational use of targeting specific therapeutic gene related to lineage commitment for clinical bone treatment.

Vascular regeneration is known to play an essential role in the repair of injured tissues mainly through accelerating the repair of vascular injury caused by vascular diseases, as well as the recovery of ischemic tissues. However, the clinical vascular regeneration is still challenging. Cell-based therapy is thought to be a promising strategy for vascular regeneration, since various cells have been identified to exert important influences on the process of vascular regeneration such as the enhanced endothelium formation on the surface of vascular grafts, and the induction of vessel-like network formation in the ischemic tissues. Here are a vast number of diverse cell-based strategies that have been extensively studied in vascular regeneration. These strategies can be further classified into three main categories, including cell transplantation, construction of tissue-engineered grafts, and surface modification of scaffolds. Cells used in these strategies mainly refer to terminally differentiated vascular cells, pluripotent stem cells, multipotent stem cells, and unipotent stem cells. The aim of this review is to summarize the reported research advances on the application of various cells for vascular regeneration, yielding insights into future clinical treatment for injured tissue/organ.

RNA interference (RNAi)-based therapy using small interfering RNA (siRNA) exhibits great potential to treat diseases. Although calcium phosphate (CaP)-based systems are attractive options to deliver nucleic acids due to their good biocompatibility and high affinity with nucleic acids, they are limited by uncontrollable particle formation and inconsistent transfection efficiencies. In this study, we developed a stable CaP nanocarrier system with enhanced intracellular uptake by adding highly cationic, glutamine-conjugated oligochitosan (Gln-OChi). CaP nanoparticles coated with Gln-OChi (CaP/Gln-OChi) significantly enhanced gene transfection and knockdown efficiency in both immortalized cell line (HeLa) and primary mesenchymal stem cells (MSCs) with minimal cytotoxicity. The osteogenic bioactivity of siRNA-loaded CaP/Gln-OChi particles was further confirmed in three-dimensional environments by using photocrosslinkable chitosan hydrogels encapsulating MSCs and particles loaded with siRNA targeting noggin, a bone morphogenetic protein antagonist. These findings suggest that our CaP/Gln-OChi nanocarrier provides an efficient and safe gene delivery system for therapeutic applications.

Purpose: Glioma-initiating cells (GIC) are glioma stem–like cells that contribute to glioblastoma (GBM) development, recurrence, and resistance to chemotherapy and radiotherapy. They have recently become the focus of novel treatment strategies. Cyclophilin A (CypA) is a cytosolic protein that belongs to the peptidyl–prolyl isomerase (PPIase) family and the major intracellular target of the immunosuppressive drug cyclosporin A (CsA). In this study, we investigate the functions of CypA and its mechanism of action in GICs' development. Experimental Design: We analyzed differences in CypA expression between primary tumors and neurospheres from the GDS database, both before and after GIC differentiation. A series of experiments was conducted to investigate the role of CypA in GIC stemness, self-renewal, proliferation, radiotherapy resistance, and mechanism. We then designed glutathione S-transferase (GST) pulldown and coimmunoprecipitation assays to detect signaling activity. Results: In this study, we demonstrated that CypA promotes GIC stemness, self-renewal, proliferation, and radiotherapy resistance. Mechanistically, we found that CypA binds β-catenin and is recruited to Wnt target gene promoters. By increasing the interaction between β-catenin and TCF4, CypA enhances transcriptional activity. Conclusions: Our results demonstrate that CypA enhances GIC stemness, self-renewal, and radioresistance through Wnt/β-catenin signaling. Due to its promotive effects on GICs, CypA is a potential target for future glioma therapy. Clin Cancer Res; 23(21); 6640–9. ©2017 AACR.

The hedgehog signaling pathway plays a critical role in bone development and regeneration. Applications of hedgehog morphogens or small molecular agonists are of interest in bone repair but constrained by low stability, high dose requirement, and nonspecific targeting in vivo. Herein, a nanoparticulate agonist as a new type of hedgehog signaling activator is developed for efficacious bone healing. The shell of nanoparticulate agonist consists of palmitic acid and oxysterol, which could modify hedgehog function and bind with the smoothened receptor to positively modulate hedgehog signaling. Meanwhile, the core is assembled with sonic hedgehog gene/polyethyleneimine complex, which could synergistically enhance hedgehog signaling with oxysterol constituents. Moreover, alendronate is introduced into nanoparticulate agonist to bind with hydroxyapatite for potential bone tissue targeting. Lastly, the nanoparticulate agonist surface is decorated with the guanidine group to overcome cell membrane barriers. The created multifunctional nanoparticulate agonist is successfully integrated onto apatite-coated three-dimensional scaffolds and demonstrates greatly improved osteogenesis in vitro and calvarial bone healing. This work suggests a novel biomaterial design to specifically promote hedgehog signaling for the treatment of bone defects.

