Dissecting cell diversity and connectivity in skeletal muscle for myogenesis.

Characterized by their slow adhering property, skeletal muscle myogenic progenitor cells (MPCs) have been widely utilized in skeletal muscle tissue engineering for muscle regeneration, but with limited efficacy. Skeletal muscle regeneration is regulated by various cell types, including a large number of rapidly adhering cells (RACs) where their functions and mechanisms are still unclear. In this study, we explored the function of RACs by co-culturing them with MPCs in a biomimetic skeletal muscle organoid system. Results showed that RACs promoted the myogenic potential of MPCs in the organoid. Single-cell RNA-Seq was also performed, classifying RACs into 7 cell subtypes, including one newly described cell subtype: teno-muscular cells (TMCs). Connectivity map of RACs and MPCs subpopulations revealed potential growth factors (VEGFA and HBEGF) and extracellular matrix (ECM) proteins involvement in the promotion of myogenesis of MPCs during muscle organoid formation. Finally, trans-well experiments and small molecular inhibitors blocking experiments confirmed the role of RACs in the promotion of myogenic differentiation of MPCs. The RACs reported here revealed complex cell diversity and connectivity with MPCs in the biomimetic skeletal muscle organoid system, which not only offers an attractive alternative for disease modeling and in vitro drug screening but also provides clues for in vivo muscle regeneration.

Knitted Silk-Collagen Scaffold Incorporated with Ligament Stem/Progenitor Cells Sheet for Anterior Cruciate Ligament Reconstruction and Osteoarthritis Prevention.

Current surgical management of anterior cruciate ligament (ACL) rupture still remains an intractable challenge in ACL regeneration due to the weak self-healing capability of ACL. Inadequate cell numbers and vascularization within the articular cavity contribute mainly to the poor prognosis. This time, we fabricated a new tissue engineering scaffold by adding ligament stem/progenitor cell (LSPC) sheets to our previous knitted silk-collagen sponge scaffold, which overcame these limitations by providing sufficient numbers of seed cells and a natural extracellular matrix to facilitate regeneration. LSPCs display excellent proliferation and multilineage differentiation capacity. Upon ectopic implantation, the knitted silk-collagen sponge scaffold incorporated with an LSPC sheet exhibited less immune cells but more fibroblast-like cells, deposited ECM and neovascularization, and better tissue ingrowth. In a rabbit model, we excised the ACL and performed a reconstructive surgery with our scaffold. Increased expression of ligament-specific genes and better collagen fibril formation could be observed after orthotopic transplantation. After 6 months, the LSPC sheet group showed better results on ligament regeneration and ligament-bone healing. Furthermore, no obvious cartilage and meniscus degeneration were observed at 6 months postoperation. In conclusion, these results indicated that the new tissue engineering scaffold can promote ACL regeneration and slow down the progression of osteoarthritis, thus suggesting its high clinical potential as an ideal graft in ACL reconstruction.

Subchondral bone osteoclasts induce sensory innervation and osteoarthritis pain.

Joint pain is the defining symptom of osteoarthritis (OA) but its origin and mechanisms remain unclear. Here, we investigated an unprecedented role of osteoclast-initiated subchondral bone remodeling in sensory innervation for OA pain. We show that osteoclasts secrete netrin-1 to induce sensory nerve axonal growth in subchondral bone. Reduction of osteoclast formation by knockout of receptor activator of nuclear factor kappa-B ligand (Rankl) in osteocytes inhibited the growth of sensory nerves into subchondral bone, dorsal root ganglion neuron hyperexcitability, and behavioral measures of pain hypersensitivity in OA mice. Moreover, we demonstrated a possible role for netrin-1 secreted by osteoclasts during aberrant subchondral bone remodeling in inducing sensory innervation and OA pain through its receptor DCC (deleted in colorectal cancer). Importantly, knockout of Netrin1 in tartrate-resistant acid phosphatase-positive (TRAP-positive) osteoclasts or knockdown of Dcc reduces OA pain behavior. In particular, inhibition of osteoclast activity by alendronate modifies aberrant subchondral bone remodeling and reduces innervation and pain behavior at the early stage of OA. These results suggest that intervention of the axonal guidance molecules (e.g., netrin-1) derived from aberrant subchondral bone remodeling may have therapeutic potential for OA pain.

