3D-Printed Porous Scaffolds of Hydrogels Modified with TGF-ß1 Binding Peptides to Promote In Vivo Cartilage Regeneration and Animal Gait Restoration.

The treatment of cartilage injury and osteoarthritis has been a classic problem for many years. The idea of in situ tissue regeneration paves a way for osteochondral repair in vivo. Herein, a hydrogel scaffold linked with bioactive peptides that can selectively adsorb transforming growth factor ß1 (TGF-ß1) was hypothesized to not only afford cell ingrowth space but also induce the endogenous TGF-ß1 recruitment for chondrogenesis promotion. In this study, bilayered porous scaffolds with gelatin methacryloyl (GelMA) hydrogels as a matrix were constructed via three-dimensional (3D) printing, of which the upper layer was covalently bound with bioactive peptides that can adsorb TGF-ß1 for cartilage repair and the lower layer was blended with hydroxyapatite for subchondral regeneration. The scaffolds showed promising therapeutic efficacy proved by cartilage and osteogenic induction in vitro and osteochondral repair of rats in vivo. In particular, the animal gait behavior was recovered after the in situ tissue regeneration, and the corresponding gait analysis demonstrated the promotion of tissue regeneration induced by the porous hydrogels with the binding peptides.

Preparation and application of quick hemostatic gauze based on biomimetic mineralized thrombin.

Thrombin is a serine protease known as activated coagulation factor II and is primarily applied as an effective local hemostatic agent. However, its clinical application is hindered by drawbacks, such as high sensitivity to the surrounding environment, instability and poor storage stability, easy inactivation, and low bioavailability. The biological functions of biomacromolecules in harsh environments can be preserved through biomineralization. Despite the success of biomimetic mineralization, limited consideration has been given to the mineral-based methods and the effect of various metal ions on enzyme activity. To explore an efficient technique for biomimetic mineralized thrombin, six kinds of ion/thrombin hybrid microflowers and two kinds of thrombin/MOF were synthesized in this work. The results showed that Zn-HNFs-G exhibits good hemostatic effect and maintains high enzymatic activity when exposed to high-temperature conditions. Meanwhile, Fe-HNFs-G, Thrombin@ZIF-8-G and Thrombin@MAF-7-G possess negligible enzyme protection.

Rational design of nonstoichiometric bioceramic scaffolds via digital light processing: tuning chemical composition and pore geometry evaluation.

Bioactive ceramics are promising candidates as 3D porous substrates for bone repair in bone regenerative medicine. However, they are often inefficient in clinical applications due to mismatching mechanical properties and compromised biological performances. Herein, the additional Sr dopant is hypothesized to readily adjust the mechanical and biodegradable properties of the dilute Mg-doped wollastonite bioceramic scaffolds with different pore geometries (cylindrical-, cubic-, gyroid-) by ceramic stereolithography. The results indicate that the compressive strength of Mg/Sr co-doped bioceramic scaffolds could be tuned simultaneously by the Sr dopant and pore geometry. The cylindrical-pore scaffolds exhibit strength decay with increasing Sr content, whereas the gyroid-pore scaffolds show increasing strength and Young's modulus as the Sr concentration is increased from 0 to 5%. The ion release could also be adjusted by pore geometry in Tris buffer, and the high Sr content may trigger a faster scaffold bio-dissolution. These results demonstrate that the mechanical strengths of the bioceramic scaffolds can be controlled from the point at which their porous structures are designed. Moreover, scaffold bio-dissolution can be tuned by pore geometry and doping foreign ions. It is reasonable to consider the nonstoichiometric bioceramic scaffolds are promising for bone regeneration, especially when dealing with pathological bone defects.

Cell-Free Bilayered Porous Scaffolds for Osteochondral Regeneration Fabricated by Continuous 3D-Printing Using Nascent Physical Hydrogel as Ink.

Cartilage is difficult to self-repair and it is more challenging to repair an osteochondral defects concerning both cartilage and subchondral bone. Herein, it is hypothesized that a bilayered porous scaffold composed of a biomimetic gelatin hydrogel may, despite no external seeding cells, induce osteochondral regeneration in vivo after being implanted into mammal joints. This idea is confirmed based on the successful continuous 3D-printing of the bilayered scaffolds combined with the sol-gel transition of the aqueous solution of a gelatin derivative (physical gelation) and photocrosslinking of the gelatin methacryloyl (gelMA) macromonomers (chemical gelation). At the direct printing step, a nascent physical hydrogel is extruded, taking advantage of non-Newtonian and thermoresponsive rheological properties of this 3D-printing ink. In particular, a series of crosslinked gelMA (GelMA) and GelMA-hydroxyapatite bilayered hydrogel scaffolds are fabricated to evaluate the influence of the spacing of 3D-printed filaments on osteochondral regeneration in a rabbit model. The moderately spaced scaffolds output excellent regeneration of cartilage with cartilaginous lacunae and formation of subchondral bone. Thus, tricky rheological behaviors of soft matter can be employed to improve 3D-printing, and the bilayered hybrid scaffold resulting from the continuous 3D-printing is promising as a biomaterial to regenerate articular cartilage.