Reconstructing segmental mandiblular defects remains a challenge in the clinic. Tissue engineering strategies provide an alternative option to resolve this problem. The objective of the present study was to determine the effects of adipose-derived stem cells (ASCs) and bone morphogenetic proteins-2 (BMP-2) in three-dimensional (3D) scaffolds on mandibular repair in a small animal model. Noggin expression levels in ASCs were downregulated by a lentiviral short hairpin RNA strategy to enhance ASC osteogenesis (ASCs(Nog-)). Chitosan (CH) and chondroitin sulfate (CS), natural polysaccharides, were fabricated into 3D porous scaffolds, which were further modified with apatite coatings for enhanced cellular responses and efficient delivery of BMP-2. The efficacy of 3D apatite-coated CH/CS scaffolds supplemented with ASCs(Nog-) and BMP-2 were evaluated in a rat critical-sized mandibular defect model. After 8 weeks postimplantation, the scaffolds treated with ASCs(Nog-) and BMP-2 significantly promoted rat mandibular regeneration as demonstrated by micro-computerized tomography, histology, and immunohistochemistry, compared with the groups treated with ASCs(Nog-) or BMP-2 alone. These results suggest that our combinatorial strategy of ASCs(Nog-)+BMP-2 in 3D apatite microenvironments can significantly promote mandibular regeneration, and these may provide a potential tissue engineering approach to repair large bony defects.

Hydrogels derived from naturally occurring polymers are attractive matrices for tissue engineering. Here, we report a biofunctional hydrogel for specific use in bone regeneration by introducing Arg–Gly–Asp (RGD)-containing cell adhesive motifs and phosphorylated serine residues, which are prevalent in the native bone extracellular matrix and known to promote osteogenesis by enhancing cell–matrix interactions and hydroxyapatite nucleation, into photopolymerizable methacrylated glycol chitosan (MeGC). Incorporation of phosphoserine into MeGC hydrogels increased the ability of the hydrogels to nucleate minerals on their surfaces. RGD incorporation enhanced cell–matrix interactions by supporting attachment, spreading, and proliferation of bone marrow stromal cells (BMSCs) encapsulated in the hydrogels. Moreover, co-modification of MeGC hydrogels with RGD and phosphoserine synergistically increased the osteogenic differentiation of encapsulated BMSCs in vitro. The bone healing capacity of the modified hydrogels was further confirmed in a mouse calvarial defect model. These findings suggest a promising hydrogel platform with a specific microenvironment tailored to promote osteogenesis for clinical bone repair.

The continuing existent challenges of conventional surgical approaches for musculoskeletal reconstruction have led to the favorite of stem cell-mediated treatments over the last decade [1]. However, the in-depth understanding of underlying therapeutic mechanisms, regulatory mechanisms of stem cell differentiation, interaction of stem cells and inductive factors/delivery vehicles, and evaluation criteria in vitro and in vivo are indispensable to the wide practice of stem cells-based therapeutics for functionally restoring the musculoskeletal tissues from bench to bedside [2]. We are pleased to present a special issue encompassing both basic and translational aspects of stem cells-related research and review work in musculoskeletal regeneration including cartilage, bone, ligament, spinal cord, and meniscus, aiming to blossom in the effective use of stem cells for clinical application.

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.

Despite advances in the knowledge of tooth morphogenesis and differentiation, relatively little is known about the aetiology and molecular mechanisms underlying supernumerary tooth formation. A small number of supernumerary teeth may be a common developmental dental anomaly, while multiple supernumerary teeth usually have a genetic component and they are sometimes thought to represent a partial third dentition in humans. Mice, which are commonly used for studying tooth development, only exhibit one dentition, with very few mouse models exhibiting supernumerary teeth similar to those in humans. Inactivation of Apc or forced activation of Wnt/β(catenin signalling results in multiple supernumerary tooth formation in both humans and in mice, but the key genes in these pathways are not very clear. Analysis of other model systems with continuous tooth replacement or secondary tooth formation, such as fish, snake, lizard, and ferret, is providing insights into the molecular and cellular mechanisms underlying succesional tooth development, and will assist in the studies on supernumerary tooth formation in humans. This information, together with the advances in stem cell biology and tissue engineering, will pave ways for the tooth regeneration and tooth bioengineering.

