Cyclic-RGD is as effective as rhBMP-2 in anterior interbody fusion of the sheep cervical spine.

STUDY DESIGN: Radiological and histological assessment of fusion status after anterior cervical discectomy and fusion (ACDF) procedure in a sheep spinal fusion model. OBJECTIVE: To evaluate the efficacy of cyclic arginine-glycine-aspartic (cRGD) in comparison with recombinant human bone morphogenetic protein-2 (rhBMP-2) on a mineralized collagen matrix (MCM). SUMMARY OF BACKGROUND DATA: A previous evaluation of MCM alone in comparison with autologous bone graft alone was not able to show an advantage on spinal fusion. The cRGD peptide sequence plays a major role in mediating cell adhesion. Studies have demonstrated enhances osteoblasts adhesion resulting in increased periimplant bone formation after implantcoating with cRGD. rhBMP-2 has already proven its ability to enhance spinal fusion. To date, no comparative in vivo evaluation of cRGD and rhBMP-2 in combination with a MCM for spinal fusion has been performed. METHODS: Twenty-four sheep (N = 8/group) underwent C3-C4 fusion. Implants: group 1: titanium cage with MCM and rhBMP-2; group 2: titanium cage with MCM and cRGD; control group: titanium cage with MCM alone. After 12 weeks fusion sites were evaluated by computed tomography to assess fusion status, bone mineral density as well as bony callus volume. Furthermore, histomorphological and histomorphometrical analysis of the fusion sites were performed. RESULTS: In comparison with the control group, cRGD, and rhBMP-2 groups showed a higher fusion rate in radiographical findings and a higher degree of interbody fusion in histomorphometrical analysis (P < 0.05). There was no significant difference in radiographical and histological parameters between the rhBMP-2 and the cRGD group. Although rhBMP-2 demonstrated ectopic prevertebral bone formations, this effect was less prominent in the cRGD group. CONCLUSION: In this animal model the combination of cRGD and a mineralized collagen matrix showed superior fusion results in comparison with the mineralized collagen alone. Further, cRGD was comparably effective to rhBMP-2 in promoting interbody fusion by demonstrating less ectopic bone formations.

Effects of gentamicin and gentamicin-RGD coatings on bone ingrowth and biocompatibility of cementless joint prostheses: an experimental study in rabbits.

Antimicrobial coatings are of interest as a means to improve infection prophylaxis in cementless joint arthroplasty. However, those coatings must not interfere with the essential bony integration of the implants. Gentamicin-hydroxyapatite (gentamicin-HA) and gentamicin-RGD (arginine-glycine-aspartate)-HA coatings have recently been shown to significantly reduce infection rates in a rabbit infection prophylaxis model. The purpose of the current study was to investigate the in vitro elution kinetics and in vivo effects of gentamicin-HA and gentamicin-RGD-HA coatings on new bone formation, implant integration and biocompatibility in a rabbit model. In vitro elution testing showed that 95% and 99% of the gentamicin was released after 12 and 24 h, respectively. The in vivo study comprised 45 rabbits in total, with six animals for each of the gentamicin-HA, gentamicin-RGD-HA group and control pure HA coating groups for the 4 week time period, and nine animals for each of the three groups for the 12 week observation period. A 2.0 mm steel K-wire with one of the coatings under test was placed in the intramedullary canal of the tibia. After 4 and 12 weeks the tibiae were harvested and three different areas (proximal metaphysis, shaft area, distal metaphysis) were assessed by quantitative and qualitative histology for new bone formation, direct implant-bone contact and the formation of multinucleated giant cells. The results exhibited high standard deviations in all subgroups. There was a trend towards better bone formation and better direct implant contact in the pure HA coating group compared with both gentamicin coatings after 4 and 12 weeks, which was, however, not statistically significant. The number of multinucleated giant cells did not differ significantly between the three groups at both time points. In summary, both gentamicin coatings with 99% release of gentamicin within 24 h revealed good biocompatibility and bony integration, which was not statistically significant different compared with pure HA coating. Limitations of the study are the high standard deviation of the results and the limited number of animals per time point.

Cages augmented with mineralized collagen and platelet-rich plasma as an osteoconductive/inductive combination for interbody fusion.

