Huilin Yang

Thermo-responsive molecularly imprinted nanogels for specific recognition and controlled release of proteins

Intelligent nanogels which respond to environmental stimuli with on/off characteristics hold great promise in a number of biomedical applications including drug/gene delivery, diagnostics and therapeutics. Here, we report the synthesis and characterization of a novel type of thermo-responsive nanogel built using the molecular imprinting technique for specific recognition and controlled release of proteins. Using lysozyme as the protein template and N-isopropylacrylamide as the major monomer, protein-imprinted spherical nanogel particles were readily prepared via aqueous precipitation polymerization with the aid of a surfactant, sodium dodecyl sulfate (SDS). Simply by adjusting the SDS amount during polymerization, the size of nanogels could be finely controlled, ranging from a few hundred down to a few dozen nanometers. Compared to non-imprinted counterparts, the lysozyme-imprinted nanogels possessed higher rebinding capacity, more rapid rebinding kinetics, and much higher specificity toward lysozyme. Importantly, both the rebinding and release characteristics of lysozyme-imprinted nanogels showed dramatic temperature-dependence, with clear on–off transition around 33 °C, i.e., the volume phase transition temperature of the thermo-responsive polymer poly(N-isopropylacrylamide). Therefore, we have developed a facile yet versatile approach to fabricate molecularly imprinted nanogels of well controlled sizes and thermo-responsive binding/release properties toward specific biomolecules, which may facilitate a broad spectrum of applications ranging from bioseparation and biosensing to drug delivery and therapeutics.

Thermo‐Responsive Hydrogel Layers Imprinted with RGDS Peptide: A System for Harvesting Cell Sheets

Cell sheet technology is a novel approach to preparing and harvesting monolayer cell sheets by using poly(N-isopropylacrylamide)(PNIPAAm)-modified surfaces as thermoresponsive cell culture substrates. At lower temperatures the cultured cells detach spontaneously as the surface of the substrate changes from hydrophobic to hydrophilic. In this way, an intact cell monolayer can be harvested non-invasively together with its underlying extracellular matrix (ECM). As a frequently used way to achieve scaffold-free tissue engineering, cell sheet technology holds great promise in cell-based regenerative medicine. Considering the limited availability of autologous cells and the timeliness requirement of clinical treatments, current attempts at improving the efficiency of thermo-responsive cell sheet harvest systems face a dilemma. On the one hand, the cell culture substrate must be very cell-adhesive to markedly promote rapid adhesion and proliferation of the limited autologous cells for a timely therapy. On the other hand, the same substrate should become very cell-repulsive after the formation of a confluent cell monolayer such that the mature cell sheet can be rapidly released without hurting the cells and their underlying ECM. Specifically, simple regulation of the chemical composition or the topography of the surface can only either promote cell adhesion or accelerate cell detachment and these surfaces commonly have very low bioactivity. Introducing cell-adhesive biomolecules by means of covalent binding or physical adsorption can improve the surface bioactivity but result in the inevitable deceleration of cell detachment and serious leakage of the biomolecules, respectively. Thus far, no single method without additional auxiliary means can effectively enhance cell adhesion during culture as well as facilitate the rapid harvest of cell sheets. To conquer this long-standing problem, we conceive of introducing cell-adhesive biomolecules to a thermo-responsive cell culture substrate in a reversible way and modulating them through temperature-dependent interactions. In this case, biomolecules can be stably immobilized on the substrate at cell culture temperature (37 8C), while they can be released as the temperature drops (e.g., 20 8C), thus facilitating both the initial cell adhesion and the final detachment of the cell sheet. With this strategy in mind, we find that the reversible interaction known as “specific binding” in noncovalent molecular imprinting is very appealing. As is well known, polymeric receptors with tailor-made recognition sites and “specific binding” properties comparable with those of natural receptors can be easily prepared by molecular imprinting. More importantly, molecularly imprinted polymers (MIPs) containing thermo-responsive recognition sites (i.e., the sites with temperature-dependent interactions between MIPs and targeted molecules) can be readily obtained using PNIPAAm-based materials. We herein report a novel system for harvesting cell sheets which relies on a PNIPAAm-based MIP hydrogel layer with thermo-responsive affinity toward specific biomolecules (Scheme 1). The commonly used cell-adhesive peptide ArgGly-Asp-Ser (RGDS) was chosen as the target biomolecule to demonstrate the proof-of-principle of our strategy. In our design, the thermo-responsive recognition sites in the MIPs were the tactic used to achieve temperature-dependent interactions between RGDS molecules and cell culture substrate. Specifically, besides the temperature-induced change of the surface wettability, the thermo-responsive recognition sites on the MIP hydrogel layer also resulted in the stable recognition and binding of RGDS at 37 8C and the triggered release of RGDS as the temperature was decreased. In contrast to the introduction of biomolecules by means of covalent binding or physical absorption, the thermo-responsive affinity of the MIPs toward RGDS not only significantly promotes cell adhesion during cell culture (37 8C) but also facilitates the detachment of cell sheets at low temperature (20 8C). To the best of our knowledge, although MIPs have exhibited some expanded bioapplications with the emergence of various biomolecule (e.g., proteins or peptides) imprinted polymers, this study is the first demonstration of molecular imprinting as a methodology to biofunctionalize thermoresponsive cell culture substrates to harvest cell sheets for potential biomedical applications. To achieve the best affinity between RGDS and the MIP matrix during cell culture, the imprinting process was performed by redox-initiated polymerization at 37 8C in [*] Dr. G. Pan, Q. Guo, Prof. Dr. H. Yang, Prof. Dr. B. Li Department of Orthopaedics The First Affiliated Hospital of Soochow University 188 Shizi Street, Suzhou, Jiangsu 215006 (China) and Orthopaedic Institute, Soochow University 708 Renmin Road, Suzhou, Jiangsu 215007 (China) E-mail: yueer@suda.edu.cn binli@suda.edu.cn