Mesenchymal stem cells (MSCs) and fibroblasts are two major seed cells for ligament tissue engineering. To understand the effects of mechanical stimulation on these cells and to develop effective approaches for cell therapy, it is necessary to investigate the biological effects of various mechanical loading conditions on cells. In this study, fibroblasts and MSCs were tested and compared under a novel Uniflex/Bioflex culture system that might mimic mechanical strain in ligament tissue. The cells were uniaxially or radially stretched with different strains (5%, 10%, and 15%) at 0.1, 0.5, and 1.0 Hz. The cell proliferation and collagen production were compared to find the optimal parameters. The results indicated that uniaxial stretch (15% at 0.5 Hz; 10% at 1.0 Hz) showed positive effects on fibroblast. The uniaxial strains (5%, 10%, and 15%) at 0.5 Hz and 10% strain at 1.0 Hz were favorable for MSCs. Radial strain did not have significant effect on fibroblast. On the contrary, the radial strains (5%, 10%, and 15%) at 0.1 Hz had positive effects on MSCs. This study suggested that fibroblasts and MSCs had their own appropriate mechanical stimulatory parameters. These specific parameters potentially provide fundamental knowledge for future cell-based ligament regeneration.

Vascular regeneration is known to play an essential role in the repair of injured tissues mainly through accelerating the repair of vascular injury caused by vascular diseases, as well as the recovery of ischemic tissues. However, the clinical vascular regeneration is still challenging. Cell-based therapy is thought to be a promising strategy for vascular regeneration, since various cells have been identified to exert important influences on the process of vascular regeneration such as the enhanced endothelium formation on the surface of vascular grafts, and the induction of vessel-like network formation in the ischemic tissues. Here are a vast number of diverse cell-based strategies that have been extensively studied in vascular regeneration. These strategies can be further classified into three main categories, including cell transplantation, construction of tissue-engineered grafts, and surface modification of scaffolds. Cells used in these strategies mainly refer to terminally differentiated vascular cells, pluripotent stem cells, multipotent stem cells, and unipotent stem cells. The aim of this review is to summarize the reported research advances on the application of various cells for vascular regeneration, yielding insights into future clinical treatment for injured tissue/organ.

Approaches to control precisely growth factor presentation to a tissue defect in a sustained fashion are of increasing interest for a number of complex tissue engineering applications. Although transforming growth factor beta-1 (TGF-β1) plays a key role in promoting chondrogenesis, the therapeutic use of TGF-β1 is limited by its inherent protein instability, requiring high amounts of the protein that can cause adverse side effects with inefficient cartilage formation. In this work, we have developed strategies to stabilize TGF-β1 signaling in the injectable, visible blue light inducible chitosan (MeGC) hydrogel system for specific use in cartilage regeneration. We successfully modulated delivery of TGF-β1 with reduced burst release in a complex biological environment of serum and cells by covalently conjugating the protein to MeGC hydrogels with preserving type II collagen, one of the major cartilaginous extracellular matrix (ECM) components. The hydrogel system supported cellular condensation and deposition of cartilaginous ECM by encapsulating adipose derived stem cells in vitro. We confirmed further the ability of these TGF-β1 functionalized hydrogel systems to promote cartilage regeneration in challenging healing environments such as in a rat partial-thickness chondral defect model which present a limited source of subchondral bone marrow elements. These results suggest a new injectable delivery modality of therapeutic agents to improve clinical cartilage repair.

Bone morphogenetic proteins (BMPs) have been widely used for bone repair in the craniofacial region. However, its high dose requirement in clinical applications revealed adverse effects and inefficient bone formation, along with high cost. Here, we report a novel osteoinductive strategy to effectively complement the osteogenic activity of BMP-2 using phenamil, a small molecule that can induce osteogenic differentiation via stimulation of BMP signaling. Treatment of adipose-derived stem cells (ASCs) with BMP-2 in combination with phenamil significantly promoted the in vitro osteogenic differentiation of ASCs. The efficacy of the combination strategy of phenamil+BMP-2 was further confirmed in a mouse calvarial defect model using scaffolds consisting of poly(lactic-co-glycolic acid) and apatite layer on their surfaces designed to slowly release phenamil and BMP-2. Six weeks after implantation, the scaffolds treated with phenamil+BMP-2 significantly promoted mouse calvarial regeneration as demonstrated by micro-computerized tomography and histology, compared with the groups treated with phenamil or BMP-2 alone. Moreover, the combination treatment reduced the BMP-2 dose without compromising calvarial healing efficacy. These results suggest promising complementary therapeutic strategies for bone repair in more efficient and cost-effective manners.