Highly biosafe biomimetic stem cell membrane-disguised nanovehicles for cartilage regeneration.

Cartilage injury is very common and results in considerable pain and osteoarthritis. Owing to its low self-renewal capability, cartilage regeneration is still a great challenge for clinicians. Stem cell therapy has been treated as the most promising treatment for cartilage regeneration in recent decades. However, increasing concerns about the potential biosafety of stem cell products such as immune rejection and neoplastic transformation restrict their further application in clinic. Herein, biomimetic stem cell membrane-disguised nanovehicles without biosafety risks are designed and prepared for cartilage regeneration. In this study, based on the disguise of the natural bone marrow mesenchymal stem cell (BMSC) membrane, Kartogenin (KGN) as a drug for cartilage regeneration was encapsulated into Fe3O4 nanoparticles as the core of biomimetic stem cell nanovehicles. In the core-shell structure of biomimetic stem cell nanovehicles, the fabricated KGN-loaded BMSC membrane-disguised Fe3O4 nanoparticles (KGN-MNPs) showed a stable hybrid structure with a uniform size and morphology in the physiological environments. Moreover, the prepared KGN-MNPs exhibited excellent biocompatibility when disguised with the natural membrane of BMSCs and good biosafety by eliminating the nuclei of BMSCs. In a cartilage defect rat model, compared with pure KGN, the intra-articularly injected KGN-MNPs were capable of regenerating an integrated organized structure with a layer of hyaline-like cartilage in a shorter time due to the retained natural activities of the BMSC membrane. In a word, KGN-MNPs as one kind of our designed biomimetic stem cell nanovehicles enable rapid and high quality cartilage regeneration, and provide a novel and standardized strategy for stem cell therapy in the future.

Electrospun PCL/Gel-aligned scaffolds enhance the biomechanical strength in tendon repair.

Tendons can transmit mechanical force from muscles to bones for movement. However, the mechanical strength of tendons is compromised after surgery, thus causing a high rate of tendon retear. Hence, the design and preparation of biodegradable materials with excellent mechanical properties have become an urgent demand for sports medicine. In this study, biomimetic polycaprolactone (PCL)/gelatin (Gel)-aligned scaffolds were fabricated for the mechanical restoration of the injured tendon in a rabbit model. The diameter of nanofibers was about 427.82 ± 56.99 nm, which was approximate to that of the native collagen fibrils; the directional consistency of the nanofibers in PCL/Gel-aligned scaffolds reached 77.33 ± 3.22%, which were ultrastructurally biomimetic. Compared to the observations for the control group, the in vitro mechanical results showed that the PCL/Gel-aligned scaffolds (P/G-A) were anisotropic in terms of failure load, tensile strength, and Young's modulus. After verifying their good cytocompatibility, the scaffolds were implanted into the rabbit patellar tendon in situ. The biomechanical properties of the repaired tendon in P/G-A reached 343.97 ± 65.30 N in failure load, 85.99 ± 16.33 MPa in tensile strength, 590.84 ± 201.87 MPa in Young's modulus, and 171.29 ± 61.50 N mm-1 in stiffness in vivo at 8 weeks post operation. In a word, our results demonstrated that P/G-A could support the regenerated tissue of injured patellar tendons to restore the biomechanical strength in a rabbit model. This suggested that the PCL/Gel-aligned scaffolds can pave a promising way to improve the healing of injured tendons in the clinic in the future.

Encapsulation of a nanoporous simvastatin-chitosan composite to enhance osteointegration of hydroxyapatite-coated polyethylene terephthalate ligaments.

