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The study presents a retrospective case study. This aim was to determine whether lactate, glucose and total protein in serum and cerebrospinal fluid (CSF) can be used as specific biochemical markers for rapid differential diagnosis between intervertebral disk herniation and spinal tumors. Canine blood (n=9) and cerebrospinal fluid (n=9) were collected for chemistry analysis in nine dogs divided into two groups: intervertebral disk herniation group (IVDHG) (n=6) and spinal tumor group (TG) (n=3). Absorption spectrophotometry was used for quantitative determination of the concentrations of lactate, glucose and total protein in serum and CSF. Lactate concentration in serum and CSF and glucose concentration in CSF were 40-60% higher in subjects in TG compared to the IVDHG group. Serum lactate concentrations in the IVDHG group were 60% lower than physiological values. The results indicate that elevated concentration of glucose, lactate and total proteins in CSF and serum are indicative of a tumor, while a decrease of serum lactate and other parameters within normal range indicate disk herniation., Ovo istraživanje predstavlja retrospektivnu studiju. Cilj ovog istraživanja bio je ustvrditi mogu li laktat, glukoza ili ukupni protein u serumu i cerebrospinalnoj tekućini biti specifični biokemijski markeri u brzoj diferencijalnoj dijagnostici kralježničnog tumora i hernijacije intervertebralnog diska. U istraživanju je korišteno 9 uzoraka pseće krvi i 9 uzoraka pseće cerebrospinalne tekućine koje su biokemijski analizirane. Ukupno 9 je pasa podijeljeno u 2 skupine: hernijacija intervertebralnog diska (IVDHG) (n=6) i skupina vertebralni tumor (TG) (n=3). Za kvantitativno određivanje koncentracija laktata, glukoze i ukupnih proteina u serumu i cerebrospinalnoj tekućini koristila se metoda apsorpcijske spektrofotometrije. Koncentracije laktatata u serumu i cerebrospnalnoj tekućini i glukoze u cerebrospinalnoj tekućini su bile povišene za 40 do 60 % u pasa s tumorom u kralježnici u usporedbi s psima iz skupine s hernijacijom intervertebralnog diska. Serumska koncentracija laktata u pasa s hernijacijom intervertebralnog diska je bila 60 % niža nego fiziološke vrijednosti. Rezultati ove studije su pokazali da povećane koncentracije glukoze, laktata i ukupnih proteina u cerebrospinalnoj tekućini upućuju na tumor u kralježnici, dok, smanjene ili fiziološke vrijednosti laktata i ostalih mjerenih pokazatelja mjerenih u serumu i cerebrospinalnoj tekućini ukazuju na hernijaciju intervertebralnog diska.

Background/Introduction: Autologous bone graft substitute (ABGS) containing rhBMP6 in autologous blood coagulum (ABC) as a BMP carrier combined with compression resistant matrix (CRM) is a novel therapeutic solution for bone regeneration. Purpose: The aim of this study was to evaluate synthetic ceramics with different particle size and chemical composition of particles as a CRM in posterolateral lumbar fusion (PLF) rabbit model. Methods: ABGS implants were prepared as follow: rhBMP6 (125 mg) was added to autologous blood (2, 5 mL) which was mixed with synthetic ceramic particles and left to coagulate. Tested ceramic particles differed in particle size (74-420 μm and 500-1700 μm) and chemical composition (TCP and biphasic ceramics containing TCP and HA in 40/60 ratio). ABGS implants (n=6 per group) were implanted bilaterally between lumbar transverse processes (L5-L6) of New Zealand White Rabbits. At the end of the follow up period (27 weeks) animals were killed and spine specimens were analysed by microCT, histology and biomechanical testing. Results: Successful spinal fusion was observed in all animals regardless of the size and chemical composition of tested ceramic particles. However, microCT analyses revealed that chemical composition of ceramics determined longevity of CRM. Specifically, TCP ceramics regardless of the particle size were significantly resorbed while particles containing large proportion of HA were only partially resorbed. Osseointegration of newly formed bone with adjacent transverse processes was observed in all specimens. Moreover, biomechanical testing revealed that adjacent transverse processes were rebridged with biomechanically competent bone. Conclusion(s): A novel ABGS containing rhBMP6 within ABC successfully promoted lumbar fusion with different ceramic particles used as a CRM. However, the chemical composition of ceramics significantly affected CRM resorbability.