Development of a centrally vascularized tissue engineering bone graft with the unique core-shell composite structure for large femoral bone defect treatment.

Great effort has been spent to promote the vascularization of tissue engineering bone grafts (TEBG) for improved therapeutic outcome. However, the thorough vascularization especially in the central region still remained as a major challenge for the clinical translation of TEBG. Here, we developed a new strategy to construct a centrally vascularized TEBG (CV-TEBG) with unique core-shell composite structure, which is consisted of an angiogenic core and an osteogenic shell. The in vivo evaluation in rabbit critical sized femoral defect was conducted to meticulously compare CV-TEBG to other TEBG designs (TEBG with osteogenic shell alone, or angiogenic core alone or angiogenic core+shell). Microfil-enhanced micro-CT analysis has been shown that CV-TEBG could outperform TEBG with pure osteogenic or angiogenic component for neo-vascularization. CV-TEBG achieved a much higher and more homogenous vascularization throughout the whole scaffold (1.52-38.91 folds, p < 0.01), and generated a unique burrito-like vascular network structure to perfuse both the central and peripheral regions of TEBG, indicating a potential synergistic effect between the osteogenic shell and angiogenic core in CV-TEBG to enhance neo-vascularization. Moreover, CV-TEBG has generated more new bone tissue than other groups (1.99-83.50 folds, p < 0.01), achieved successful bridging defect with the formation of both cortical bone like tissue externally and cancellous bone like tissue internally, and restored approximately 80% of the stiffness of the defected femur (benchmarked to the intact femur). It has been further observed that different bone regeneration patterns occurred in different TEBG implants and closely related to their vascularization patterns, revealing the potential profound influence of vascularization patterns on the osteogenesis pattern during defect healing.

Transplantation of BDNF Gene Recombinant Mesenchymal Stem Cells and Adhesive Peptide-modified Hydrogel Scaffold for Spinal Cord Repair.

INTRODUCTION: Mesenchymal Stem Cells (MSCs) are promising candidates for nerve tissue engineering. Brain Derived Neurotrophic Factor (BDNF) secreted by MSCs can function to increase neural differentiation and relieve inflammation response. Gene transfection technology is an efficient strategy to increase the secretion levels of cytokines and enhance cellular functions. However, transfection and in vivo gene expression of environmentally sensitive stem cells have been one of the most challenging subjects due to the requirement in both safety and transfection efficiency. In this study, gene transfection technology was applied to prepare BDNF gene recombinant MSCs based on our previously reported liposomal vector ScreenFect® A. To improve cellular survival and gene expression after in situ implantation of MSCs, an adhesive peptide modified hydrogel scaffold was constructed using hyaluronic acid. The scaffold was optimized and modified with an adhesive peptide PPFLMLLKGSTR. The transfected MSCs exhibited improved cellular survival and sustained gene expression in the three-Dimentional (3D) scaffold in vitro. Compared to untransfected MSCs, gene recombinant MSCs effectively improved spinal tissue integrity, inhibited glial scar formation and alleviated inflammatory response. These effects were found discounted when cells were implanted without the scaffold. CONCLUSION: The study developed a promising implantation system for therapy of severe spinal cord injury and provided the first understanding of Screenfect® A about its functions on stem cell therapy for nerve tissue repair as well as three-dimentional gene expression.

Exogenous stromal derived factor-1 releasing silk scaffold combined with intra-articular injection of progenitor cells promotes bone-ligament-bone regeneration.

