A novel method for quantification of cefazolin local tissue concentration in blood, fat, synovium, and bone marrow using liquid chromatography - mass spectrometry.

To be effective, the concentration of antibiotic used must exceed the minimum inhibitory concentration (MIC) against infecting organisms at and in the surgical site. Few studies follow antibiotic levels for tissues that are manipulated during surgery. The aim of this work was to develop and validate a novel LC-MS method as well as an efficient extraction technique for the quantification of cefazolin in local tissues and whole blood. This method uses the same efficient extraction method across multiple tissue types affected by orthopedic surgery: blood, fat, synovium, and bone marrow. The ability to quantify cefazolin in these tissues will help identify surgical techniques and antibiotic dosing protocols that better protect patients from infection. The internal standard, 13C2,15N-cefazolin, co-elutes with cefazolin, and was used in calibration curves and tissue extracts as well as for cefazolin recovery and matrix effects. The protocol was rigorously tested, including measurements of reproducibility and calibration curve quality. The recovery of the extraction method ranges from 94% to 113% across all sample types. There is little to no matrix effect on cefazolin signal (98-120%). The developed method was used to determine cefazolin concentrations in tissues of 10 patients undergoing a total knee replacement. Cefazolin blood concentrations were approximately 500 times higher than in adipose, synovium, and bone marrow tissues. This clinical data shows that although the minimum inhibitory concentration is largely surpassed in blood, the concentration of cefazolin in fat, synovium, and bone marrow could be insufficient during a knee replacement. This method of cefazolin quantification will help surgeons optimize antibiotic concentrations in the local tissues during knee replacement surgery and potentially reduce serious post-surgical infections.

Analgesia and functional outcome after total knee arthroplasty: periarticular infiltration vs continuous femoral nerve block.

BACKGROUND: Capacity to ambulate represents an important milestone in the recovery process after total knee arthroplasty (TKA). The purpose of this study was to determine the analgesic effect of two analgesic techniques and their impact on functional walking capacity as a measure of surgical recovery. METHODS: Forty ASA II-III subjects undergoing TKA were enrolled in a randomized, double-blind, single-centre study receiving 48 h postoperative analgesia with either periarticular infiltration of local anaesthetic (Group I) or continuous femoral nerve block (Group F). Breakthrough pain relief was achieved with patient-controlled analgesia (PCA) morphine. The main outcome was postoperative morphine consumption. Early (postoperative days 1-3) and late (6 weeks) functional walking capacity (2 and 6 min walk tests, 2MWT and 6MWT, respectively), degree of physical activity (CHAMPS), health-related quality of life (SF-12), and clinical indicators of knee function (WOMAC, Knee Society evaluation, and range of motion) were measured. RESULTS: Patients in Group F used the PCA less (P=0.02) to achieve adequate analgesia. Postoperative 2MWT was similar in both groups (P=0.27). Six weeks after surgery, recovery of 6MWT, physical activity, and knee function were significantly improved in Group F (P<0.05). Preoperative walking capacity, physical activity and early total walking time were the independent predictors of early recovery. Distance and time spent walking were the predictors of functional walking exercise capacity at 6 weeks after surgery. CONCLUSIONS: Femoral block is associated with lower opioid consumption and a better recovery at 6 weeks than periarticular infiltration. Early postoperative activity measures (2MWT and walking time) were proved to be possible indicators of knee function recovery at 6 weeks after surgery.

Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin.

The monovalent ionophore monensin inhibits the secretion of both procollagen and fibronectin from human fibroblasts in culture. The distribution of these proteins in control and inhibited (5 x 10(-7) M monensin) cells has been studied by immunofluorescence microscopy. In control cells, both antigens are present throughout the cytoplasm and in specific deposits in a region adjacent to the nucleus, which we identify as a Golgi zone by electron microscopy. Treatment of cells with monensin causes intracellular accumulation of procollagen and fibronectin, initially in the juxta-nuclear region and also subsequently in peripheral regions. Electron microscope studies reveal that in such cells the juxta-nuclear Golgi zone becomes filled with a new population of smooth-membraned vacuoles and that normal Golgi complexes are not found. Immunocytochemically detected procollagen and fibronectin are localized in the region of these vacuoles, whereas more peripheral deposits correspond to the dilated cisternae of rough endoplasmic reticulum, which are also caused by monensin. Procollagen and fibronectin are often codistributed in these peripheral deposits. Accumulation of exportable proteins in Golgi-related vacuoles is consistent with previous analyses of the monensin effect. The subsequent development of dilated rough endoplasmic reticulum also containing accumulated proteins may indicate that there is an additional blockade at the exit from the endoplasmic reticulum, or that the synthesized proteins exceed the capacity of the Golgi compartment and that their accumulation extends into the endoplasmic reticulum.