The aim of this study was to investigate ectopic bone formation and osseointegration in the posterolateral spinal fusion (PLF) model in New Zealand white rabbits using a novel innovative autologous bone graft substitute (ABGS) containing recombinant human Bone Morphogenetic Protein 6 (rhBMP6), autologous blood coagulum (ABC) and tricalcium-phosphate (TCP) or biphasic (composed of 80% TCP and 20% hydroxyapatite) granulate bioceramics as compression resistant matrix (CRM). Blood samples were collected from rabbit ear marginal vein into tubes without an anticoagulant substance in a volume of 2500 µl. rhBMP6 (125 µg) was mixed with blood and bioceramics. The ABGS implants (n=6 per group) were implanted bilateraly between tranverse processes of the lumbar vertebrae L5-L6 following exposition and decortication of transverse processes. All animals were euthanized on day 50 after surgery. To visualize new ectopic bone formation, lumbar spine was scanned by µCT. The success of spinal fusion was analysed on µCT sections through the anterior and posterior transverse process. Furthermore, lumbar spine was palpated and the mobility of fused transverse processes was tested. Total fusion success rate was 90, 9% and the same result was obtained by both analysis of spinal fusion on µCT sections and by palpatory mobility testing. µCT analyses revealed that an extensive amount of newly formed bone was present in both experimental groups and that there was no significant difference among experimental groups regardless the type of bioceramics used (biphasic bioceramics vs tri-calcium phosphate bioceramics). Bone formation and osseointegration were confirmed on histological sections through newly formed bone between transverse processes. Successful spinal fusion between adjacent transverse processes was confirmed radiologically (µCT sections and µCT analyses), by palpatory segmental mobility test and on histological sections. Therefore, an ABGS containing rhBMP6, ABC and bioceramic granulate might be an innovative and original biological approach for achieving a successful lumbar spine fusion.

In the present study, we evaluated an autologous bone graft substitute (ABGS) composed of recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) used as a physiological carrier for new bone formation in spine fusion sheep models. The application of ABGS included cervical cage for use in the anterior lumbar interbody fusion (ALIF), while for the posterolateral lumbar fusion (PLF) sheep model allograft devitalized bone particles (ALLO) were applied with and without use of instrumentation. In the ALIF model, ABGS (rhBMP6/ABC/cage) implants fused significantly when placed in between the L4-L5 vertebrae as compared to control (ABC/cage) which appears to have a fibrocartilaginous gap, as examined by histology and micro CT analysis at 16 weeks following surgery. In the PLF model, ABGS implants with or without ALLO showed a complete fusion when placed ectopically in the gutter bilaterally between two decorticated L4-L5 transverse processes at a success rate of 88% without instrumentation and at 80% with instrumentation; however the bone volume was 50% lower in the instrumentation group than without, as examined by histology, radiographs, micro CT analyses and biomechanical testing at 27 weeks following surgery. The newly formed bone was uniform within ABGS implants resulting in a biomechanically competent and histologically qualified fusion with an optimum dose in the range of 100 μg rhBMP6 per mL ABC, while in the implants that contained ALLO, the mineralized bone particles were substituted by the newly formed remodeling bone via creeping substitution. These findings demonstrate for the first time that ABGS (rhBMP6/ABC) without and with ALLO particles induced a robust bone formation with a successful fusion in sheep models of ALIF and PLF, and that autologous blood coagulum (ABC) can serve as a preferred physiological native carrier to induce new bone at low doses of rhBMP6 and to achieve a successful spinal fusion.