STUDY DESIGN: After anterior cervical discectomy, fusion was radiologically, biomechanically, and histologically assessed in a sheep spine fusion model. OBJECTIVE: To evaluate the efficacy of a platelet-rich plasma (PRP) application combined with a mineralized collagen matrix (MCM) as an alternative to autologous cancellous iliac crest bone grafts in a spine fusion model. SUMMARY OF BACKGROUND DATA: PRP has the ability to stimulate bone and tissue healing. MCM is a recently developed osteoconductive material. Up to now, no comparative evaluation of PRP in combination with a MCM at the cervical spine has been performed in vivo. METHODS: Twenty-four sheep (N = 8/group) underwent C3/4 discectomy and fusion: group 1, titanium cage filled with autologous cancellous iliac crest bone graft; group 2, titanium cage filled with MCM; and group 3, titanium cage filled with MCM and PRP. Radiographic evaluation was performed before surgery and after 1, 2, 4, 8, and 12 weeks, respectively. After 12 weeks, fusion sites were evaluated using functional radiographic views and quantitative computed tomographic scans to assess bone mineral density. Furthermore, histomorphologic and histomorphometrical analyses were performed to evaluate fusion. RESULTS: In comparison with the titanium cage group filled with autologous cancellous iliac crest bone grafts representing the control group, MCM-alone group showed a slightly lower fusion rate in the radiographic and the histomorphometrical analysis. The addition of PRP could not enhance this finding. There was no significant difference between MCM and MCM + PRP group in radiologic and histologic findings. CONCLUSION: The MCM alone is not able to replace autologous bone grafts. Early activation of the platelets by calcium, which is released from mineralized collagen, could be the reason for the insufficient osteoinductive effect of PRP. In consequence, the combined application of mineralized collagen and PRP had no significant osteoinductive effect in this model.

Compound management beyond efficiency.

Codeveloping alongside chemistry and in vitro screening, compound management was one of the first areas in research recognizing the need for efficient processes and workflows. Material management groups have centralized, automated, miniaturized and, importantly, found out what not to do with compounds. While driving down cost and improving quality in storage and processing, researchers still face the challenge of interfacing optimally with changing business processes, in screening groups, and with external vendors and focusing on biologicals in many companies. Here we review our strategy to provide a seamless link between compound acquisition and screening operations and the impact of material management on quality of the downstream processes. Although this is driven in part by new technologies and improved quality control within material management, redefining team structures and roles also drives job satisfaction and motivation in our teams with a subsequent positive impact on cycle times and customer feedback.

New working paradigms in research laboratories.

Work in research laboratories, especially within centralised functions in larger organisations, is changing fast. With easier access to external providers and Contract Research Organisations, and a focus on budgets and benchmarking, scientific expertise has to be complemented with operational excellence. New concepts, globally shared projects and restricted resources highlight the constraints of traditional operating models working from Monday to Friday and nine to five. Whilst many of our scientists welcome this new challenge, organisations have to enable and foster a more business-like mindset. Organisational structures, remuneration, as well as systems in finance need to be adapted to build operations that are best-in-class rather than merely minimising negative impacts of current organisational structures.

Comparison of new bone formation, implant integration, and biocompatibility between RGD-hydroxyapatite and pure hydroxyapatite coating for cementless joint prostheses--an experimental study in rabbits.

This is the first work to report on additional Arginin-Glycin-Aspartat (RGD) coating on precoated hydroxyapatite (HA) surfaces regarding new bone formation, implant bone contact, and biocompatibility compared to pure HA coating and uncoated stainless K-wires. There were 39 rabbits in total with 6 animals for the RGD-HA and HA group for the 4 week time period and 9 animals for each of the 3 implant groups for the 12 week observation. A 2.0 K-wire either with RGD-HA or with pure HA coating or uncoated was placed into the intramedullary canal of the tibia. After 4 and 12 weeks, the tibiae were harvested and three different areas of the tibia were assessed for quantitative and qualitative histology for new bone formation, direct implant bone contact, and formation of multinucleated giant cells. Both RGD-HA and pure HA coating showed statistically higher new bone formation and implant bone contact after 12 weeks than the uncoated K-wire. There were no significant differences between the RGD-HA and the pure HA coating in new bone formation and direct implant bone contact after 4 and 12 weeks. The number of multinucleated giant did not differ significantly between the RGD-HA and HA group after both time points. Overall, no significant effects of an additional RGD coating on HA surfaces were detected in this model after 12 weeks.

Additive effect of RGD coating to functionalized titanium surfaces on human osteoprogenitor cell adhesion and spreading.

