Decellularized allogeneic cartilage paste with human costal cartilage and crosslinked hyaluronic acid-carboxymethyl cellulose carrier augments microfracture for improved articular cartilage repair.

Articular cartilage lacks natural healing abilities and necessitates surgical treatments for injuries. While microfracture (MF) is a primary surgical approach, it often results in the formation of unstable fibrocartilage. Delivering hyaline cartilage directly to defects poses challenges due to the limited availability of autologous cartilage and difficulties associated with allogeneic cartilage delivery. We developed a decellularized allogeneic cartilage paste (DACP) using human costal cartilage mixed with a crosslinked hyaluronic acid (HA)-carboxymethyl cellulose (CMC) carrier. The decellularized allogeneic cartilage preserved the extracellular matrix and the nanostructure of native hyaline cartilage. The crosslinked HA-CMC carrier provided shape retention and moldability. In vitro studies confirmed that DACP did not cause cytotoxicity and promoted migration, proliferation, and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. After 6 months of implantation in rabbit knee osteochondral defects, DACP combined with MF outperformed MF alone, demonstrating improved gait performance, defect filling, morphology, extracellular matrix deposition, and biomechanical properties similar to native cartilage. Thus, DACP offers a safe and effective method for articular cartilage repair, representing a promising augmentation to MF. STATEMENT OF SIGNIFICANCE: Directly delivering hyaline cartilage to repair articular cartilage defects is an ideal treatment. However, current allogeneic cartilage products face delivery challenges. In this study, we developed a decellularized allogeneic cartilage paste (DACP) by mixing human costal cartilage with crosslinked hyaluronic acid (HA)-carboxymethyl cellulose (CMC). DACP preserves extracellular matrix components and nanostructures similar to native cartilage, with HA-CMC ensuring shape retention and moldability. Our study demonstrates improved cartilage repair by combining DACP with microfracture, compared to microfracture alone, in rabbit knee defects over 6 months. This is the first report showing better articular cartilage repair using decellularized allogeneic cartilage with microfracture, without the need for exogenous cells or bioactive substances.

Comparing Verum and Sham Acupuncture in Fibromyalgia Syndrome: A Systematic Review and Meta-Analysis.

OBJECTIVES: Acupuncture is often used for relieving symptoms of fibromyalgia syndrome (FMS). Our aim is to ascertain whether verum acupuncture is more effective than sham acupuncture in FMS. METHODS: We collected RCTs to investigate the effects of verum acupuncture and sham acupuncture on pain, sleep quality, fatigue, and general status in FMS patients. The databases used for data retrieval were PubMed, Central Cochrane, EMBASE, PsycINFO, CNKI, VIP, OASIS, KoreaMed, and RISS. Selection/exclusion from the retrieved records was performed according to prespecified criteria, and the final selected records were assessed according to the Cochrane risk of bias tool. The results of the included trials were synthesized on the basis of outcomes, and subgroup analysis depended on the type of add-on sham acupuncture that was performed. RESULTS: Ten RCTs (690 participants) were eligible, and eight RCTs were eventually included in the meta-analysis. The synthesis showed a sizable effect of verum acupuncture compared with sham acupuncture on pain relief (standardized mean difference (SMD) -0.49, Z = 3.26, P=0.001; I 2 = 59%), improving sleep quality (SMD -0.46, Z = 3.24, P=0.001; I 2 = 0%), and reforming general status (SMD -0.69, Z = 6.27, P < 0.00001; I 2 = 4%). However, efficacy on fatigue was insignificant (SMD -0.10, Z = 0.51, P=0.61; I 2 = 46%). When compared with a combination of simulation and improper location of needling, the effect of verum acupuncture for pain relief was the most obvious. CONCLUSIONS: Verum acupuncture is more effective than sham acupuncture for pain relief, improving sleep quality, and reforming general status in FMS posttreatment. However, evidence that it reduces fatigue was not found.

Degradation and Remodeling of Epitaxially Grown Collagen Fibrils.