Posterolateral lumbar fusion (PLF) is a commonly performed surgical procedure for the treatment of pathological conditions of the lumbosacral spine. In the present study, we evaluated an autologous bone graft substitute (ABGS) containing rhBMP6 in autologous blood coagulum (ABC) and synthetic ceramics used as compression resistant matrix (CRM) in the rabbit PLF model. In the pilot PLF rabbit experiment, we tested four different CRMs (BCP 500-1700 μm, BCP 1700-2500 μm and two different TCP in the form of slabs) which were selected based on achieving uniform ABC distribution. Next, ABGS implants composed of 2.5 mL ABC with 0.5 g ceramic particles (TCP or BCP (TCP/HA 80/20) of particle size 500-1700 μm) and 125 μg rhBMP6 (added to blood or lyophilized on ceramics) were placed bilaterally between transverse processes of the lumbar vertebrae (L5- L6) following exposition and decortication in 12 New Zealand White Rabbits observed for 7 weeks following surgery. Spinal fusion outcome was analysed by μCT, palpatory segmental mobility testing and selected specimens were either tested biomechanically (three-point bending test) and/or processed histologically. The total fusion success rate was 90.9% by both μCT analyses and by palpatory segmental mobility testing. The volume of newly formed bone between experimental groups with TCP or BCP ceramics and the different method of rhBMP6 application was comparable. The newly formed bone and ceramic particles integrated with the transverse processes on histological sections resulting in superior biomechanical properties. The results were retrospectively found superior to allograft devitalized mineralized bone as a CRM as reported previously in rabbit PLF. Overall, this novel ABGS containing rhBMP6, ABC and the specific 500-1700 μm synthetic ceramic particles supported new bone formation for the first time and successfully promoted posterolateral lumbar fusion in rabbits.

Prvi pisani dokazi korištenja životinja u pokusima potječu još iz Staroga vijeka. Sve do otkrića anestetika životinje su u pokusima proživljavale nezamislive patnje. Prvi akt koji je zabranjivao okrutnost prema životinjama u pokusu donesen je u Britanskom parlamentu 1876. godine. Sredinom dvadesetog stoljeća znanstvenici Russell i Burch u svome su djelu “Načela humanih pokusnih tehnika” postavili temelje nove primijenjene znanstvene discipli- ne koja je trebala poboljšati postupke u radu s laboratorijskim životinjama, a ujedno i pri- donijeti kvaliteti znanstvenog rada u kojem se te životinje koriste. Raščlamba metoda kojima bi se mogli isključiti neljudski postupci prema životinjama u pokusu rezultirala je stvaranjem koncepta 3R načela: Replacement (zamjena), Re- duction (smanjenje) i Refinement (poboljšanje) s ultimativnim ciljem sadržanim u tek jednoj rije- či‒čovječnost. Razvojem znanosti, tehnologije, ali i evolucijom svijesti kako znanstvenika, tako i šire društvene zajednice, 3R načela svakod- nevno se razvijaju, tako da možemo govoriti i o 11R načelima jer se uz tri prethodno navede- na danas još navode i Reliability (pouzdanost), Relevance (pogodnost‒prikladnost), Reproduci- bility (ponovljivost), Rehabilitation (oporavak), Responsibility (odgovornost), Respect (poštova- nje), Redundancy avoidance (izbjegavanje nepo- trebnih ponavljanja) i Regulation (zakonitost postupaka). 3R načelo razvijalo se tijekom pro- teklih pedeset godina osiguravajući okvir za izvođenje humanih pokusnih postupaka pre- ma životinjama, a prvi se puta sam naziv „Na- čela zamjene, smanjenja i poboljšanja“ uvodi u zakonodavstvo Europske unije kroz Direktivu 2010/63/EU. Godine 2010. donesena je Baselska deklaracija kojoj je cilj ujediniti napore znan- stvene zajednice u nastojanjima daljnjeg una- prjeđivanja primjene etičkih principa poput 3R načela u pokusima na životinjama sa svrhom postizanja pozitivnog dijaloga znanstvene za- jednice sa zainteresiranom javnošću. Danas projekt “Odgovorno istraživanje i inovacije” (Responsible Research and Innovation, RRI) Eu- ropskog okvirnog programa Obzor 2020 potiče suradnju i dijalog među istraživačima, građa- nima, političarima, itd. tijekom cijelog procesa istraživanja kako bi se taj proces i njegovi isho- di bolje uskladili s vrijednostima, potrebama i očekivanjima društva., The first written evidence of the use of animals in experiments originated in the Ancient world. Until the discovery of anaesthetics, experimental animals experienced unimaginable suffering. The first act prohibiting cruelty to animals was passed by the British Parliament in 1876. In the mid-20th century, scientists Russell and Burch in their work The Principles of Humane Experimental Technique laid the foundations of a new applied science discipline aimed at improving procedures in working with laboratory animals, while also contributing to the quality of the scientific work in which these animals were used. The analysis of methods to exclude inhumane treatment of animals in experiments resulted in the creation of the concept of the 3R Principle: Replacement, Reduction and Refinement, with the ultimate goal contained in a single word ‒ humaneness. With the development of science, technology, and the evolution of consciousness within the sciences and the general public, the 3R Principle has evolved rapidly, and today has been expanded into the 11R principle, in which Reliability, Relevance Reproducibility, Rehabilitation, Responsibility, Respect, Redundancy avoidance, and Regulation were added to the original 3R principles. Over the past 50 years, the 3R principle has developing into a framework for performing experimental techniques on animals, and for the first time the Principles of Replacement, Reduction and Refinement have been introduced into the European Union legislation through Directive 2010/63/EU. In 2010, the Basel Declaration was adopted, aiming to unite the efforts of the scientific community with the goal for further improvement of the application of ethical principles such as the 3R principles in animal experiments with the aim of achieving positive dialogue between the scientific community and the interested public. Today, the project “European Responsibility Research and Innovation” (RRI) within the European framework programme Horizon 2020 encourages co-operation and dialogue among researchers, citizens, politicians, and others throughout the research process, to better align this process and its outcomes with the values, needs and expectations of society.