A Comparison of the Proximal Femoral Nail Antirotation Device and Dynamic Hip Screw in the Treatment of Unstable Pertrochanteric Fracture

This prospective randomized study compared the outcome of elderly patients with an unstable pertrochanteric fracture, treated with a proximal femoral nail antirotation device (PFNA; n = 51) or a dynamic hip screw (DHS; n = 55). All patients in the DHS group and nine in the PFNA group had open reductions. Incisions were significantly shorter for the PFNA than the DHS group. Blood loss and the number of patients requiring postoperative blood transfusions were significantly greater, but operation and fluoroscopy times were significantly shorter, for the DHS versus the PFNA group. Time to mobilization with a frame was significantly shorter in the PFNA group, and post-operative complications were more common in the DHS group. Poor fracture reduction led to three revisions. All fractures in both groups united during follow-up. The PFNA allowed earlier mobilization and faster recovery than the DHS. The PFNA is a highly acceptable, minimally invasive implant for unstable fractures.

Melatonin reverses H2O2‐induced premature senescence in mesenchymal stem cells via the SIRT1‐dependent pathway

Mesenchymal stem cells (MSCs) represent an attractive source for stem cell‐based regenerative therapy, but they are vulnerable to oxidative stress‐induced premature senescence in pathological conditions. We previously reported antioxidant and antiarthritic effects of melatonin on MSCs against proinflammatory cytokines. In this study, we hypothesized that melatonin could protect MSCs from premature senescence induced by hydrogen peroxide (H2O2) via the silent information regulator type 1 (SIRT1)‐dependent pathway. In response to H2O2 at a sublethal concentration of 200 μm, human bone marrow‐derived MSCs (BM‐MSCs) underwent growth arrest and cellular senescence. Treatment with melatonin before H2O2 exposure cannot significantly prevent premature senescence; however, treatment with melatonin subsequent to H2O2 exposure successfully reversed the senescent phenotypes of BM‐MSCs in a dose‐dependent manner. This result was made evident by improved cell proliferation, decreased senescence‐associated β‐galactosidase activity, and the improved entry of proliferating cells into the S phase. In addition, treatment with 100 μm melatonin restored the osteogenic differentiation potential of BM‐MSCs that was inhibited by H2O2‐induced premature senescence. We also found that melatonin attenuated the H2O2‐stimulated phosphorylation of p38 mitogen‐activated protein kinase, decreased expression of the senescence‐associated protein p16INK4α, and increased SIRT1. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked melatonin‐mediated antisenescence effects. Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin, suggesting that melatonin reversed the senescence in cells through the SIRT1‐dependent pathway. Together, these findings lay new ground for understanding oxidative stress‐induced premature senescence and open perspectives for therapeutic applications of melatonin in stem cell‐based regenerative medicine.