Anterior cruciate ligament (ACL) is one of the most difficult tissues to heal once injured. Ligament regeneration and tendon-bone junction healing are two major goals of ACL reconstruction. This study aimed to investigate the synergistic therapeutic effects of Stromal cell-derived factor 1 (SDF-1)-releasing collagen-silk (CSF) scaffold combined with intra-articular injection of ligament-derived stem/progenitor cells (LSPCs) for ACL regeneration and the amelioration in the long-term complication of osteoarthritis (OA). The stem cell recruitment ability of CSF scaffold and the multipotency, particularly the tendon forming ability of LSPCs from rabbits were characterized in vitro, while the synergistic effect of the CSF scaffold and LSPCs for ACL regeneration and OA amelioration were investigated in vivo at 1, 3, and 6 months with a rabbit ACL reconstruction model. The CSF scaffold was used as a substitute for the ACL, and LSPCs were injected into the joint cavity after 7 days of the ACL reconstruction. CSF scaffold displayed a controlled release pattern for the encapsulated protein for up to 7 days with an increased stiffness in the mechanical property. LSPCs, which exhibited highly I Collagen and CXCR4 expression, were attracted by SDF-1 and successfully relocated into the CSF scaffold at 1 month in vivo. At 3 and 6 months post-treatment, the CSF scaffold combined with LSPCs (CSFL group) enhanced the regeneration of ACL tissue, and promoted bone tunnel healing. Furthermore, the OA progression was impeded efficiently. Our findings here provided a new strategy that using stem cell recruiting CSF scaffold with tissue-specific stem cells, could be a promising solution for ACL regeneration. STATEMENT OF SIGNIFICANCE: In this study, we developed a silk scaffold with increased stiffness and SDF-1 controlled release capacity for ligament repair. This advanced scaffold transplantation combined with intra-articular injection of LSPCs (which was isolated from rabbit ligament for the first time in this study) promoted the regeneration of both the tendinous and bone tunnel portion of ACL. This therapeutic strategy also ameliorated cartilage degeneration and reduced the severity of arthrofibrosis. Hence, combining LSPCs injection with SDF-1-releasing silk scaffold is demonstrated as a therapeutic strategy for ACL regeneration and OA treatment in the clinic.

Concise Review: Stem Cell Fate Guided By Bioactive Molecules for Tendon Regeneration.

Tendon disorders, which are commonly presented in the clinical setting, disrupt the patients' normal work and life routines, and they damage the careers of athletes. However, there is still no effective treatment for tendon disorders. In the field of tissue engineering, the potential of the therapeutic application of exogenous stem cells to treat tendon pathology has been demonstrated to be promising. With the development of stem cell biology and chemical biology, strategies that use inductive tenogenic factors to program stem cell fate in situ are the most easily and readily translatable to clinical applications. In this review, we focus on bioactive molecules that can potentially induce tenogenesis in adult stem cells, and we summarize the various differentiation factors found in comparative studies. Moreover, we discuss the molecular regulatory mechanisms of tenogenesis, and we examine the various challenges in developing standardized protocols for achieving efficient and reproducible tenogenesis. Finally, we discuss and predict future directions for tendon regeneration. Stem Cells Translational Medicine 2018;7:404-414.

Silk fibroin-chondroitin sulfate scaffold with immuno-inhibition property for articular cartilage repair.

The demand of favorable scaffolds has increased for the emerging cartilage tissue engineering. Chondroitin sulfate (CS) and silk fibroin have been investigated and reported with safety and excellent biocompatibility as tissue engineering scaffolds. However, the rapid degradation rate of pure CS scaffolds presents a challenge to effectively recreate neo-tissue similar to natural articular cartilage. Meanwhile the silk fibroin is well used as a structural constituent material because its remarkable mechanical properties, long-lasting in vivo stability and hypoimmunity. The application of composite silk fibroin and CS scaffolds for joint cartilage repair has not been well studied. Here we report that the combination of silk fibroin and CS could synergistically promote articular cartilage defect repair. The silk fibroin (silk) and silk fibroin/CS (silk-CS) scaffolds were fabricated with salt-leaching, freeze-drying and crosslinking methodologies. The biocompatibility of the scaffolds was investigated in vitro by cell adhesion, proliferation and migration with human articular chondrocytes. We found that silk-CS scaffold maintained better chondrocyte phenotype than silk scaffold; moreover, the silk-CS scaffolds reduced chondrocyte inflammatory response that was induced by interleukin (IL)-1ß, which is in consistent with the well-documented anti-inflammatory activities of CS. The in vivo cartilage repair was evaluated with a rabbit osteochondral defect model. Silk-CS scaffold induced more neo-tissue formation and better structural restoration than silk scaffold after 6 and 12weeks of implantation in ICRS histological evaluations. In conclusion, we have developed a silk fibroin/ chondroitin sulfate scaffold for cartilage tissue engineering that exhibits immuno-inhibition property and can improve the self-repair capacity of cartilage. STATEMENT OF SIGNIFICANCE: Severe cartilage defect such as osteoarthritis (OA) is difficult to self-repair because of its avascular, aneural and alymphatic nature. Current scaffolds often focus on providing sufficient mechanical support or bio-mimetic structure to promote cartilage repair. Thus, silk has been adopted and investigated broadly. However, inflammation is one of the most important factors in OA. But few scaffolds for cartilage repair reported anti-inflammation property. Meanwhile, chondroitin sulfate (CS) is a glycosaminoglycan present in the natural cartilage ECM, and has exhibited a number of useful biological properties including anti-inflammatory activity. Thus, we designed this silk-CS scaffold and proved that this scaffold exhibited good anti-inflammatory effects both in vitro and in vivo, promoted the repair of articular cartilage defect in animal model.