Titanium-based biomaterials for endosseous implants have found widespread applications in the orthopedic, maxillofacial, and dental domains. Indeed, the surface characteristics such as their chemical modification control considerably the cellular response and, subsequently, the quality and the quantity of new-formed bone around the implant. In this study, human osteoprogenitor (HOP) cell adhesion on different titanium surfaces functionalized with hydroxyapatite (HA), type I collagen, or Arg-Gly-Asp (RGD)-containing peptides is investigated by the quartz crystal resonators and by confocal laser scanning microscopy (CLSM) for the imaging of focal contact formation. Data obtained by quartz crystal resonator technique revealed that RGD-containing peptides alone increase HOP cell adhesion in early time period of culture. Moreover, association of RGD-containing peptides with either type I collagen or with HA layers induces an additive effect on HOP cell adhesion compared to Ti-Coll or Ti-HA. CLSM shows both the area of focal contact by cell unit and the cytoskeleton network organization to differ according to the surfaces. Interestingly, association of RGD-containing peptides with HA layers induces an additive effect on focal contact formation on HOP cells compared to Ti-HA alone. These data confirm that an RGD peptide effect occurs in the early time of culture, which is beneficial for osteoblast to spreading, differentiation, and survival.

Helping science to succeed: improving processes in R&D.

Bringing drugs to the market remains a costly and, until now, often unpredictable challenge. Although understanding the underlying science is key to further progress, our imperfect knowledge of disease and complex biological systems leaves excellence in execution as the most tangible lever to sustain our serendipitous approach to drug discovery. The problems encountered in pharmaceutical R&D are not unique, but to learn from other industries it is important to recognise similarity, rather than differences, and to advance industrialisation of R&D beyond technology and automation. Tools like Lean and Six Sigma, already applied to increase business excellence across diverse organisations, can equally be introduced to pharmaceutical R&D and offer the potential to transform operations without large-scale investment.

Biomimetic calcium phosphate composite coating of dental implants.

PURPOSE: The aim of the present study was to test the hypothesis that calcium phosphate coating of titanium screw-type implants enhances peri-implant bone formation in the jaw. MATERIALS AND METHODS: Ten adult female foxhounds received experimental titanium screw-type implants in the mandible 3 months after removal of all premolar teeth. Four types of implants were evaluated in each animal: implants with machined titanium surface (the control group), implants coated with collagen I (the collagen-only group), implants with a composite coating of calcium phosphate and mineralized collagen I (the composite group), and implants with calcium phosphate (hydroxyapatite [HA]) coating (the HA-only group). Peri-implant bone regeneration was assessed histomorphometrically after 1 and 3 months in 5 dogs each by measuring bone-implant contact (BIC) and the volume density of the newly formed peri-implant bone (BVD). RESULTS: After 1 month, BIC was significantly enhanced only in the group of implants with composite coating of calcium phosphate and mineralized collagen (P = .038). Volume density of the newly formed peri-implant bone was significantly higher in all coated implants after 1 month. No significant difference from baseline was found in BIC for the collagen-only and HA-only groups, but BVD was significantly higher in implants with composite coating (P = .041). After 3 months, BIC and BVD were significantly higher in all coated implants than in the controls with machined surfaces. CONCLUSION: It was concluded that composite coating of dental screw-type implant surfaces using calcium phosphate and collagen can enhance BIC and peri-implant bone formation.

The application of fluorescence lifetime readouts in high-throughput screening.

Measurement of fluorescence lifetime is a well-established technique, which has recently been introduced into the portfolio of assay formats used in high-throughput screening (HTS). This investigation establishes appropriate conditions for using lifetime measurements to reduce the impact of compound interference effects during large-scale HTS of corporate screening files. Experimental data on mixtures of standard fluorophores and interfering compounds (from 5 HTS campaigns) have been combined with a theoretical model to identify the minimum data quality required, defined by the photon count in the peak channel, for discrimination of biological activity. Single-component fluorophore lifetimes can be recovered with an error of 1%, with a peak photon count of 10(2), but the same accuracy with a 2-component decay requires a peak photon count of 10(3). When a 3rd component is introduced, the minimum peak count increases to 10(4). The influence of scattered light on lifetime determination was investigated using an emulsion (diameters 25-675 nm). The measured decays of interfering compounds, identified as autofluorescent, show that the vast majority have a very short lifetime that can readily be resolved from the reporter fluorophore, using appropriate data-fitting methods.

Data visualization during the early stages of drug discovery.