Fibronectin glycosylation modulates fibroblast adhesion and spreading.

The role of the carbohydrate residues of fibronectin concerning the specificities of that glycoprotein to interact with fibroblastic cell surfaces, gelatin, and heparin was examined. Tunicamycin was used to produce carbohydrate-depleted fibronectin; it was synthesized by cultured fibroblasts. Unglycosylated and glycosylated fibronectins were analyzed for their ability to bind gelatin and heparin, using affinity columns. Fibronectin-coated surfaces were used to quantitatively measure cell adhesion and spreading. The results showed that the lack of carbohydrates significantly increased the interaction of the protein with gelatin and markedly enhanced its ability to promote adhesion and spreading of fibroblasts. In contrast, the binding of fibronectin to heparin was not influenced by glycosylation. The composite data indicate that the Asn-linked oligosaccharides of fibronectin act as modulators of biological functions of the glycoprotein.

Cross-linking of collagen.

The formation of collagen cross-links is attributable to the presence of two aldehyde-containing amino acids which react with other amino acids in collagen to generate difunctional, trifunctional, and tetrafunctional cross-links. A necessary prerequisite for the development of these cross-links is that the collagen molecules be assembled in the naturally occurring fibrous polymer. Once this condition is met, cross-linking occurs in a spontaneous, progressive fashion. The chemical structures of the cross-links dictate that very precise intermolecular alignments must occur in the collagen polymer. This seems to be a function of each specific collagen because the relative abundance of the different cross-links varies markedly, depending upon the tissue of origin of the collagen.

Polyphenolic Substance of Mytilus edulis: Novel Adhesive Containing L-Dopa and Hydroxyproline.

The fouling marine mussel Mytilus edulis attaches itself to various substrates by spinning byssal threads, the adhesive discs of which are rich in the amino acid 3,4-dihydroxyphenylalanine (dopa). An acid-soluble protein was extracted and purified from the phenol gland located in the byssus-secreting foot of the animal. This protein is highly basic and contains large amounts of lysine, dopa, and 3- and 4-hydroxyproline. The composition of this protein and its sticky tendencies in vitro strongly suggest that it contributes to byssal adhesion.

Characterization of bone ingrowth and interface mechanics of a new porous 3D printed biomaterial: an animal study.

AIMS: The purpose of this study was to evaluate the biological fixation of a 3D printed porous implant, with and without different hydroxyapatite (HA) coatings, in a canine model. MATERIALS AND METHODS: A canine transcortical model was used to evaluate the characteristics of bone ingrowth of Ti6Al4V cylindrical implants fabricated using laser rapid manufacturing (LRM). At four and 12 weeks post-implantation, we performed histological analysis and mechanical push-out testing on three groups of implants: a HA-free control (LRM), LRM with precipitated HA (LRM-PA), and LRM with plasma-sprayed HA (LRM-PSHA). RESULTS: Substantial bone ingrowth was observed in all LRM implants, with and without HA, at both time periods. Bone ingrowth increased from 42% to 52% at four weeks, to 60% to 65% at 12 weeks. Mechanical tests indicated a minimum shear fixation strength of 20 MPa to 24 MPa at four weeks, and 34 MPa to 40 MPa at 12 weeks. There was no significant difference in the amount of bone ingrowth or in the shear strength between the three implant types at either time period. CONCLUSION: At four and 12 weeks, the 3D printed porous implants exhibited consistent bone ingrowth and high mechanical shear strength. Based on the results of this study, we confirmed the suitability of this novel new additive manufacturing porous material for biological fixation by bone ingrowth. Cite this article: Bone Joint J 2019;101-B(6 Supple B):62-67.

Evidence for altered synthesis of type II collagen in patients with osteoarthritis.

There is evidence to suggest that the synthesis of type II collagen is increased in osteoarthritis (OA). Using an immunoassay, we show that the content of the C-propeptide of type II procollagen (CPII), released extracellularly from the newly synthesized molecule, is directly related to the synthesis of this molecule in healthy and osteoarthritic articular cartilages. In OA cartilage, CPII content is often markedly elevated (mean 7.6-fold), particularly in the mid and deep zones, reaching 29.6% of the content in newborn. Synthesis is also directly related to total collagen II content in OA, suggesting its importance in maintaining collagen content and cartilage structure. The release of CPII from cartilage is correlated directly with cartilage content. However, the increase in CPII in OA cartilage is not reflected in serum, where a significant reduction is observed. Together these studies provide evidence for alterations in procollagen II synthesis in vivo in patients with OA.