INTRODUCTION­: The extracellular matrix (ECM) in the tumor microenvironment contains high densities of collagen that are highly aligned, resulting in directional migration called contact guidance that facilitates efficient migration out of the tumor. Cancer cells can remodel the ECM through traction force controlled by myosin contractility or proteolytic activity controlled by matrix metalloproteinase (MMP) activity, leading to either enhanced or diminished contact guidance. METHODS­: Recently, we have leveraged the ability of mica to epitaxially grow aligned collagen fibrils in order to assess contact guidance. In this article, we probe the mechanisms of remodeling of aligned collagen fibrils on mica by breast cancer cells. RESULTS­: We show that cells that contact guide with high fidelity (MDA-MB-231 cells) exert more force on the underlying collagen fibrils than do cells that contact guide with low fidelity (MTLn3 cells). These high traction cells (MDA-MB-231 cells) remodel collagen fibrils over hours, pulling so hard that the collagen fibrils detach from the surface, effectively delaminating the entire contact guidance cue. Myosin or MMP inhibition decreases this effect. Interestingly, blocking MMP appears to increase the alignment of cells on these substrates, potentially allowing the alignment through myosin contractility to be uninhibited. Finally, amplification or dampening of contact guidance with respect to a particular collagen fibril organization is seen under different conditions. CONCLUSIONS­: Both myosin II contractility and MMP activity allow MDA-MB-231 cells to remodel and eventually destroy epitaxially grown aligned collagen fibrils.

Functional Production of Catalytic Domains of Human MMPs in Escherichia coli Periplasm.

Due to their central roles in tumor growth and invasion, milligram-level amounts of active MMPs are frequently required for cancer research and development of chemical or biological MMP inhibitors. Here we describe methods for functional production of catalytic domains of MMPs (cdMMPs) in E. coli periplasm without refolding or activation process. We demonstrate applications of this straightforward approach for cdMMP-9, cdMMP-14, and cdMMP-14 mutants.

Direct expression of active human tissue inhibitors of metalloproteinases by periplasmic secretion in Escherichia coli.

BACKGROUND: As regulators of multifunctional metalloproteinases including MMP, ADAM and ADAMTS families, tissue inhibitors of metalloproteinases (TIMPs) play a pivotal role in extracellular matrix remodeling, which is involved in a wide variety of physiological processes. Since abnormal metalloproteinase activities are related to numerous diseases such as arthritis, cancer, atherosclerosis, and neurological disorders, TIMPs and their engineered mutants hold therapeutic potential and thus have been extensively studied. Traditional productions of functional TIMPs and their N-terminal inhibitory domains (N-TIMPs) rely on costly and time-consuming insect and mammalian cell systems, or tedious and inefficient refolding from denatured inclusion bodies. The later process is also associated with heterogeneous products and batch-to-batch variation. RESULTS: In this study, we developed a simple approach to directly produce high yields of active TIMPs in the periplasmic space of Escherichia coli without refolding. Facilitated by disulfide isomerase (DsbC) co-expression in protease-deficient strain BL21 (DE3), N-TIMP-1/-2 and TIMP-2 which contain multiple disulfide bonds were produced without unwanted truncations. 0.2-1.4 mg purified monomeric TIMPs were typically yielded per liter of culture media. Periplasmically produced TIMPs exhibited expected inhibition potencies towards MMP-1/2/7/14, and were functional in competitive ELISA to elucidate the binding epitopes of MMP specific antibodies. In addition, prepared N-TIMPs were fully active in a cellular context, i.e. regulating cancer cell morphology and migration in 2D and 3D bioassays. CONCLUSION: Periplasmic expression in E. coli is an excellent strategy to recombinantly produce active TIMPs and N-TIMPs.

Identification of highly selective MMP-14 inhibitory Fabs by deep sequencing.