Multidimensional compound optimization is a new paradigm in the drug discovery process, yielding efficiencies during early stages and reducing attrition in the later stages of drug development. The success of this strategy relies heavily on understanding this multidimensional data and extracting useful information from it. This paper demonstrates how principled visualization algorithms can be used to understand and explore a large data set created in the early stages of drug discovery. The experiments presented are performed on a real-world data set comprising biological activity data and some whole-molecular physicochemical properties. Data visualization is a popular way of presenting complex data in a simpler form. We have applied powerful principled visualization methods, such as generative topographic mapping (GTM) and hierarchical GTM (HGTM), to help the domain experts (screening scientists, chemists, biologists, etc.) understand and draw meaningful decisions. We also benchmark these principled methods against relatively better known visualization approaches, principal component analysis (PCA), Sammon's mapping, and self-organizing maps (SOMs), to demonstrate their enhanced power to help the user visualize the large multidimensional data sets one has to deal with during the early stages of the drug discovery process. The results reported clearly show that the GTM and HGTM algorithms allow the user to cluster active compounds for different targets and understand them better than the benchmarks. An interactive software tool supporting these visualization algorithms was provided to the domain experts. The tool facilitates the domain experts by exploration of the projection obtained from the visualization algorithms providing facilities such as parallel coordinate plots, magnification factors, directional curvatures, and integration with industry standard software.

A novel method for analyzing [Ca2+] flux kinetics in high-throughput screening.

Driven by multiparameter fluorescence readouts and the analysis of kinetic responses from biological assay systems, the amount and complexity of high-throughput screening data are constantly increasing. As a consequence, the reduction of data to a simple number, reflecting a percentage activity/inhibition, is no longer an adequate approach because valuable additional information, for example, about compound-or process-induced artifacts, is lost. Time series data such as the transient calcium flux observed after activation of Gq-coupled G protein-coupled receptors (GPCRs), are especially challenging with respect to quantity of data; typically, responses are followed for several minutes. Based on measurements taken on the fluorometric imaging plate reader, the authors have introduced a mathematical model to describe the time traces of cellular calcium fluxes mediated by the activation of GPCRs. The model describes the time series using 13 parameters, reducing the amount of data by 90% while guiding the detection of compound-induced artifacts as well as the selection of compounds for further characterization.

Analysing the output from primary screening.

From a perspective of process knowledge and enhancement, the analysis of the results of biological screening should not be limited to the outcome of specific projects, but additionally encompass a process centric view. Summarising outcomes across multiple projects is a powerful tool to gain a greater understanding of biological screening that will also enable optimisation of the strategy for specific projects or target classes. We have analysed a set of 73,651 compounds with reproducible (confirmed) results from 63 high-throughput screening (HTS) campaigns to reveal the underlying trends in the population of active compounds. We have focused on the overall physico-chemical profile of compound populations derived from biological screening since the in vivo activity of drug molecules is the result of physico-chemical and structural properties of the compound.

The effects of combined gentamicin-hydroxyapatite coating for cementless joint prostheses on the reduction of infection rates in a rabbit infection prophylaxis model.

Infections remain a critical issue in total joint arthroplasty. Addition of antibiotics to bone cement was shown to significantly improve antimicrobial prophylaxis in cemented joint arthroplasty. In cementless joint arthroplasty a comparable prophylaxis by local antibiotics has not been possible yet. The aim of the current study was to investigate the antimicrobial effect of two different gentamicin-hydroxyapatite (HA) coatings for cementless prostheses in a rabbit infection model. Staphylococcus aureus with a dose of 10(7) CFUs was inoculated into the intramedullary canal of the tibia of 30 rabbits followed by the implantation of standard steel HA K-wires (n=10), steel K-wires coated with a gentamicin-HA combination (n=10), and steel K-wires coated with a gentamicin-RGD-HA combination (n=10), respectively. The animals were sacrificed after 28 days and clinical, histological and microbiological assessment on the bone and on the removed K-wire itself by agar plating and DNA-pulsed field gel electrophoresis were carried out to detect infection. There was a statistically significant reduction of infection rates by both gentamicin-coating types (0 infections in both groups) compared to standard HA coating (7 infections in 8 animals; 2 animals were lost due to acute diarrhea) (p<0.001). An excellent correlation between agar plating testing results of the K-wires and of the bone samples was found. Detailed histology showed cortical lysis, abscess and sequester formation in the infected animals. Both gentamicin-coating types showed significant improvement of infection prophylaxis compared to standard HA coating and, therefore, this coating technology could help to improve infection prophylaxis in cementless total joint arthroplasty. In further studies biocompatibility of the coatings has to be assessed.