Alignment of collagen fiber in knitted silk scaffold for functional massive rotator cuff repair.

Rotator cuff tear is one of the most common types of shoulder injuries, often resulting in pain and physical debilitation. Allogeneic tendon-derived decellularized matrices do not have appropriate pore size and porosity to facilitate cell infiltration, while commercially-available synthetic scaffolds are often inadequate at inducing tenogenic differentiation. The aim of this study is to develop an advanced 3D aligned collagen/silk scaffold (ACS) and investigate its efficacy in a rabbit massive rotator cuff tear model. ACS has similar 3D alignment of collagen fibers as natural tendon with superior mechanical characteristics. Based on ectopic transplantation studies, the optimal collagen concentration (10mg/ml), pore diameter (108.43±7.25µm) and porosity (97.94±0.08%) required for sustaining a stable macro-structure conducive for cellular infiltration was determined. Within in vitro culture, tendon stem/progenitor cells (TSPCs) displayed spindle-shaped morphology, and were well-aligned on ACS as early as 24h. TSPCs formed intercellular contacts and deposited extracellular matrix after 7days. With the in vivo rotator cuff repair model, the regenerative tendon of the ACS group displayed more conspicuous native microstructures with larger diameter collagen fibrils (48.72±3.75 vs. 44.26±5.03nm) that had better alignment and mechanical properties (139.85±49.36vs. 99.09±33.98N) at 12weeks post-implantation. In conclusion, these findings demonstrate the positive efficacy of the macroporous 3D aligned scaffold in facilitating rotator cuff tendon regeneration, and its practical applications for rotator cuff tendon tissue engineering. STATEMENT OF SIGNIFICANCE: Massive rotator cuff tear is one of the most common shoulder injuries, and poses a formidable clinical challenge to the orthopedic surgeon. Tissue engineering of tendon can potentially overcome the problem. However, more efficacious scaffolds with good biocompatibility, appropriate pore size, favorable inductivity and sufficient mechanical strength for repairing massive rotator cuff tendon injuries need to be developed. In this study, we developed a novel macroporous 3D aligned collagen/silk scaffold, and demonstrated that this novel scaffold enhanced the efficacy of rotator cuff tendon regeneration by inducing aligned supracellular structures similar to natural tendon, which in turn enhanced cellular infiltration and tenogenic differentiation of stem/progenitor cells from both the tendon itself and surrounding tissues. Hence, it can potentially be a clinically useful application for tendon tissue engineering.

Isolation of Live Premature Senescent Cells Using FUCCI Technology.

Cellular senescence plays an important role in diverse biological processes such as tumorigenesis and organismal aging. However, lack of methods to specifically identify and isolate live senescent cells hampers the precise understanding of the molecular mechanisms regulating cellular senescence. Here, we report that utilization of fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology allows isolation of live premature senescent cells induced by doxorubicin treatment. Exposure of human foreskin fibroblasts (HFFs) to a low dose of doxorubicin led to cellular senescent phenotypes including formation of γ-H2AX and 53BP1 foci indicative of DNA damage, decreased cell proliferation and increased senescence-associated ß-galactosidase (SA-ß-gal) activity. Importantly, doxorubicin-induced senescent cells were arrested at S/G2/M phases of cell cycle which can be reported by a construct encoding a fragment of hGeminin fused with monomeric Azami-Green (mAG-hGeminin). Flow cytometric sorting of GFP(+) cells from doxorubicin-treated HFFs carrying mAG-hGeminin reporter enabled isolation and enrichment of live senescent cells in the culture. Our study develops a novel method to identify and isolate live premature senescent cells, thereby providing a new tool to study cellular senescence.