PURPOSE: This study was designed to evaluate the in vitro and in vivo biocompatibility and osteointegration of plasma-sprayed hydroxyapatite (HA)-coated polyethylene terephthalate (PET) ligaments encapsulated with a simvastatin (SV)-chitosan (CS) composite. METHODS: This study compared the in vitro and in vivo bone responses to three different PET ligaments: SV/CS/PET-HA, CS/PET-HA and PET-HA. A field emission scanning electron microscope was used to characterize the morphology, and the in vitro SV release profile was analyzed. MC3T3 cells were cocultured with SV/CS/PET-HA, CS/PET-HA and PET-HA to test their biocompatibility using CCK-8 tests. Osteogenic differentiation was investigated by the expression of marker genes using qPCR. Osteointegration was performed by implanting the PET ligaments into the proximal tibia bone tunnels of male Sprague-Dawley rats for 3 weeks and 6 weeks. The bone-implant interface was evaluated by micro-computed tomography (micro-CT) and histological analysis. RESULTS: The characteristic nanoporous structures mainly formed on the surface of the plasma-sprayed HA particles in the SV/CS/PET-HA and CS/PET-HA groups. The SV release test showed that the sustained release of simvastatin lasted for 25 days in the SV/CS/PET-HA group. The in vitro studies demonstrated that the SV/CS/PET-HA ligaments induced osteogenic differentiation in the MC3T3 cells, with higher mRNA expression levels of collagen-1, bone morphogenetic protein-2, osteocalcin and alkaline phosphatase than those in the CS/PET-HA and PET-HA ligament groups. The in vivo tests showed that both micro-CT analysis (bone mineral density and bone volume per total volume) and histological analysis (bone implant contact and interface area) revealed significantly higher peri-implant bone formation and less interface area in the SV/CS/PET-HA group than in the other groups. CONCLUSION: The SV-CS composite nanoporous structure was associated with the improved biocompatibility and osteogenic differentiation in vitro and enhanced osteointegration process in vivo of plasma-sprayed HA-coated PET ligaments.

[Research progress of porous tantalum in bone tissue engineering].

Objective: To review the basical research progress of porous tantalum in bone tissue engineering. Methods: The related basical research in fabrication, cytobiology, and surface modification of porous tantalum was reviewed and analyzed. Results: The outstanding physiochemical properties of porous tantalum granted its excellent performance in biocompatibility and osteointegration, as well as promoting cartilage and tendon tissue restoration. However, the clinical utilization of porous tantalum is somehow greatly limited by the complex and rigid commercial fabrication methods and extraordinary high cost. Along with the publication of novel fabrication and surface modification technology, the application of porous tantalum will be more extensive, the promotion in bone tissue regeneration will be more prominent. Conclusion: Porous tantalum has advantage in bone defect restoration, and significant breakthrough technology is needed in fabrication methods and surface modification.

The effects and mechanisms of SLC34A2 on tumorigenicity in human non-small cell lung cancer stem cells.

A novel paradigm in tumor biology suggests that non-small cell lung cancer (NSCLC) growth is driven by lung cancer stem cell-like cells (LCSCs), but molecular mechanisms regulating tumorigenic and self-renewal potential of LCSCs are still unclear. Here, we aim to investigate biological function of SLC34A2 in regulating tumorigenicity of LCSCs and its underlying mechanisms. Our findings testified that CD166(+) cells which were derived from fresh primary NSCLC samples displayed stem cell-like features. Fluorescence-activated cell sorting (FACS) analysis showed the presence of a variable fraction of CD166 cells in 15 out of 15 NSCLC samples. Significantly, CD166(+) LCSCs from primary NSCLC tumors expressed high level of SLC34A2 which was required for CD166(+) LCSCs tumorigenic and self-renewal potential. In NSCLC patient cohort, increased SLC34A2 expression correlated with histology, which suggests a potential role of SLC34A2 in CD166(+) LCSCs. Furthermore, Wnt/ß-catenin pathway and Bmi1 were found necessary for tumorigenicity and self-renewal capacity of CD166(+) LCSCs by a series in vitro and in vivo experiments. Then, our study indicated that SLC34A2 regulated Bmi1 to promote tumorigenic and self-renewal potential of CD166(+) LCSCs through Wnt/ß-catenin pathway. In this study, the characterization of molecular basis of SLC34A2 in CD166(+) LCSCs not only allows for better understanding of the mechanisms regulating tumorigenicity of this specific population of NSCLC cells but also provides insight into the gradual improvement of more effective cancer therapies against this disease.

[Etanercept inhibits synovial inflammation and reduces the expression of adhesion related molecules in synovial tissues of patients with rheumatoid arthritis].