BMP2 and BMP7, which use bovine Achilles tendon-derived absorbable collagen sponge and bovine bone collagen as scaffold, respectively, have been approved as bone graft substitutes for orthopedic and dental indications. Here, we describe an osteoinductive autologous bone graft substitute (ABGS) that contains recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) scaffold. The ABGS is created as an injectable or implantable coagulum gel with rhBMP6 binding tightly to plasma proteins within fibrin meshwork, as examined by dot-blot assays, and is released slowly as an intact protein over 6 to 8 days, as assessed by ELISA. The biological activity of ABGS was examined in vivo in rats (Rattus norvegicus) and rabbits (Oryctolagus cuniculus). In a rat subcutaneous implant assay, ABGS induced endochondral bone formation, as observed by histology and micro-CT analyses. In the rabbit ulna segmental defect model, a reproducible and robust bone formation with complete bridging and restoration of the defect was observed, which is dose dependent, as determined by radiographs, micro-CT, and histological analyses. In ABGS, ABC scaffold provides a permissive environment for bone induction and contributes to the use of lower doses of rhBMP6 compared with BMP7 in bovine bone collagen as scaffold. The newly formed bone undergoes remodeling and establishes cortices uniformly that is restricted to implant site by bridging with host bone. In summary, ABC carrier containing rhBMP6 may serve as an osteoinductive autologous bone graft substitute for several orthopedic applications that include delayed and nonunion fractures, anterior and posterior lumbar interbody fusion, trauma, and nonunions associated with neurofibromatosis type I.

The antibiotic 5-nitrofurantoin (5-NF) has been used widely for the treatment of urosepsis in children during the last 20 years. Recent experimentation suggests the need for reevaluating its genotoxic potential. Because of possible differences in the metabolism and clearance of 5-NF in young and adult animals, we conducted a study to determine whether micronuclei caused by 5-NF were age-related. The in vivo micronucleus (MN) assay was performed on 3- and 8-week-old mice given single intraperitoneal injections of 5, 10, and 50 mg/kg 5-NF. Blood samples from the tail vein were taken before injection (baseline) and at 48, 96, 168, and 336 hr (2 weeks) after the treatment. One thousand reticulocytes were analyzed for micronuclei from each animal. Compared to similar baseline values for young and adult mice, 5-NF caused a significant increase in MN frequency in both age groups. The mean MN frequency in the young animals was higher than in the adult animals for each dose and sampling time. MN frequencies remained significantly elevated in young animals even 2 weeks after exposure to 5-NF. The results of the study confirm the genotoxic potential of 5-NF in young and adult animals, and indicate that young animals are more sensitive to the genotoxic effects of 5-NF than adult mice and that the response in young mice persists for a significantly longer time. These findings may be related to poorly developed mechanisms of xenobiotic detoxification and renal elimination in young animals.