Improving the design and analysis of high-throughput screening technology comparison experiments using statistical modeling.

Contemporary small-molecule drug discovery frequently involves the screening of large compound files as a core activity. Subsequently cost, speed, and safety become critical issues. In order to meet this need, numerous technologies have been developed to allow mix and measure approaches, facilitate miniaturization, and to increase speed and to minimize the use of potentially hazardous reagents such as radioactive materials. However, despite the on-paper advantages of these new technologies, risks can remain undefined. For example, the question of whether the novel method will facilitate identification of active chemical series in a way that is comparable with conventional methods arises. In order to address this question, we have taken the approach of carrying out experiments to directly compare the output of high-throughput screens using a given novel approach and a traditional method. The concordance between the screening methods can then be determined via comparison of the numbers and structures of the active molecules identified. This article describes the approach taken in our laboratory to minimize variability in such experiments and shows data that exemplifies the general result of lower than expected concordance. Statistical modeling was subsequently used to facilitate this interpretation. The model used beta-distribution function to generate a real-activity frequency relationship with added normal random error and occasional outliers to represent assay variability. Hence, the effect of assay parameters such as the threshold, the number of real actives, and the number of outliers and the standard deviation could readily be explored. The model was found to describe the data reasonably and moreover was found to be of great utility when it came to planning further optimal experiments. A key conclusion from the model was that concordance between screening methods could appear poor even when one approach is compared with itself. This occurs simply because the result is a function of assay threshold, standard deviation and the true compound % activity. In response to this finding we have adopted alternative experimental designs that more reliably measure the concordance between screening methods.

Effect of immobilized bone morphogenic protein 2 coating of titanium implants on peri-implant bone formation.

The aim of the present study was to test the hypothesis that immobilization of bone morphogenic protein (BMP2) on the surface of titanium implants can enhance peri-implant bone formation. Ten adult female foxhounds received experimental titanium screw implants in the mandible 3 months after removal of all premolar teeth. Three types of implant surfaces were evaluated in each animal: (i) implants with machined titanium surface, (ii) implants coated with collagen I, (iii) implants coated with collagen I, chondroitin sulphate (CS) and BMP2. Peri-implant bone regeneration was assessed using histomorphometry after 1 and 3 months in five dogs each by measuring bone-implant contact (BIC) and the volume density of the newly formed peri-implant bone (BVD). After 1 month, there was no significant enhancement in BIC values but volume density of the newly formed peri-implant bone was significantly higher in the two groups of coated implants. No significant difference was found between collagen and BMP2 coating. After 3 months, BIC was significantly higher in both collagen and BMP2-coated implants compared with implants with machined surfaces. Peri-implant BVD was also significantly increased in coated implants in comparison with machined surfaces. It was concluded that collagen coating of dental screw implants can enhance BIC and peri-implant bone formation. Addition of BMP2 does not increase peri-implant bone formation in the present application.

The effect of cyclo-DfKRG peptide immobilization on titanium on the adhesion and differentiation of human osteoprogenitor cells.

This study takes place in the field of development of a bioactive surface of titanium alloys. In this paper, titanium was functionalized with cyclo-DfKRG peptide by coating or grafting using different anchors (thiol or phosphonate) as spacers between the surface and the peptide. Cell adhesion, and differentiation of human osteoprogenitor (HOP) cells arising from human bone marrow were investigated. Our results seem to demonstrate that cyclo-DfKRG peptide coating with a phosphonate anchor and grafting procedure contributes to higher cell adhesion and a strong ALP and Cbfa1 mRNA expression, after 10 days of cell seeding. At the contrary, this peptide coated with a thiol anchor stimulates differentiation of HOP within 3 days of culture.

Evaluating real-life high-throughput screening data.

High-throughput screening (HTS) is the result of a concerted effort of chemistry, biology, information technology, and engineering. Many factors beyond the biology of the assay influence the quality and outcome of the screening process, yet data analysis and quality control are often focused on the analysis of a limited set of control wells and the calculated values derived from these wells. Taking into account the large number of variables and the amount of data generated, multiple views of the screening data are necessary to guarantee quality and validity of HTS results. This article does not aim to give an exhaustive outlook on HTS data analysis but tries to illustrate the shortfalls of a reductionist approach focused on control wells and give examples for further analysis.

Development and automation of a 384-well cell fusion assay to identify inhibitors of CCR5/CD4-mediated HIV virus entry.

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/beta-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of beta-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturization to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen approximately 650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z' consistently above 0.6, which compares favourably with less complex biochemical assays.