Characterization of Post-Transcriptionally Suppressed Transgene Expression That Confers Resistance to Tobacco Etch Virus Infection in Tobacco.

Tobacco lines expressing transgenes that encode tobacco etch virus (TEV) coat protein (CP) mRNA with or without nonsense codons give rise to TEV-resistant tissues that have reduced levels of TEV CP mRNA while maintaining high levels of transgene transcriptional activity. Two phenotypes for virus resistance in the lines containing the transgene have been described: immune (no virus infection) and recovery (initial systemic symptoms followed by gradual recovery over several weeks). Here, we show that at early times in development, immune lines are susceptible to TEV infection and accumulate full-length CP mRNA. Therefore, immune lines also exhibit meiotic resetting, as is seen in the recovery lines, providing molecular evidence for a common mechanism of gene silencing and virus resistance in both cases. We also investigated the characteristics of two sets of low molecular weight RNAs that appear only in silenced tissue. One set has nearly intact 5[prime] ends, lacks poly(A) tails, and is associated with polyribosomes; the second set contains the 3[prime] end of the mRNA. Treating silenced leaf tissue with cycloheximide resulted in decreased levels of full-length mRNA and an increase in the levels of the low molecular weight RNAs, supporting a cytoplasmic decay mechanism that does not require ongoing translation. Surprisingly, mRNA from the transgene containing nonsense codons was associated with more ribosomes than expected, possibly resulting from translation from a start codon downstream of the introduced translational stop codons. We present a hypothesis for transgene/viral RNA degradation in which RNA degradation occurs in the cytoplasm while in association with polyribosomes.

Faithful degradation of soybean rbcS mRNA in vitro.

The mRNA encoding the soybean rbcS gene, SRS4, is degraded into a set of discrete lower-molecular-weight products in light-grown soybean seedlings and in transgenic petunia leaves. The 5'-proximal products have intact 5' ends, lack poly(A) tails, lack various amounts of 3'-end sequences, and are found at higher concentrations in the polysomal fraction. To study the mechanisms of SRS4 mRNA decay more closely, we developed a cell-free RNA degradation system based on a polysomal fraction isolated from soybean seedlings or mature petunia leaves. In the soybean in vitro degradation system, endogenous SRS4 mRNA and proximal product levels decreased over a 6-h time course. When full-length in vitro-synthesized SRS4 RNAs were added to either in vitro degradation system, the RNAs were degraded into the expected set of proximal products, such as those observed for total endogenous RNA samples. When exogenously added SRS4 RNAs already truncated at their 3' ends were added to either system, they too were degraded into the expected subset of proximal products. A set of distal fragments containing intact 3' ends and lacking various portions of 5'-end sequences were identified in vivo when the heterogeneous 3' ends of the SRS4 RNAs were removed by oligonucleotide-directed RNase H cleavage. Significant amounts of distal fragments which comigrated with the in vivo products were also observed when exogenous SRS4 RNAs were degraded in either in vitro system. These proximal and distal products lacking various portions of their 3' and 5' sequences, respectively, were generated in essentially a random order, a result supporting a nonprocessive mechanism. Tagging of the in vitro-synthesized RNAs on their 5' and 3' ends with plasmid vector sequences or truncation of the 3' end had no apparent effect on the degradation pattern. Therefore, RNA sequences and/or structures in the immediate vicinity of each 3' end point may be important in the degradation machinery. Together, these data suggest that SRS4 mRNA is degraded by a stochastic mechanism and that endonucleolytic cleavage may be the initial event. These plant in vitro systems should be useful in identifying the cis- and trans-acting factors involved in the degradation of mRNAs.

Degradation of the soybean ribulose-1,5-bisphosphate carboxylase small-subunit mRNA, SRS4, initiates with endonucleolytic cleavage.