Matrix metalloproteinase (MMP)-14 is an important target for cancer treatment due to its critical roles in tumor invasion and metastasis. Previous failures of all compound-based broad-spectrum MMP inhibitors in clinical trials suggest that selectivity is the key for a successful therapy. With inherent high specificity, monoclonal antibodies (mAbs) therefore arise as attractive inhibitors able to target the particular MMP of interest. As a routine screening method, enzyme-linked immunosorbent assays (ELISA) have been applied to panned phage libraries for the isolation of mAbs inhibiting MMP-14. However, because of suboptimal growth conditions and insufficient antibody expression associated with monoclonal ELISA, a considerable number of potentially inhibitory clones might not be identified. Taking advantage of next-generation sequencing (NGS), we monitored enrichment profiles of millions of antibody clones along three rounds of phage panning, and identified 20 Fab inhibitors of MMP-14 with inhibition IC50 values of 10-4,000 nM. Among these inhibitory Fabs, 15 were not found by monoclonal phage ELISA. Particularly, Fab R2C7 exhibited an inhibition potency of 100 nM with an excellent selectivity to MMP-14 over MMP-9. Inhibition kinetics and epitope mapping suggested that as a competitive inhibitor, R2C7 directly bound to the vicinity of the MMP-14 catalytic site. This study demonstrates that deep sequencing is a powerful tool to facilitate the systematic discovery of mAbs with protease inhibition functions. Biotechnol. Bioeng. 2017;114: 1140-1150. © 2017 Wiley Periodicals, Inc.

Generation of inhibitory monoclonal antibodies targeting matrix metalloproteinase-14 by motif grafting and CDR optimization.

Matrix metalloproteinase-14 (MMP-14) plays important roles in cancer metastasis, and the failures of broad-spectrum MMP compound inhibitors in clinical trials suggested selectivity is critical. By grafting an MMP-14 specific inhibition motif into complementarity determining region (CDR)-H3 of antibody scaffolds and optimizing other CDRs and the sequences that flank CDR-H3, we isolated a Fab 1F8 showing a binding affinity of 8.3 nM with >1000-fold enhancement on inhibition potency compared to the peptide inhibitor. Yeast surface display and fluorescence-activated cell sorting results indicated that 1F8 was highly selective to MMP-14 and competed with TIMP-2 on binding to the catalytic domain of MMP-14. Converting a low-affinity peptide inhibitor into a high potency antibody, the described methods can be used to develop other inhibitory antibodies of therapeutic significance.

Selective function-blocking monoclonal human antibody highlights the important role of membrane type-1 matrix metalloproteinase (MT1-MMP) in metastasis.

The invasion-promoting MT1-MMP is a cell surface-associated collagenase with a plethora of critical cellular functions. There is a consensus that MT1-MMP is a key protease in aberrant pericellular proteolysis in migrating cancer cells and, accordingly, a promising drug target. Because of high homology in the MMP family and a limited success in the design of selective small-molecule inhibitors, it became evident that the inhibitor specificity is required for selective and successful MT1-MMP therapies. Using the human Fab antibody library (over 1.25×109 individual variants) that exhibited the extended, 23-27 residue long, VH CDR-H3 segments, we isolated a panel of the inhibitory antibody fragments, from which the 3A2 Fab outperformed others as a specific and potent, low nanomolar range, inhibitor of MT1-MMP. Here, we report the in-depth characterization of the 3A2 antibody. Our multiple in vitro and cell-based tests and assays, and extensive structural modeling of the antibody/protease interactions suggest that the antibody epitope involves the residues proximal to the protease catalytic site and that, in contrast with tissue inhibitor-2 of MMPs (TIMP-2), the 3A2 Fab inactivates the protease functionality by binding to the catalytic domain outside the active site cavity. In agreement with the studies in metastasis by others, our animal studies in acute pulmonary melanoma metastasis support a key role of MT1-MMP in metastatic process. Conversely, the selective anti-MT1-MMP monotherapy significantly alleviated melanoma metastatic burden. It is likely that further affinity maturation of the 3A2 Fab will result in the lead inhibitor and a proof-of-concept for MT1-MMP targeting in metastatic cancers.

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries.

Proteases are frequent pharmacological targets, and their inhibitors are valuable drugs in multiple pathologies. The catalytic mechanism and the active-site fold, however, are largely conserved among the protease classes, making the development of the selective inhibitors exceedingly challenging. In our departure from the conventional strategies, we reviewed the structure of known camelid inhibitory antibodies, which block enzyme activities via their unusually long, convex-shaped paratopes. We synthesized the human Fab antibody library (over 1.25 × 109 individual variants) that carried the extended, 23- to 27-residue, complementarity-determining region (CDR)-H3 segments. As a proof of principle, we used the catalytic domain of matrix metalloproteinase-14 (MMP-14), a promalignant protease and a drug target in cancer, as bait. In our screens, we identified 20 binders, of which 14 performed as potent and selective inhibitors of MMP-14 rather than as broad-specificity antagonists. Specifically, Fab 3A2 bound to MMP-14 in the vicinity of the active pocket with a high 4.8 nM affinity and was similarly efficient (9.7 nM) in inhibiting the protease cleavage activity. We suggest that the convex paratope antibody libraries described here could be readily generalized to facilitate the design of the antibody inhibitors to many additional enzymes.