Scaffolds derived from naturally occurring polysaccharides have attracted significant interest in bone tissue engineering due to their excellent biocompatibility and hydrophilic nature favorable for cell attachment. In this study, we developed composite chitosan (CH) scaffolds containing anionic carbohydrate, such as chondroitin 4-sulfate (CS) or alginate (AG), with biomimetic apatite layer on their surfaces, and investigate their capacity to deliver progenitor cells (bone marrow stromal cells, BMSC) and model proteins with net-positive (histone) and net-negative charge (bovine serum albumin, BSA). The incorporation of CS or AG in CH scaffolds increased compressive modulus of the scaffolds and enhanced apatite formation. Initial burst release of histone was significantly higher than that of BSA from CH scaffold, while the addition of CS or AG in the scaffolds significantly reduced the initial burst release of histone, indicating strong electrostatic interaction between histone and negatively charged CS or AG. The apatite layer created on scaffold surfaces significantly reduced the initial burst release of both BSA and histone. Furthermore, apatite-coated scaffolds enhanced spreading, proliferation, and osteogenic differentiation of BMSC seeded on the scaffolds compared to non-coated scaffolds as assessed by live/dead and alamarBlue assays, scanning electron microscopy (SEM), alkaline phosphatase (ALP) activity, and Picrosirius red staining. This study suggests that apatite-coated CH/CS composite scaffolds have the potential as a promising osteogenic system for bone tissue engineering applications.

This was the first study to use genipin to cross-link collagen and chitosan., In this study, genipin-cross-linked collagen/chitosan biodegradable porous scaffolds were prepared for articular cartilage regeneration. The influence of chitosan amount and genipin concentration on the scaffolds physicochemical properties was evaluated. The morphologies of the scaffolds were characterized by scanning electron microscope (SEM) and cross-linking degree was investigated by ninhydrin assay. Additionally, the mechanical properties of the scaffolds were assessed under dynamic compression. To study the swelling ratio and the biostability of the collagen/chitosan scaffold, in vitro tests were also carried out by immersion of the scaffolds in PBS solution or digestion in collagenase, respectively. The results showed that the morphologies of the scaffolds underwent a fiber-like to a sheet-like structural transition by increasing chitosan amount. Genipin cross-linking remarkably changed the morphologies and pore sizes of the scaffolds when chitosan amount was less than 25%. Either by increasing the chitosan ratio or performing cross-linking treatment, the swelling ratio of the scaffolds can be tailored. The ninhydrin assay demonstrated that the addition of chitosan could obviously increase the cross-linking efficiency. The degradation studies indicated that genipin cross-linking can effectively enhance the biostability of the scaffolds. The biocompatibility of the scaffolds was evaluated by culturing rabbit chondrocytes in vitro. This study demonstrated that a good viability of the chondrocytes seeded on the scaffold was achieved. The SEM analysis has revealed that the chondrocytes adhered well to the surface of the scaffolds and contacted each other. These results suggest that the genipin-cross-linked collagen/chitosan matrix may be a promising formulation for articular cartilage scaffolding., Key Projects in the National Science and Technology Pillar Program in the Eleventh Five-year Plan Period. Grant Number: 2006BA116B04, Guangdong Natural Science Foundation. Grant Number: 07300602, Natural Science Foundation Team Project of Guangdong. Grant Number: 4205786, State Key Program of National Natural Science of China. Grant Number: 50732003, National Basic Research Program of China. Grant Number: 2005CB623902