Human Cartilage-Derived Progenitor Cells From Committed Chondrocytes for Efficient Cartilage Repair and Regeneration.

UNLABELLED: Articular cartilage is not a physiologically self-renewing tissue. Injury of cartilage often progresses from the articular surface to the subchondral bone, leading to pathogenesis of tissue degenerative diseases, such as osteoarthritis. Therapies to treat cartilage defects using autologous chondrocyte-based tissue engineering have been developed and used for more than 20 years; however, the challenge of chondrocyte expansion in vitro remains. A promising cell source, cartilage stem/progenitor cells (CSPCs), has attracted recent attention. Because their origin and identity are still unclear, the application potential of CSPCs is under active investigation. Here we have captured the emergence of a group of stem/progenitor cells derived from adult human chondrocytes, highlighted by dynamic changes in expression of the mature chondrocyte marker, COL2, and mesenchymal stromal/stem cell (MSC) marker, CD146. These cells are termed chondrocyte-derived progenitor cells (CDPCs). The stem cell-like potency and differentiation status of CDPCs were determined by physical and biochemical cues during culture. A low-density, low-glucose 2-dimensional culture condition (2DLL) was critical for the emergence and proliferation enhancement of CDPCs. CDPCs showed similar phenotype as bone marrow mesenchymal stromal/stem cells but exhibited greater chondrogenic potential. Moreover, the 2DLL-cultured CDPCs proved efficient in cartilage formation both in vitro and in vivo and in repairing large knee cartilage defects (6-13 cm(2)) in 15 patients. These findings suggest a phenotype conversion between chondrocytes and CDPCs and provide conditions that promote the conversion. These insights expand our understanding of cartilage biology and may enhance the success of chondrocyte-based therapies. SIGNIFICANCE: Injury of cartilage, a non-self-repairing tissue, often progresses to pathogenesis of degenerative joint diseases, such as osteoarthritis. Although tissue-derived stem cells have been shown to contribute to tissue renewal and homeostasis, the derivation, biological function, and application potential of stem/progenitor cells found in adult human articular cartilage are incompletely understood. This study reports the derivation of a population of cartilage stem/progenitor cells from fully differentiated chondrocytes under specific culture conditions, which have the potential to reassume their chondrocytic phenotype for efficient cartilage regeneration. These findings support the possibility of using in vitro amplified chondrocyte-derived progenitor cells for joint cartilage repair.

Apoptotic transition of senescent cells accompanied with mitochondrial hyper-function.

Defined as stable cell-cycle arrest, cellular senescence plays an important role in diverse biological processes including tumorigenesis, organismal aging, and embryonic development. Although increasing evidence has documented the metabolic changes in senescent cells, mitochondrial function and its potential contribution to the fate of senescent cells remain largely unknown. Here, using two in vitro models of cellular senescence induced by doxorubicin treatment and prolonged passaging of neonatal human foreskin fibroblasts, we report that senescent cells exhibited high ROS level and augmented glucose metabolic rate concomitant with both morphological and quantitative changes of mitochondria. Furthermore, mitochondrial membrane potential depolarized at late stage of senescent cells which eventually led to apoptosis. Our study reveals that mitochondrial hyper-function contributes to the implementation of cellular senescence and we propose a model in which the mitochondrion acts as the key player in promoting fate-determination in senescent cells.

[Development of human embryonic stem cell platforms for human health-safety evaluation].

The human embryonic stem cells (hESCs) serve as a self-renewable, genetically-healthy, pluripotent and single source of all body cells, tissues and organs. Therefore, it is considered as the good standard for all human stem cells by US, Europe and international authorities. In this study, the standard and healthy human mesenchymal progenitors, ligament tissues, cardiomyocytes, keratinocytes, primary neurons, fibroblasts, and salivary serous cells were differentiated from hESCs. The human cellular health-safety of NaF, retinoic acid, 5-fluorouracil, dexamethasone, penicillin G, adriamycin, lead acetate PbAc, bisphenol A-biglycidyl methacrylate (Bis-GMA) were evaluated selectively on the standardized platforms of hESCs, hESCs-derived cardiomyocytes, keratinocytes, primary neurons, and fibroblasts. The evaluations were compared with those on the currently most adopted cellular platforms. Particularly, the sensitivity difference of PM2.5 toxicity on standardized and healthy hESCs derived fibroblasts, currently adopted immortalized human bronchial epithelial cells Beas-2B and human umbilical vein endothelial cells (HUVECs) were evaluated. The RESULTS showed that the standardized hESCs cellular platforms provided more sensitivity and accuracy for human cellular health-safety evaluation.