Direct production of functional matrix metalloproteinase--14 without refolding or activation and its application for in vitro inhibition assays.

Human matrix metalloproteinase (MMP)-14, a membrane-bound zinc endopeptidase, is one of the most important cancer targets because it plays central roles in tumor growth and invasion. Large amounts of active MMP-14 are required for cancer research and the development of chemical or biological MMP-14 inhibitors. Current methods of MMP-14 production through refolding and activation are labor-intensive, time-consuming, and often associated with low recovery rates, lot-to-lot variation and heterogeneous products. Here, we report direct production of the catalytic domain of MMP-14 in the periplasmic space of Escherichia coli. 0.5 mg/L of functional MMP-14 was produced without tedious refolding or problematic activation process. MMP-14 prepared by simple periplasmic treatment can be readily utilized to evaluate the potencies of chemical and antibody-based inhibitors. Furthermore, co-expression of both MMP-14 and antibody Fab fragments in the periplasm facilitated inhibitory antibody screening by avoiding purification of MMP-14 or Fabs. We expect this MMP-14 expression strategy can expedite the development of therapeutic drugs targeting MMPs with biological significance.

Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion.

Matrix metalloproteinases (MMPs) are extracellular matrix (ECM) degrading enzymes and have complex and specific regulation networks. This includes activation interactions, where one MMP family member activates another. ECM degradation and MMP activation can be initiated by several different stimuli including changes in ECM mechanical properties or intracellular contractility. These mechanical stimuli are known enhancers of metastatic potential. MMP-14 facilitates local ECM degradation and is well known as a major mediator of cell migration, angiogenesis and invasion. Recently, function blocking antibodies have been developed to specifically block MMP-14, providing a useful tool for research as well as therapeutic applications. Here we utilize a selective MMP-14 function blocking antibody to delineate the role of MMP-14 as an activator of other MMPs in response to changes in cellular contractility and ECM stiffness. Inhibition using function blocking antibodies reveals that MMP-14 activates soluble MMPs like MMP-2 and -9 under various mechanical stimuli in the pancreatic cancer cell line, Panc-1. In addition, inhibition of MMP-14 abates Panc-1 cell extension into 3D gels to levels seen with non-specific pan-MMP inhibitors at higher concentrations. This strengthens the case for MMP function blocking antibodies as more potent and specific MMP inhibition therapeutics.

Development of a periplasmic FRET screening method for protease inhibitory antibodies.

Proteases play critical roles in numerous physiological processes and thus represent one of the largest families of potential pharmaceutical targets. Previous failure of broad-spectrum small molecule inhibitors toward tumorigenic metalloproteinases in clinical trials emphasizes that selectivity is the key for a successful protease-inhibition therapy. With exquisite specificity, antibody-based inhibitors are emerging as promising therapeutics. However, the majority of current antibody selection technologies are based on binding and not on inhibition. Here, we report the development of a function-based inhibitory antibody screening method, which combines a simple periplasmic preparation and an ultra sensitive FRET assay, both processes are amenable to high-throughput applications. Using this method, inhibitory antibodies can be rapidly distinguished from non-inhibitory clones with satisfactory Z-factors. Coupled with ELISA, this method also provides a fast semi-quantitative estimation of IC50 values without antibody purification. We expect this technology to greatly facilitate the generation of highly selective biologic inhibitors, targeting many proteases that are important to medical research and therapeutic development.

Silaffin peptides as a novel signal enhancer for gravimetric biosensors.