A co-culture strategy has been developed in this study wherein rabbit synovial mesenchymal stem cells (SMSCs) are co-cultured with growth factor (GF) transfected articular chondrocytes. Toward this end, both SMSCs and early passage rabbit articular chondrocytes that had been adenovirally transduced with transforming growth factor-beta 3 (TGF-beta3) gene were separately encapsulated in alginate beads and co-cultured in the same pool of chondrogenic medium. The chondrocytes act as transfected companion cells (TCCs) providing GF supply to induce chondrogenic differentiation of SMSCs that play the role of therapeutic progenitor cells (TPCs). Against the same TCC based TGF-beta3 release profile, the co-culture was started at different time points (Day 0, Day 10 and Day 20) but made to last for identical periods of exposure (30 days) so that the exposure conditions could be optimized in terms of initiation and duration. Transfection of TCCs prevents the stem cell based TPCs from undergoing the invasive procedure. It also prevents unpredictable complications in the TPCs caused by long-term constitutive over-expression of a GF. The adenovirally transfected TCCs exhibit a transient GF expression which results in a timely termination of GF supply to the TPCs. The TCC-sourced transgenic TGF-beta3 successfully induced chondrogenesis in the TPCs. Real-time PCR results show enhanced expression of cartilage markers and immuno/histochemical staining for Glycosaminoglycans (GAG) and Collagen II also shows abundant extracellular matrix (ECM) production and chondrogenic morphogenesis in the co-cultured TPCs. These results confirm the efficacy of directing stem cell differentiation towards chondrogenesis and cartilage tissue formation by co-culturing them with GF transfected chondrocytes.

In this study, transgenic effects of combination of transforming growth factor (TGF) beta3 and shRNA silencing type I collagen (Col I) on chondrogenesis of synovium-derived mesenchymal stem cells (SMSCs) were evaluated. SMSCs were infected with recombinant adenoviruses encoding TGF beta 3 (Ad-TGF beta 3) and/or anti-Col I shRNA (Ad-shRNA) separately, simultaneously (Ad-combination), or conjugately (Ad-double, mediated by one vector encoding both). The transduced SMSCs were encapsulated in alginate hydrogel and cultured for 30 days in chondrogenic medium. The expression of cartilaginous extracellular matrix components was investigated by quantitative real-time RT-PCR (qRT-PCR) and histological staining. qRT-PCR showed an up-regulation in chondrocytes marker genes such as type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP) in Ad-TGF beta 3, Ad-double, and Ad-combination groups on day 30. Whereas, Ad-TGF beta 3 treatment induced significant elevation in Col I, which could be largely resisted by anti-Col I shRNA functionality. Histological and immunohistochemical staining results were consistent with our qRT-PCR data. These results demonstrate that the application of combinational adenoviral vector-mediated transgenic TGF beta 3 and shRNA targeting Col I possesses the potential in promoting the chondrogenic differentiation of SMSCs as well as inhibiting the formation of fibrocartilage.

Synovium-derived mesenchymal stem cells (SMSC), a novel line of stem cells, are regarded as a promising cell source for cartilage tissue engineering. The goal of this study was to investigate rabbit SMSC coupled with injectable gellan hydrogels for in vitro engineered cartilage. SMSC were isolated from rabbit synovial tissue, amplified to passage 4 in monolayer, and encapsulated in injectable gellan hydrogels, constructs of which were cultured in chondrogenic medium supplemented with TGF-beta1, TGF-beta3 or BMP-2 for up to 42 days. The quality of the constructs was assessed in terms of cell proliferation and chondrocytic gene/protein expression using WST-1 assay, real-time RT-PCR, biochemical analysis, histology and immunohistochemical analysis. Results indicate that the viability of SMSC in hydrogels treated with TGF-beta1, TGF-beta3 and BMP-2 remained high at culture time. The constructs formed cartilaginous tissue with the expression of chondrocytic genes (collagen type II, aggrecan, biglycan, SOX 9) and cartilaginous matrix (sulphated glycosaminoglycan and collagen) as early as 21 days in culture. Both TGF-beta1 and TGF-beta3 treated SMSC-laden hydrogels showed more chondrogenesis compared with BMP-2 treated SMSC-laden hydrogels. It demonstrates that injectable SMSC-laden gels, when treated with TGF-beta1, TGF-beta3 or BMP-2, are highly competent for in vitro engineered cartilage formation, which lays a foundation for their potential application in clinical cartilage repair.

Ever since synovium-derived mesenchymal stem cells (SMSCs) were first identified and successfully isolated in 2001, as a brand new member in MSC families, they have been increasingly regarded as a promising therapeutic cell species for musculoskeletal regeneration, particularly for reconstructions of cartilage, bones, tendons, and muscles. Besides the general multipotency in common among the MSC community, SMSCs excel other sourced MSCs in higher ability of proliferation and superiority in chondrogenesis. This review summarizes the latest advances in SMSC-related studies covering their specific isolation methodologies, biological insights, and practical applications in musculoskeletal therapeutics of which an emphasis is cast on engineered chondrogenesis.