Pharmacological Regulation of In Situ Tissue Stem Cells Differentiation for Soft Tissue Calcification Treatment.

Calcification of soft tissues, such as heart valves and tendons, is a common clinical problem with limited therapeutics. Tissue specific stem/progenitor cells proliferate to repopulate injured tissues. But some of them become divergent to the direction of ossification in the local pathological microenvironment, thereby representing a cellular target for pharmacological approach. We observed that HIF-2alpha (encoded by EPAS1 inclined form) signaling is markedly activated within stem/progenitor cells recruited at calcified sites of diseased human tendons and heart valves. Proinflammatory microenvironment, rather than hypoxia, is correlated with HIF-2alpha activation and promoted osteochondrogenic differentiation of tendon stem/progenitor cells (TSPCs). Abnormal upregulation of HIF-2alpha served as a key switch to direct TSPCs differentiation into osteochondral-lineage rather than teno-lineage. Notably, Scleraxis (Scx), an essential tendon specific transcription factor, was suppressed on constitutive activation of HIF-2alpha and mediated the effect of HIF-2alpha on TSPCs fate decision. Moreover, pharmacological inhibition of HIF-2alpha with digoxin, which is a widely utilized drug, can efficiently inhibit calcification and enhance tenogenesis in vitro and in the Achilles's tendinopathy model. Taken together, these findings reveal the significant role of the tissue stem/progenitor cells fate decision and suggest that pharmacological regulation of HIF-2alpha function is a promising approach for soft tissue calcification treatment.

Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality.

The repair of injured tendons remains a formidable clinical challenge because of our limited understanding of tendon stem cells and the regulation of tenogenesis. With single-cell analysis to characterize the gene expression profiles of individual cells isolated from tendon tissue, a subpopulation of nestin+ tendon stem/progenitor cells (TSPCs) was identified within the tendon cell population. Using Gene Expression Omnibus datasets and immunofluorescence assays, we found that nestin expression was activated at specific stages of tendon development. Moreover, isolated nestin+ TSPCs exhibited superior tenogenic capacity compared to nestin- TSPCs. Knockdown of nestin expression in TSPCs suppressed their clonogenic capacity and reduced their tenogenic potential significantly both in vitro and in vivo. Hence, these findings provide new insights into the identification of subpopulations of TSPCs and illustrate the crucial roles of nestin in TSPC fate decisions and phenotype maintenance, which may assist in future therapeutic strategies to treat tendon disease.

Cartilage stem/progenitor cells are activated in osteoarthritis via interleukin-1ß/nerve growth factor signaling.

INTRODUCTION: Interleukin-1ß (IL-1ß) and nerve growth factor (NGF) are key regulators in the pathogenesis of inflammatory arthritis; specifically, IL-1ß is involved in tissue degeneration and NGF is involved in joint pain. However, the cellular and molecular interactions between IL-1ß and NGF in articular cartilage are not known. Cartilage stem/progenitor cells (CSPCs) have recently been identified in osteoarthritic (OA) cartilage on the basis of their migratory properties. Here we hypothesize that IL-1ß/NGF signaling is involved in OA cartilage degeneration by targeting CSPCs. METHOD: NGF and NGF receptor (NGFR: TrkA and p75NTR) expression in healthy and OA human articular cartilage and isolated chondrocytes was determined by immunostaining, qRT-PCR, flow cytometry and western blot. Articular cartilage derived stem/progenitor cells were collected and identified by stem/progenitor cell characteristics. 3D-cultured CSPC pellets and cartilage explants were treated with NGF and NGF neutralizing antibody, and extracellular matrix changes were examined by sulfated glycosaminoglycan (GAG) release and MMP expression and activity. RESULTS: Expression of NGF, TrkA and p75NTR was found to be elevated in human OA cartilage. Cellular changes upon IL-1ß and/or NGF treatment were then examined. NGF mRNA and NGFR proteins levels were upregulated in cultured chondrocytes exposed to IL-1ß. NGF was chemotactic for cells isolated from OA cartilage. Cells isolated on the basis of their chemotactic migration towards NGF demonstrated stem/progenitor cell characteristics, including colony-forming ability, multi-lineage differentiation potential, and stem cell surface markers. The effects of NGF perturbation in cartilage explants and 3D-cultured CSPCs were next analyzed. NGF treatment resulted in extracellular matrix catabolism indicated by increased sGAG release and MMP expression and activity; conversely, treatment with NGF neutralizing antibody inhibited increased MMP levels, and enhanced tissue inhibitor of matrix metalloprotease-1 (TIMP1) expression in OA cartilage explants. NGF blockade with neutralizing antibody also affected cartilage matrix remodeling in 3D-CSPC pellet cultures. CONCLUSION: Our results strongly suggest that NGF signaling is a contributing factor in articular cartilage degeneration in OA, which likely targets a specific subpopulation of progenitor cells, the CSPCs, affecting their migratory and matrix remodeling activities. These findings provide novel cellular/signaling therapeutic targets in osteoarthritic cartilage.