Protection against titanium particle induced osteolysis by cannabinoid receptor 2 selective antagonist

Osteolysis and subsequent aseptic loosening are the most common causes of failure of total joint arthroplasty. Osteolysis is initiated by inflammatory response to wear debris, resulting in localized, osteoclastic peri-implant bone loss. However, there were no effective measures for prevention and treatment of periprosthetic osteolysis. The aim of the current study was to determine whether CB2 selective antagonist (AM630) inhibits wear debris-induced osteolysis in a murine osteolysis model. Titanium (Ti) particles were introduced into established air pouches on BALB/c mice, followed by implantation of calvaria bone from syngeneic littermates. AM630 was given to mice intraperitoneally 2 days before Ti particles introduction and maintained until the sacrifice of the mice. Mice without drug treatment, as well as mice injected with saline alone, were included. Each group contains 10 mice. Pouch tissues were harvested 14 days after bone implantation for histological and molecular analysis. Ti particles stimulation significantly increased CB2 expression. However, less CB2 was observed in AM630 treatment group. AM630 inhibited Ti particle-induced osteolysis associated gene activity of RANK, RANKL and CPK, and diminished RANKL expression in Ti particle stimulated pouches. AM630 markedly reduced the number of TRAP+ cells in pouch tissues. In conclusion, this study provides the evidence that blockage of CB2 with AM630 can markedly reduce Ti particle induced osteolysis in a murine air pouch model. This finding points to the possibility that CB2 selective antagonists like AM630 may have potential value for prevention and treatment of wear particle induced osteolysis.

Surface biofunctional drug-loaded electrospun fibrous scaffolds for comprehensive repairing hypertrophic scars.

Incorporation of bioactive drugs and biofunctionalization of polyester fibrous scaffolds are essential means to improve their bio-functions and histocompatibility for regenerative medicine. However, it is still a challenge to biofunctionalize such drug carriers via traditional biochemical methods while maintaining their properties without changes in drug activity and loading ratio. Here, we demonstrated a facile approach for biofunctionalization of PLGA fibrous scaffolds with various molecules (i.e., PEG polymer, RGD peptide and bFGF growth factor for cell repellent, adhesion and proliferation, respectively) via mussel-Inspired poly(dopamine) (PDA) coating in aqueous solution. By virtue of the mild and efficient nature of this approach, the drug-loaded PLGA fibers could be easily biofunctionalized and showed negligible effects on the scaffold properties, especially drug activity and loading ratio. Further, in vivo study showed that, a ginsenoside-Rg3-loaded fibrous scaffold functionalized with bFGF growth factor could not only promote the early-stage wound healing in rabbit ear wounds (bio-signal from bFGF), but also inhibit later-stage hypertrophic scars formation (release of Rg3 drug). Therefore, the mussel-inspired method for bio-modification provides a facile and effective strategy to combine drug and bio-function in one system, thus facilitating a synergistic effect of drug-therapy and bio-signal when such biomaterial is used for regenerative medicine.

Comparison of the Minimally Invasive and Standard Medial Parapatellar Approaches for Total Knee Arthroplasty: Systematic Review and Meta-Analysis

This systematic literature review analysed the efficacy of minimally invasive subvastus (SV) and midvastus (MV) approaches, compared with the standard medial parapatellar (MP) approach, for total knee arthroplasty. Fixed- and random-effect meta-analyses were performed to pool the results of primary studies assessing the mean difference of each clinical outcome. Length of hospital stay was significantly different between the MP and SV approaches, but not between the MP and MV approaches. Blood loss was significantly higher following MP compared with SV. The number of days to perform a straight-leg raise was significantly longer following MP than either MV or SV. In the first 6 months postsurgery, MV was associated with a better range of motion (ROM) than MP. No significant difference in ROM between MP and SV was apparent at 1 year postsurgery. Quadriceps muscle strength recovered significantly more slowly following MP compared with SV. Future studies should assess the efficacy of the quadriceps-sparing approach and compare minimally invasive approaches, assessing intrinsic postoperative differences.