Bone Morphogenetic Proteins (BMPs) are growth and differentiation factors within the TGFβ superfam- ily of proteins. They induce ectopic and orthotopic endochondral bone formation and are involved in the regulation of cell proliferation, differentiation, apoptosis and mesenchymal- epithelial interactions in critical morphogenetic processes of tissues beyond bone. BMP2 and BMP7 osteogenic devices have been approved for enhancing healing in patients with long bone defects and anterior spinal fusion proce- dures. However, due to a high price and various serious adverse events including heterotopic ossifica- tion, retrograde ejaculation and pain their clinical use have been limited. In this review we discuss the BMP discovery, biology and their use in clinical studies with particular reference to the newly developed BMP6 based autologous bone graft substitute (ABGS). A novel ABGS consisting of an autologous bone coagulum (ABC) carrier with dispersed BMP6 to initiate the differentiation of mesenchymal cells into endochondral bone. The ABC met the conditions for an optimal delivery system for BMP6 due to han- dling simplicity, without an immunogenic and inflammatory response at the implantation site. Addition of allograft or synthetic ceramics to ABGS demonstrated in animal models significantly increased volume and better microarchitecture of the newly formed bone. The first clinical study was conducted in patients with distal radial fractures (Phase I study) and the second in patients undergoing high tibial osteotomy (Phase I/II study) and no serious adverse events have been observed. Finally, in the ongoing OSTEO- proSPINE study ABGS enforced with allograft bone is evaluated in patients with chronic back pain due to degenerative disc diseases. The novel ABGS bone mimetic is a major breakthrough and contribution to bone biology and regenerative medicine of skeletal repair.

Because of neurotropic and neurovirulent properties of mumps virus, neurovirulence testing of live attenuated vaccine is required by most national regulatory organisations. Such testing is preformed in monkeys (Ph.Eu. 01/2008:20618-Test for neurovirulence of live virus vaccines). But results obtained from these tests do not necessarily distinguish among the neurovirulent strain from those that are not. As a part of an international collaborative study we investigated the neurovirulence of three vaccine strains (JL5, Urabe AM9 and L-Zagreb) and two wild–type mumps viruses isolated in Croatia (9218/Zg98 and MuVi/Zagreb.HRV/28.12) by the neurovirulence assay in newborn rats. The results obtained by alternative assay on two vaccine strains (JL5 and Urabe AM9) correspond to the results obtained by the National Institute for Biological Standards (NIBSC) in the United Kingdom and the Food and Drug Administration in the United Station. Strain L-Zagreb also showed reduced neurovirulent properties as expected because it is a vaccine strain. Wild-type mumps virus isolates showed high neurovirulence. These results indicate as that the test in newborn rats is suitable for assessing the neurovirulence of mumps viruses. The test is robust, reproducible and follows the 3R principles.

Continuous quality control of venoms and antivenoms is necessary for a successful production of the only specific treatment of victims of venomous snake bites in Europe. Control is based on two in vivo tests: (a) the venom lethal toxicity assay (determination of median lethal dose of venom or LD50) and (b) test for determining the neutralization potency (effectiveness) of produced antivenoms (determination of mean effective antivenom dose or ED50). Both these tests, which cause suffering, pain and death of the experimental animals and also require a large number of animals, were identified by ECVAM (European Centre for Validation of Alternative Methods) as assays that are necessary to be replaced with alternative methods. Snake venoms are complex mixtures of more than a hundred mostly proteins and peptides, and non-protein compounds with different biochemical and pharmacological effects. Such complex mixtures are used as an antigen for animal immunization in antivenom production. For the development of in vitro tests, it is necessary to identify the components that contribute to the venom’s overall toxicity or specificity of antibodies that effectively neutralize them. So far several molecules, mainly from the family of metalloproteinases (haemorrhagins, the dominant cause of the human envenomation pathology) and phospholipase A2 (ammodytoxins, the most toxic molecules in the venom known to date) have been isolated and characterized from the European vipers’ venom. However, there were no literature data for their immunogenicity. Our study showed that ammodytoxin content, determined by two developed biochemical methods, HPLC and the "sandwich" ELISA, highly correlated with the lethal toxicity of the whole viper venom. Further we examined the role of ammodytoxin (Atx)- and haemorrhagin (H)-specific antibodies in the venom lethal toxicity neutralization. The results showed that functional anti-Atx antibodies were only partially involved in the neutralization of the venom toxicity. On the other hand, functional anti-H antibodies did not provide protection at all. Developed methods for anti-Atx or anti-H determination, due to only limited involvement of the mentioned antibodies in venom toxicity neutralization, cannot be a substitute for the in vivo assays. Deeper insight into snake venom composition at the molecular level and the participation of the particular components in the venom toxicity has yet to be done before solving this task. On the other hand, Atx content determination in different venom batches could be a good screening method in the selection of the best antigen for immunisation.