3D-Printed Atsttrin-Incorporated Alginate/Hydroxyapatite Scaffold Promotes Bone Defect Regeneration with TNF/TNFR Signaling Involvement.

High expression levels of pro-inflammatory tumor necrosis factor (TNF)-α within bone defects can decelerate and impair bone regeneration. However, there are few available bone scaffolds with anti-inflammatory function. The progranulin (PGRN)-derived engineered protein, Atsttrin, is known to exert antagonistic effects on the TNF-α function. Hence, this study investigates whether 3D-printed Atsttrin-incorporated alginate(Alg)/hydroxyapatite(nHAp) scaffolds can facilitate bone healing through affecting the TNF/TNFR signaling. A 3D bioprinting system is used to fabricate Atsttrin-Alg/nHAp composite scaffolds, and the Atsttrin release from this scaffold is characterized, followed by evaluation of its efficacy on bone regeneration both in vitro and in vivo. The 3D-printed Atsttrin-Alg/nHAp scaffold exhibits a precisely defined structure, can sustain Atsttrin release for at least 5 days, has negligible cytotoxicity, and supports cell adhesion. Atsttrin can also attenuate the suppressive effects of TNF-α on BMP-2-induced osteoblastic differentiation in vitro. The 3D-printed Atsttrin-Alg/nHAp scaffold significantly reduces the number of TNF-α positive cells within wound sites, 7 days after post-calvarial defect surgery. Additionally, histological staining and X-ray scanning results also show that the 3D-printed Atsttrin-Alg/nHAp scaffold enhances the regeneration of mice calvarial bone defects. These findings thus demonstrate that the precise structure and anti-inflammatory properties of 3D-printed Atsttrin-Alg/nHAp scaffolds may promote bone defect repair.

Nanoparticle delivery of stable miR-199a-5p agomir improves the osteogenesis of human mesenchymal stem cells via the HIF1a pathway.

Elucidating the regulatory mechanisms of osteogenesis of human mesenchymal stem cell (hMSC) is important for the development of cell therapies for bone loss and regeneration. Here we showed that hsa-miR-199a-5p modulated osteogenic differentiation of hMSCs at both early and late stages through HIF1a pathway. hsa-miR-199a expression was up-regulated during osteogenesis for both of two mature forms, miR-199a-5p and -3p. Over-expression of miR-199a-5p but not -3p enhanced differentiation of hMSCs in vitro, whereas inhibition of miR-199a-5p reduced the expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and mineralization. Furthermore, over-expression of miR-199a enhanced ectopic bone formation in vivo. Chitosan nanoparticles were used for delivery of stable modified hsa-miR-199a-5p (agomir) both in vitro and in vivo, as a proof-of-concept for stable agomir delivery on bone regeneration. The hsa-mir199a-5p agomir were mixed with Chitosan nanoparticles to form nanoparticle/hsa-mir199a-5p agomir plasmid (nanoparticle/agomir) complexes, and nanoparticle/agomir complexes could improve the in vivo regeneration of bone. Further mechanism studies revealed that hypoxia enhanced osteogenesis at early stage and inhibited osteogenesis maturation at late stage through HIF1a-Twist1 pathway. At early stage of differentiation, hypoxia induced HIF1a-Twist1 pathway to enhance osteogenesis by up-regulating miR-199a-5p, while at late stage of differentiation, miR-199a-5p enhanced osteogenesis maturation by inhibiting HIF1α-Twist1 pathway.