Application of biomimetic silica formation to gravimetric biosensors has been conducted for the first time. As a model system, silaffin peptides fused with green fluorescent protein (GFP) were immobilized on a gold quartz crystal resonator for quartz crystal microbalances using a self-assembled monolayer. When a solution of silicic acid was supplied, silica particles were successfully deposited on the Au surface, resulting in a significant change in resonance frequency (i.e., signal enhancement) with the silaffin-GFP. However, frequency was not altered when bare GFP was used as a control. The novel peptide enhancer is advantageous because it can be readily and quantitatively conjugated with sensing proteins using recombinant DNA technology. As a proof of concept, this study shows that the silaffin domains can be employed as a novel and efficient biomolecular signal enhancer for gravimetric biosensors.

Coexpression of molecular chaperones to enhance functional expression of anti-BNP scFv in the cytoplasm of Escherichia coli for the detection of B-type natriuretic peptide.

Molecular chaperones are a ubiquitous family of cellular proteins that mediate the correct folding of other target polypeptides. In our previous study, the recombinant anti-BNP scFv, which has promising applications for diagnostic, prognostic, and therapeutic monitoring of heart failure, was expressed in the cytoplasm of Escherichia coli. However, when the anti-BNP scFv was expressed, 73.4% of expressed antibodies formed insoluble inclusion bodies. In this study, molecular chaperones were coexpressed with anti-BNP scFv with the goal of improving the production of functional anti-BNP in the cytoplasm of E. coli. Five sets of molecular chaperones were assessed for their effects on the production of active anti-BNP scFv. These sets included the following: trigger factor (TF); groES/groEL; groES/groEL/TF; dnaK/dnaJ/grpE; groES/groEL/dnaK/dnaJ/grpE. Of these chaperones, the coexpression of anti-BNP scFv with the groES/groEL chaperones encoded in plasmid pGro7 exhibited the most efficient functional expression of anti-BNP scFv as an active form. Coexpressed with the groES/groEL chaperones, 64.9% of the total anti-BNP scFv was produced in soluble form, which is 2.4 times higher scFv than that of anti-BNP scFv expressed without molecular chaperones, and the relative binding activity was 1.5-fold higher. The optimal concentration of L-arabinose required for induction of the groES/groEL chaperone set was determined to be 1.0 mM and relative binding activity was 3.5 times higher compared with that of no induction with L-arabinose. In addition, soluble anti-BNP scFv was increased from 11.5 to 31.4 µg/ml with optimized inducer concentration (1.0 mM L-arabinose) for the coexpression of the groES/groEL chaperones. These results demonstrate that the functional expression of anti-BNP scFv can be improved by coexpression of molecular chaperones, as molecular chaperones can identify and help to refold improperly folded anti-BNP scFv.

Oriented immobilization of antibody fragments on Ni-decorated single-walled carbon nanotube devices.

We herein demonstrate that Ni-decorated single-walled carbon nanotube field effect transistors (SWNT-FETs) combined with antibody fragments can be used as effective biosensing platforms. Nanoscales Ni particles 20 to 60 nm in diameter were formed on the sidewalls of SWNT-FETs using an electrochemical method. Carcinoembryonic antigen (CEA)-binding single chain variable fragments (scFvs) with a hexahistidine tag [(his)(6)] were synthesized using genetic engineering, and ordered immobilization of anti-CEA ScFvs on Ni nanoparticles was achieved by exploiting the specific interaction between hexahistidine and Ni. Whereas randomly oriented anti-CEA scFvs did not impart a noticeable change of conductance upon addition of CEA, a clear increase in conductance was observed using Ni-decorated SWNT-FETs functionalized with engineered scFvs.

A novel route for immobilization of proteins to silica particles incorporating silaffin domains.

In the diatom Cylindrotheca fusiformis, modified peptides called silaffin polypeptides are responsible for silica deposition in vivo at ambient conditions. Recently, it was discovered that the synthetic R5 peptide, the repeat unit of silaffin polypeptide without post-translational modification, was capable of precipitating silica in vitro and at ambient conditions. Herein, chimeric proteins were generated by incorporating synthetic silaffin R5 peptides and related unmodified silaffin domains (R1-R7) from Cylindrotheca fusiformis onto green fluorescent protein (GFP) by recombinant DNA technology and their ability to cause silicification was also examined. GFP chimeric proteins showed silicification at very low concentrations (600-700 microg/mL) when compared with adding excess amounts of R5 peptides (10 mg/mL) as previously reported. Sensitive to pH conditions, only the GFP-R1 chimera showed silicification activity at pH 8.0. The protein immobilization efficiencies of these chimeras were unexpectedly high ranging from 75 to 85%, with the R1 silaffin-protein construct showing excellent immobilization efficiency and a constant molar ratio of silica to protein ranging from 250 to 350 over a wide pH range. The average silica particle sizes had a tendency to decrease as pH increased to basic conditions. This study demonstrated the production of nanoscale immobilized protein, fabricated via silaffin-fused proteins.