OBJECTIVE: To observe the changes of synovial inflammation score and expression of related molecular markers in patients with rheumatoid arthritis treated with tumor necrosis factor (TNF) antagonist etanercept. METHODS: Sixteen patients with rheumatoid arthritis received synovectomy in the knee under arthroscopy, of which 8 patients had been treated with etanercept before surgery (etanercept group) and the other 8 patients were given no etanercept or other biologics (non-biological agent group). The synovial tissues obtained from surgery were subjected to HE staining and immunohistochemical staining respectively, to assess Rooney's inflammation score and detect the expressions of proliferating cell nuclear antigen (PCNA), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and cadherin-11. RESULTS: Rooney's score in etanercept group was significantly lower than that in non-biological agent group. The expressions of PCNA and cadherin-11 in synovial lining and sublining layers significantly decreased in etanercept group. Expressions of VCAM-1 and ICAM-1 had no significant difference in either synovial lining or sublining layer between the two groups. Clinical inflammatory markers including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), platelet count (PLT) and disease activity score in 28 joints (DAS28) had no statistical correlation with Rooney's inflammation score. CONCLUSION: Etanercept could effectively inhibit proliferation of synoviocytes and infiltration of lymphocytes in synovium of rheumatoid arthritis, and decrease the expressions of proliferation-and adhesion-related molecular markers, which histologically alleviated the synovial inflammation of rheumatoid arthritis. Clinical inflammatory markers might not fully reflect histological changes in the local synovial tissue.

Cadherin-11 involves in synovitis and increases the migratory and invasive capacity of fibroblast-like synoviocytes of osteoarthritis.

OBJECTIVE: To detect the expression of cadherin-11 and its correlation with synovitis in osteoarthritis (OA), to explore the mechanism of over expression of cadherin-11 and its role in migratory or invasive capacity of fibroblast-like synoviocytes (FLS). METHODS: Synovitis severity was recorded according to Krenn's scoring system in 25 osteoarthritis patients undergoing total knee arthroplasty. Cadherin-11 expression in OA synoviums and its correlation with synovitis score and systemic inflammation markers were explored. After induction with Interleukin-1 beta (IL-1ß) or tumor necrosis factor-alpha (TNF-α),cadherin-11 expression on OA FLS was assessed by qPCR and western blot,the capacity of migration and invasion of OA FLS was tested by transwell assay, and matrix metalloproteinase-2 production was assessed with ELISA as cadherin-11 expression was up regulated after infection with cadherin-11 cDNA-containing lentivirus, also when cadherin-11 expression was knocked down after infection with cadherin 11 shRNA containing lentivirus. RESULTS: Cadherin-11 expression in OA synovium showed significant differences among different grades of synovitis. Cadherin-11 in the lining layer was positively correlated with hyperplasia of the lining layer, density of the resident cells, inflammatory infiltrate, total synovitis score and D-dimer. Cadherin-11 in the sublining layer was positively correlated with the density of the resident cells, inflammatory infiltrate, total synovitis score and erythrocyte sedimentation rate. IL-1ß or TNF-α could up-regulate cadherin-11 expression on OA FLS at transcriptional and protein level. Over expression of cadherin-11 increased migratory or invasive capacity of OA FLS, while cadherin-11 knock down reduced migratory or invasive capacity and MMP-2 production in OA FLS. CONCLUSION: The over expression of cadherin-11 in osteoarthritis is positively correlated with synovitis severity, and can be driven by proinflammatory cytokines on OA FLS; cadherin-11 increases migratory or invasive capacity and MMP-2 production of fibroblast-like synoviocytes of osteoarthritis.

Osmolarity influences chondrocyte repair after injury in human articular cartilage.

BACKGROUND: The purpose was to determine the influence of irrigation solution osmolarity on articular chondrocytes survival and metabolic state following mechanical injury. METHODS: Osteochondral explants were harvested from patients undergoing total knee arthroplasty for osteoarthritis and then cut through their full thickness to establish mechanical injury models. Cartilage explants were incubated in irrigation solutions (saline and balanced salt) with different osmolarities (180, 280, 380, 580 mOsm/L) for 2 h. The percentage of cell death (100 × number of dead cells/number of dead and live cells) was quantified with the laser confocal microscopy. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed to detect apoptosis index of injured cartilage. The contents of proteoglycan elution were determined by spectrophotometer at 530 nm, and HIF-1α and type II collagen mRNA yields were quantified with real-time PCR. RESULTS: In situ dead chondrocytes were mainly localized to the superficial tangential region of injured cartilage edge after mechanical injury. The percentage of cell death was decreased, and proteoglycan elution was gradually reduced with the increasing of osmolarity. The apoptosis indices of TUNEL assay in different osmolarities had no significant difference (P = 0.158). HIF-1α and type II collagen mRNA yields were the least for chondrocytes exposed to 180 mOsm/L medium and were the greatest for chondrocytes exposed to 380 mOsm/L medium. Compared with the saline group, the cell death of superficial zone was significantly decreased (P = 0.001) and contents of proteoglycan elution were also significantly decreased (P = 0.045) in the balanced salt. HIF-1α (P = 0.017) and type II collagen (P = 0.034) mRNA yields in the chondrocytes exposed to the balanced salt were significantly more than the saline group. CONCLUSION: The osmolarity of irrigation solutions plays an important role in the survival and metabolic state of chondrocytes following mechanical injury, and the chondrocyte death is not caused by apoptosis. Increasing osmolarity of irrigation solutions may be chondroprotective with decreasing the chondrocyte death, reducing inhibition of metabolism and proteoglycan elution, ultimately preventing cartilage degeneration and promoting integrative repair.