Intra-articular transplantation of atsttrin-transduced mesenchymal stem cells ameliorate osteoarthritis development.

Osteoarthritis (OA) remains an intractable clinical challenge. Few drugs are available for reversing this degenerative disease, although some promising candidates have performed well in preclinical studies. Tumor necrosis factor α (TNFα) has been identified as a crucial effector modulating OA pathogenesis. This study aimed to investigate the therapeutic effects of Atsttrin, a novel TNFα blocker, on OA treatment. We developed genetically modified mesenchymal stem cells (MSCs) that expressed recombinant Atsttrin (named as MSC-Atsttrin). Expression levels of ADAMTS-5, MMP13, and iNOS of human chondrocytes were analyzed when cocultured with MSC-GFP/Atsttrin. OA animal models were induced by anterior cruciate ligament transection, and MSC-GFP/Atsttrin were injected into the articular cavity 1 week postsurgery. The results showed that MSC-Atsttrin significantly suppressed TNFα-driven up-regulation of matrix proteases and inflammatory factors. Intra-articular injection of MSC-Atsttrin prevented the progression of degenerative changes in the surgically induced OA mouse model. Additionally, levels of detrimental matrix hydrolases were significantly diminished. Compared with nontreated OA samples at 8 weeks postsurgery, the percentages of MMP13- and ADAMTS-5-positive cells were significantly reduced from 91.33% ± 9.87% to 24.33% ± 5.7% (p < .001) and from 91.33% ± 7.1% to 16.67% ± 3.1% (p < .001), respectively. Our results thus indicated that suppression of TNFα activity is an effective strategy for OA treatment and that intra-articular injection of MSCs-Atsttrin could be a promising therapeutic modality.

Down-regulation of Rac GTPase-activating protein OCRL1 causes aberrant activation of Rac1 in osteoarthritis development.

OBJECTIVE: Chondrocyte hypertrophy and mineralization are considered to be important pathologic factors in osteoarthritis (OA). We previously reported that Rac1 was aberrantly activated to promote chondrocyte hypertrophy, mineralization, and expression of matrix metalloproteinase 13 and ADAMTS in OA. However, the underlying mechanism of aberrant Rac1 activation in OA is unclear. The present study was undertaken to identify the specific molecular regulator controlling Rac1 activity in OA, as well as to investigate its function in chondrocyte hypertrophy, mineralization, and OA development. METHODS: Expression levels of 28 upstream regulators of Rac1 activity, including 8 GTPase-activating proteins (GAPs) and 20 guanine nucleotide exchange factors, in OA and normal cartilage were assessed by quantitative polymerase chain reaction. Chondrocytes were transduced with lentiviral vectors encoding OCRL1, GAP, non-GAP, CA-Rac1, and DN-Rac1, either alone or in combination. Alkaline phosphatase staining was used as a marker of chondrocyte hypertrophy. Rac1 activity was analyzed by pulldown assay. Finally, OA was established in mice by surgical transection of the anterior cruciate ligament and cutting of the medial meniscus. The mice were injected intraarticularly with OCRL1-encoding lentivirus, and whole joints were assessed histologically 6 weeks after surgery. RESULTS: OCRL1 was abundantly expressed in normal cartilage and was the only significantly down-regulated RacGAP in OA cartilage. Overexpression of OCRL1 inhibited interleukin-1ß-induced Rac1 activity, chondrocyte hypertrophy, and expression of hypertrophy-related genes. Conversely, knockdown of OCRL1 elevated Rac1 activity and promoted chondrocyte hypertrophy and mineralization. Further, OCRL1 modulated Rac1 activity via its GAP domain. Finally, intraarticular injection of OCRL1-encoding lentivirus protected against destruction and degeneration of cartilage in the mouse OA model. CONCLUSION: OCRL1 acts as a RacGAP in cartilage to impede chondrocyte hypertrophy and OA development through modulating Rac1 activity. This regulatory pathway might provide potential targets for the development of new therapies for OA.