Analiza cerebrospinalne tekućine u pasa s kralježničnim tumorom i hernijacijom intervertebralnog diska

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

3R Principle for the welfare protection of animals used for scientific purposes - yesterday, today, tomorrow

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

Mast Cell Tumor in Dogs – Incidence and Histopathological Characterization

Incidence of mast cell tumors, their distribution according to sex, breed, age and localisation in Croatia is not established yet. Also, the statistical significance of the various histopathological parameters according to Patnaik’s scheme, in the diagnostics of the tumor grade was not performed. Investigation analysed mast cell tumors histopathologicaly characterized at the Department of General Pathology and Pathological Morphology of the Veterinary Faculty Zagreb from January 1st 2002 to Dezember 31st 2006. Sex, age, breed, localisation and tumor grade of each animal with tumor was recorded and statisticaly evaluated. Each histopathological variable was scored and compared with tumor grade. In the analyzed period, totally 1630 tumors were recorded, and mast cell tumors were found in 106 animals or in 6.5% of all cases. With statistically significant difference, this tumor was found in more cases in male dogs, and average age was 6.96 years. Boxers and retrievers were most frequent breeds, and localization was mostly on legs. Grade I tumor was found in 15.09%, grade II in 44.34% and grade III in 28.3% of animals. There were no significant correlations between tumor grade and age, breed, sex or localization. Considering the lesion scores compared with grade, statistically significant differences were found in cell shape, number of nucleoli, anisocytosis, anisokariosis, karyomegaly, mitoses, necroses, hemorrhages, cellularity, cell borders and collagenolysis

Autologous blood coagulum containing rhBMP6 induces new bone formation to promote anterior lumbar interbody fusion (ALIF) and posterolateral lumbar fusion (PLF) of spine in sheep

resent 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 denuded 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 g 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) serves as a preferred physiological native carrier to induce new bone at low doses of rhBMP6 and to achieve a successful spinal fusion

Koštani morfogenetski proteini (BMP): Od otkrića do razvoja nove autologne koštane naprave koja se sastoji od rekombinantnog humanog BMP6 u autolognom krvnom ugrušku kao nosaču

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.Koštani morfogenetski proteini (BMP) čine grupu čimbenika rasta i diferencijacije unutar TGFβ nado- bitelji. Oni induciraju stvaranje ektopične i ortotopične endohondralne kosti te su uključeni u regulaciju stanične proliferacije, diferencijacije, apoptoze i mezenhimalno-epitelne interakcije u važnim tkivnim morfogenetskim procesima izvan koštanog sustava. Koštane naprave koje sadrže BMP2 i BMP7 pro- tein odobrene su za poboljšanje koštanog cijeljenja kod pacijenata s defektima dugih cjevastih kostiju i kod prednje spinalne fuzije kralježnice. Međutim, zbog visoke cijene i mnogobrojnih nuspojava koje su uključivale pojavu heterotopičnih osifikacija, retrogradnu ejakulaciju i bol, njihova je klinička prim- jena ograničena. U ovom smo preglednom radu raspravili otkriće BMP molekula, njihovu biologiju i primjenu u kliničkim studijama s posebnim osvrtom na nedavno otkrivenu novu autolognu koštanu napravu (ABGS) koja sadrži BMP6. Novi ABGS sastoji se od nosača autolognog koaguluma (ABC) s otopljenim BMP6 koji je ključan za pokretanje diferencijacije mezenhimalnih stanica u smjeru stvaranja endohondralne kosti. ABC je ispunio sve potrebne uvjete za formulaciju optimalnog nosača za BMP6 isključivo zbog jednostavnosti priprave i primjene te odsustva imunogenog i upalnog odgovora na mjestu implantacije. Uz dodatak alografta ili sintetičke keramike što je potvrđeno na životinjskim modelima došlo je do značajnog povećanja volumena te poboljšanja mikroarhitekture novonastale kosti. Prvo kliničko ispitivanje provedeno je na pacijentima s distalnim prijelomima radijusa (faza I studije), a drugo na pacijentima koji su podvrgnuti visokoj osteotomiji tibije (faza I/II studije) bez uočenih ozbiljnih nuspojava. Trenutno je u tijeku studija OSTEOproSPINE u kojoj se testira učinkovitost ABGS u kom- binaciji s koštanim alograftom u bolesnika s kroničnim bolovima u leđima uzrokovanim degenerativnim promjenama intervertebralnog diska. Nova ABGS koštana naprava značajna je prekretnica i napredak u području koštane biologije te regenerativne medicine koštanog sustava

3R Principle for the welfare protection of animals used for scientific purposes - yesterday, today, tomorrow

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

Mast Cell Tumor in Dogs – Incidence and Histopathological Characterization

Incidence of mast cell tumors, their distribution according to sex, breed, age and localisation in Croatia is not established yet. Also, the statistical significance of the various histopathological parameters according to Patnaik’s scheme, in the diagnostics of the tumor grade was not performed. Investigation analysed mast cell tumors histopathologicaly characterized at the Department of General Pathology and Pathological Morphology of the Veterinary Faculty Zagreb from January 1st 2002 to Dezember 31st 2006. Sex, age, breed, localisation and tumor grade of each animal with tumor was recorded and statisticaly evaluated. Each histopathological variable was scored and compared with tumor grade. In the analyzed period, totally 1630 tumors were recorded, and mast cell tumors were found in 106 animals or in 6.5% of all cases. With statistically significant difference, this tumor was found in more cases in male dogs, and average age was 6.96 years. Boxers and retrievers were most frequent breeds, and localization was mostly on legs. Grade I tumor was found in 15.09%, grade II in 44.34% and grade III in 28.3% of animals. There were no significant correlations between tumor grade and age, breed, sex or localization. Considering the lesion scores compared with grade, statistically significant differences were found in cell shape, number of nucleoli, anisocytosis, anisokariosis, karyomegaly, mitoses, necroses, hemorrhages, cellularity, cell borders and collagenolysis

Evaluation of synthetic ceramics as compression resistant matrix to promote osteogenesis of autologous blood coagulum containing recombinant human bone morphogenetic protein 6 in rabbit posterolateral lumbar fusion model

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