Inhibition of titanium particle-induced inflammatory osteolysis through inactivation of cannabinoid receptor 2 by AM630.

Wear particle could induce inflammatory osteolysis and is the primary pathological factor for aseptic loosening. Although it is known that cannabinoid receptor 2 (CB2) inhibits osteoclast differentiation, the effect on inflammatory osteolysis induced by wear particles remains unclear. This study examined the effect of CB2 in the regulation of osteoclast differentiation in a murine macrophage cell line (RAW264.7), which has been shown to be stimulated by titanium (Ti) particles and receptor activator of the NF-kappaB ligand (RANKL). Results showed that CB2 expression in RAW cells cultured with Ti particles and RANKL. CB2 inactivation by AM630, a CB2 selective antagonist, effectively inhibited osteoclastogenesis in the differentiation medium system. AM630 treatment (> or =100 nM) significantly reduced the number of tartrate-resistant acid phosphatase-positive cells when compared with the control. Real-time reverse transcription polymerase chain reaction analysis revealed that AM630 (100 nM) inhibited mRNA expression of RANK and cathepsin K in RAW cells stimulated by Ti particles and RANKL. Moreover, enzyme-linked immunosorbent assay showed that AM630 (100 nM) reduced protein expression of interleukin-1beta and tumor necrosis factor-alpha in RAW cells cultured with Ti particles. In addition, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide revealed that AM630 had no toxic effect on RAW cells. These results suggested that CB2 inactivation by AM630 could provide a promising therapeutic target for treating or preventing aseptic loosening.

Effects of a Single Dose of Methylprednisolone versus Three Doses of Rosiglitazone on Nerve Growth Factor Levels after Spinal Cord Injury

Acute spinal cord lesions result in dramatic changes in neuronal function. Studies have shown that the peroxisome proliferator-activated receptor-γ agonist, rosiglitazone, has neuroprotective properties. The effect of rosiglitazone after acute spinal cord injury was examined in the present study. Rats were subjected to laminectomy only; laminectomy with spinal cord contusion injury; laminectomy with contusion injury plus 30 mg/kg body weight methylprednisolone administered 5 min after surgery; or laminectomy with contusion injury plus 2 mg/kg body weight rosiglitazone administered intra-peritoneally 5 min, 6 h and 24 h after surgery. Both drugs increased neurotrophin gene and protein expression 24 h after injury compared with injured rats without drug treatment. Rosiglitazone increased neurotrophin expression at 7 days to a greater extent than methylprednisolone. Early functional recovery was observed in rats treated with rosiglitazone. The greater increase in rosiglitazone-induced nerve growth factor expression soon after injury could explain, at least in part, the improved recovery of motor function compared with methylprednisolone or saline.

Protection against titanium particle-induced osteoclastogenesis by cyclooxygenase-2 selective inhibitor.

Wear particle-induced osteoclastogenesis is the most common cause of aseptic loosening in total joint arthroplasty. Although cyclooxygenase (COX)-2, an inducible regulator of prostaglandin E2 (PGE2) synthesis, is known to be involved in osteoclast differentiation, its effect on osteoclastogenesis in response to wear particles remains unclear. In this study, we investigated the role of COX-2 in the regulation of osteoclast differentiation in the osteoclast precursor cell line RAW264.7 stimulated with titanium (Ti) particles. The results showed COX-2 expression in the early stages of RAW264.7 differentiation when stimulated with receptor activator of nuclear factor kappa B ligand (RANKL) and Ti particles. Blockade of COX-2 by celecoxib, a COX-2 selective inhibitor, effectively reduced the expression of PGE2 and inhibited differentiation of RAW264.7 cells into tartrate-resistant acid phosphatase-positive (TRAP+) osteoclastic cells. Quantitative real-time polymerase chain reaction revealed that celecoxib inhibited mRNA expression of RANK, cathepsin K (CPK), TRAP, and the nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells stimulated by Ti particles and RANKL. Moreover, exogenous PGE2 reversed the inhibitory effects of celecoxib. These results provide direct evidence that COX-2 dependent PGE2 induced by RANKL and Ti particles is required for osteoclastogenesis and suggests that reduced production of PGE2 by inactivation of COX-2 would provide a promising therapeutic target for the treatment of osteoclastogenesis induced by wear particles.