The degradation of the soybean SRS4 mRNA, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase, yields a set of proximal (5' intact) and distal (3' intact) products both in vivo and in vitro. These products are generated by endonucleolytic cleavages that occur essentially in a random order, although some products are produced more rapidly than others. Comparison of sizes of products on Northern (RNA) blots showed that the combined sizes of pairs of proximal and distal products form contiguous full-length SRS4 mRNAs. When the 3' ends of the proximal products and the 5' ends of the distal products were mapped by S1 nuclease and primer extension assays, respectively, both sets of ends mapped to the same sequences within the SRS4 mRNA. A small in vitro-synthesized RNA fragment containing one cleavage site inhibited cleavage of all major sites, equivalently consistent with one enzymatic activity generating the endonucleolytic cleavage products. These products were rich in GU nucleotides, but no obvious consensus sequence was found among several cleavage sites. Preliminary evidence suggested that secondary structure could play a role in site selection. The structures of the 5' ends of the proximal products and the 3' ends of the distal products were examined. Proximal products were found with approximately equal frequency in both m7G cap(+) and m7G cap(-) fractions, suggesting that the endonucleolytic cleavage events occurred independently of the removal of the 5' cap structure. Distal products were distributed among fractions with poly(A) tails ranging from undetectable to greater than 100 nucleotides in length, suggesting that the endonucleolytic cleavage events occurred independently of poly(A) tail shortening. Together, these data support a stochastic endonuclease model in which an endonucleolytic cleavage event is the initial step in SRS4 mRNA degradation.

A physical vapor deposition method for controlled evaluation of biological response to biomaterial chemistry and topography.

The purpose of this study was to characterize a technique to effectively mask surface chemistry without modifying surface topography. A thin layer of titanium was deposited by physical vapor deposition (PVD) onto different biomaterial surfaces. Commercially pure titanium disks were equally divided into three groups. Disks were either polished to a mirror finish, grit blasted with alumina particles, or grit blasted and subsequently plasma sprayed with a commercial grade of hydroxyapatite (HA). A subgroup of each of these treatment types was further treated by masking the entire disk surface with a thin layer of commercially pure titanium deposited by PVD. A comparison of surface topography and chemical composition was carried out between disks within each treatment group. Canine marrow cells were seeded on all disk surfaces to determine the stability of the PVD Ti mask under culture conditions. The PVD process did not significantly alter the surface topography of any samples. The thin titanium layer completely masked the underlying chemistry of the plasma sprayed HA surface and the chemistry of the plasma vapor deposited titanium layer did not differ from that of the commercially pure titanium disks. Aliquots obtained from the media during culture did not indicate any significant differences in Ti concentration amongst the Ti and Ti-masked surfaces. The PVD application of a Ti layer on HA coatings formed a stable, durable, and homogenous layer that effectively masked the underlying surface chemistry without altering the surface topography.

Mechanical characterization of structurally porous biomaterials built via additive manufacturing: experiments, predictive models, and design maps for load-bearing bone replacement implants.

Porous biomaterials can be additively manufactured with micro-architecture tailored to satisfy the stringent mechano-biological requirements imposed by bone replacement implants. In a previous investigation, we introduced structurally porous biomaterials, featuring strength five times stronger than commercially available porous materials, and confirmed their bone ingrowth capability in an in vivo canine model. While encouraging, the manufactured biomaterials showed geometric mismatches between their internal porous architecture and that of its as-designed counterpart, as well as discrepancies between predicted and tested mechanical properties, issues not fully elucidated. In this work, we propose a systematic approach integrating computed tomography, mechanical testing, and statistical analysis of geometric imperfections to generate statistical based numerical models of high-strength additively manufactured porous biomaterials. The method is used to develop morphology and mechanical maps that illustrate the role played by pore size, porosity, strut thickness, and topology on the relations governing their elastic modulus and compressive yield strength. Overall, there are mismatches between the mechanical properties of ideal-geometry models and as-manufactured porous biomaterials with average errors of 49% and 41% respectively for compressive elastic modulus and yield strength. The proposed methodology gives more accurate predictions for the compressive stiffness and the compressive strength properties with a reduction of the average error to 11% and 7.6%. The implications of the results and the methodology here introduced are discussed in the relevant biomechanical and clinical context, with insight that highlights promises and limitations of additively manufactured porous biomaterials for load-bearing bone replacement implants. STATEMENT OF SIGNIFICANCE: In this work, we perform mechanical characterization of load-bearing porous biomaterials for bone replacement over their entire design space. Results capture the shift in geometry and mechanical properties between as-designed and as-manufactured biomaterials induced by additive manufacturing. Characterization of this shift is crucial to ensure appropriate manufacturing of bone replacement implants that enable biological fixation through bone ingrowth as well as mechanical property harmonization with the native bone tissue. In addition, we propose a method to include manufacturing imperfections in the numerical models that can reduce the discrepancy between predicted and tested properties. The results give insight into the use of structurally porous biomaterials for the design and additive fabrication of load-bearing implants for bone replacement.

Conservative non-pharmacological treatment options are not frequently used in the management of hip osteoarthritis.