Effect of epidermal growth factor in preimplantation development of porcine cloned embryos.

In this study, we determined the expression of epidermal growth factor (EGF) and its receptor (EGFr) gene, and the effect of exogenous EGF supplementation on preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos. In vitro matured gilt oocytes were fertilized with frozen-thawed semen in vitro or reconstructed with fetal fibroblasts by SCNT. In Experiment 1, total RNA was isolated from oocytes, preimplantation SCNT, or in vitro fertilization (IVF) embryos. The expression of EGF and EGFr mRNA was determined using reverse transcription-polymerase chain reaction (RT-PCR). In SCNT and IVF embryos, the EGF mRNA was detected in oocytes, 2-cell, 4-cell, 8-cell, morulae, and blastocysts, while EGFr mRNA was detected in oocytes, 2-cell, morulae, and blastocysts. In Experiment 2, SCNT embryos at 1-cell stage were cultured in North Carolina State University (NCSU)-23 medium supplemented with different concentrations of EGF (0.1, 1, or 10 ng/ml). Supplementing with 10 ng/ml EGF improved cleavage rate (82.8% vs. 76.8%, P<0.05), but not the rate of blastocyst formation compared to the control. At all concentrations, EGF increased (P<0.05) the total cell number in blastocysts (range 50.5-53.7 vs. 43.9). In Experiment 3, EGF (10 ng/ml) was added to NCSU-23 medium at the morula stage. The EGF did not affect blastocyst formation, total cell number in blastocysts or the ratio of inner cell mass (ICM) to total cell number. In conclusion, we demonstrated that EGF and EGFr mRNA are expressed in porcine IVF and SCNT preimplantation embryos, and that EGF increased the quality of blastocysts by increasing total cell numbers in porcine SCNT embryos.

The role of gonadotropin-releasing hormone (GnRH) and its receptor in development of porcine preimplantation embryos derived from in vitro fertilization.

This study was performed to investigate the expression of embryo-derived gonadotropin-releasing hormone (GnRH) and its receptor, and to determine the role of GnRH in porcine preimplantation embryos. In Experiment 1, porcine blastocysts derived from in vitro fertilization (IVF) and cultured in North Carolina State University (NCSU)-23 medium were subjected to reverse transcription polymerase chain reaction (RT-PCR) amplification with specific primers for GnRH and its receptor. The results showed that GnRH and its receptor were expressed in porcine IVF blastocysts. In order to investigate the role of GnRH in embryo development, porcine IVF embryos were cultured in NCSU-23 supplemented with different concentrations (0, 0.1, 1, or 10 microM) of a GnRH agonist (leuprolide, Experiment 2) or GnRH antagonist (antide, Experiment 3). Supplementing the culture medium with 0.1 or 1 microM leuprolide increased the rate of blastocyst formation (28.5 or 27.6% versus 20.2%) and mean total cell number (129 versus 104) compared to the control group. In contrast, antide significantly decreased the rate of blastocyst formation [12.6% (0.1 microM), 10.2% (1.0 microM), or 8.9% (10.0 microM) versus 22.8% (control)] and total cell number [69 (1 microM) or 68 (10 microM) versus 104 (control)]. In Experiment 4, porcine IVF embryos were cultured in NCSU-23 medium containing 1 microM antide plus 1 microM leuprolide. The embryotrophic effect of GnRH agonist was reversed by co-supplementing with GnRH antagonist. In conclusion, the present study demonstrated that supplementing a culture medium with GnRH agonist can improve blastocyst formation and the quality of porcine IVF embryos, and that this action was mediated through GnRH receptors.