[Expression of cadherin-11 in rheumatoid arthritis synovium and its correlation with inflammation].

OBJECTIVE: To explore the pattern of cadherin-11 expression and its relationship with synovial inflammation in rheumatoid arthritis (RA), and study the regulatory effects of cytokines on cadherin-11 expression on RA fibroblast-like synoviocytes (RAFLS). METHODS: Synovium samples were obtained from 28 RA patients who were undergoing total knee replacement. After HE staining, synovitis score was determined according to Rooney's inflammation score system. The expression of cadherin-11 in RA synovium was semi-quantified by immunohistochemical staining, and its correlations with Rooney's inflammation score and systematic inflammatory markers were analyzed statistically. After induction with transforming growth factor ß (TGF-ß) or interleukin 17 (IL-17) at a series of concentrations, the expression of cadherin-11 on RAFLS was assessed by real time guantitative-PCR and Western blotting. RESULTS: There was no significant difference in cadherin-11 expression in RA synovium among different levels of C-reactive protein. Cadherin-11 in the lining and sublining layers were positively correlated with D-dimer, synovial lining layer hyperplasia, proliferating blood vessels, perivascular infiltration of lymphocytes, focal aggregation of lymphocytes and diffuse infiltration of lymphocytes; cadherin-11 in the lining layer was negatively correlated with interstitial fibrosis. TGF-ß or IL-17 stimulation could up-regulate cadherin-11 expression on RAFLS at mRNA and protein levels. CONCLUSION: The over-expression of cadherin-11 in RA was correlated with synovial lining layer hyperplasia, proliferating blood vessels and infiltration of lymphocytes. Cadherin-11 expression on RAFLS could be induced by TGF-ß and IL-17 induction.

Working conditions of bipolar radiofrequency on human articular cartilage repair following thermal injury during arthroscopy.

BACKGROUND: The thermal injury during bipolar radiofrequercy results in chondrocyte death that limits cartilage repair. The purpose was to determine the effects of various factors of bipolar radiofrequency on human articular cartilage after thermal injury, offering suitable working conditions for bipolar radiofrequency during arthroscopy. METHODS: Osteochondral explants from 28 patients undergoing total knee arthroplasty (TKA) in Department of Orthopaedic, Peking University Reople's Hospital from October 2013 to May 2014, were harvested and treated using bipolar radiofrequency in a light contact mode under the following conditions: various power setting of levels 2, 4 and 6; different durations of 2 seconds, 5 seconds and 10 seconds; irrigation with fluids of different temperatures of 4°C, 22°C, and 37°C; two different bipolar radiofrequency probes ArthroCare TriStar 50 and Paragon T2. The percentage of cell death and depth of cell death were quantified with laser confocal microscopy. The content of proteoglycan elution at different temperatures was determined by spectrophotometer at 530 nm. RESULTS: Chondrocyte mortality during the treatment time of 2 seconds and power setting of level 2 was significantly lower than that with long duration or in higher level groups (time: P = 0.001; power: P = 0.001). The percentage of cell death after thermal injury was gradually reduced by increasing the temperature of the irrigation solutions (P = 0.003), the depth of dead chondrocytes in the 37°C solution group was significantly less than those in the 4°C and 22°C groups (P = 0.001). The proteoglycan elution was also gradually reduced by increasing the temperature (P = 0.004). Compared with the ArthroCare TriStar 50 group, the percentage of cell death in the Paragon T2 group was significantly decreased (P = 0.046). CONCLUSIONS: Thermal chondroplasty with bipolar radiofrequency resulted in defined margins of chondrocyte death under controlled conditions. The least cartilage damage during thermal chondroplasty could be achieved with lower power, shorter duration, suitable temperature of irrigation solutions and chondroprotective probes. The recommendations for the use of bipolar radiofrequency to minimize cartilage damage could be achieved with a power setting of level 2, treatment duration of 2 seconds, suitable fluid temperature (closer to body temperature of 37°C) and chondroprotective Paragon T2 probes.