Osteoarthritis (OA) is the most frequent joint disorder in seniors. Systematic reviews suggest that conservative treatment is effective and preferred in mild-moderate cases. The objective of this study was to examine the proportion of patients receiving physiotherapy, exercise or walking aids, and to explore factors associated with their prescription. We conducted a retrospective survey of patients about to undergo total hip arthroplasty for hip osteoarthritis. Patients were asked about past prescriptions for cane use, physiotherapy and exercise. Of 161 patients (36.6% male, mean age 68.7+/-10.1 years), 76% were prescribed a cane (adherence=86.2%). The main reason for not using a cane was vanity. Of the 28.0% patients prescribed physiotherapy, 73.3% received exercises compared to only 2.6% of non-physiotherapy patients. Patients who were older or worked in manual labour were more likely to be prescribed a cane and less likely to be prescribed physiotherapy or exercises. Men were less likely than women to be prescribed all three, but only cane use was statistically significant across genders. In conclusion, physiotherapy and exercise are not commonly prescribed in patients with hip OA.

Evolutionary divergence of the necroptosis effector MLKL.

The pseudokinase, MLKL (mixed-lineage kinase domain-like), is the most terminal obligatory component of the necroptosis cell death pathway known. Phosphorylation of the MLKL pseudokinase domain by the protein kinase, receptor interacting protein kinase-3 (RIPK3), is known to be the key step in MLKL activation. This phosphorylation event is believed to trigger a molecular switch, leading to exposure of the N-terminal four-helix bundle (4HB) domain of MLKL, its oligomerization, membrane translocation and ultimately cell death. To examine how well this process is evolutionarily conserved, we analysed the function of MLKL orthologues. Surprisingly, and unlike their mouse, horse and frog counterparts, human, chicken and stickleback 4HB domains were unable to induce cell death when expressed in murine fibroblasts. Forced dimerization of the human MLKL 4HB domain overcame this defect and triggered cell death in human and mouse cell lines. Furthermore, recombinant proteins from mouse, frog, human and chicken MLKL, all of which contained a 4HB domain, permeabilized liposomes, and were most effective on those designed to mimic plasma membrane composition. These studies demonstrate that the membrane-permeabilization function of the 4HB domain is evolutionarily conserved, but reveal that execution of necroptotic death by it relies on additional factors that are poorly conserved even among closely related species.

HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death.

Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.

Structures of the asparagine-linked sugar chains of laminin.

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-beta-galactosidase, endo-beta-N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: bi-, tri- and tetraantennary complex-type oligosaccharides; polylactosaminyl side chains containing Gal(beta 1----4)GlcNAc(beta 1----3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, alpha-galactose, beta-galactose and beta-N-acetylglucosamine; alpha-galactosyl residues linked to N-acetyllactosamine sequences; high-mannose-type oligosaccharides. These results, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.

Inhibitory effects of tunicamycin on procollagen biosynthesis and secretion.

Chick embryo cells were briefly exposed to the antibiotic, tunicamycin. Pre-exposed cells, compared to control cultures, showed a severe, progressive inhibition of the incorporation of glucosamine and mannose into total cellular macromolecules. Inhibition of the incorporation of glycine, leucine and proline was also progressive but not as marked as for the carbohydrates. Cellular secretion of all macromolecules was severely impaired. while comparison of the procollagens showed no difference in their subunit size or in their degree of glycosylation; the intracellular content of procollagen polypeptides was similar for both types of cells. In vitro studies showed that tunicamycin selectively inhibited glucosamine, but not mannose, incorporation into macromolecules. The composite results indicate that tunicamycin effectively inhibits protein synthesis, protein glycosylation and protein secretion in chick embryo cells.

Zoledronic acid causes enhancement of bone growth into porous implants.

The effect of zoledronic acid on bone ingrowth was examined in an animal model in which porous tantalum implants were placed bilaterally within the ulnae of seven dogs. Zoledronic acid in saline was administered via a single post-operative intravenous injection at a dose of 0.1 mg/kg. The ulnae were harvested six weeks after surgery. Undecalcified transverse histological sections of the implant-bone interfaces were imaged with backscattered scanning electron microscopy and the percentage of available pore space that was filled with new bone was calculated. The mean extent of bone ingrowth was 6.6% for the control implants and 12.2% for the zoledronic acid-treated implants, an absolute difference of 5.6% (95% confidence interval, 1.2 to 10.1) and a relative difference of 85% which was statistically significant. Individual islands of new bone formation within the implant pores were similar in number in both groups but were 69% larger in the zoledronic acid-treated group. The bisphosphonate zoledronic acid should be further investigated for use in accelerating or enhancing the biological fixation of implants to bone.