Preparation of natural eutectic solvents and their application in cryoprotection of cell lines

Prirodna eutektična otapala (eng. Natural Deep Eutectic Solvents, NADES) su nova generacija ekološki prihvatljivih otapala sa mogućim primjenama u različitim industrijskim područjima. Definiraju se kao smjesa dviju ili više komponenata koje se međusobno mogu povezati vodikovim vezama te koje u određenom omjeru imaju niže talište nego pojedinačne komponente smjese. S obzirom na njihovu široku primjenu, u ovom radu ispitana je mogućnost njihove primjene kao krioprotektanata na staničnoj liniji HEK 293T. U ovom radu pripravljena su dva različita NADES-a u različitim molarnim odnosima (kolin klorid:glukoza, prolin:glukoza) te s različitim udjelima vode. Učinkovitost ovih NADES-a kao krioprotektanata uspoređena je sa standardnim krioprotektantima, glicerolom i dimetil sulfoksidom. Istraživanje je pokazalo da testirani spojevi nisu dostojna zamjena standardnim kriprotektantima.Natural deep eutectic solvents (NADES) are new generation of environmentally friendly solvents with potential application in various industrial fields. They are defined as a mixture of two or more components connected to each other by hydrogen bonds. At a certain ratio of their building components, NADES can have lower melting points than the individual components of the mixture. Considering their widespread application, NADES are tested as cryoprotectants on cell line HEK 293T. Two different NADES are made for this experiment; they are consisted of proline/glucose and choline chloride/glucose in different molar ratio and with different percentage of water content. Efficiency of those NADES are compared with standard cryoprotectants, glycerol and dimethyl sulfoxide. The results showed that NADES cannot serve as an appropriate alternative to the standard cryoprotectants

A simple rodent subcutaneous assay for identification of new osteoinductive molecules: The key method for screening of novel bone regeneration implants

Treatment of large bone defects and degenerative diseases of the spine is among the most challeng- ing and still unresolved issues in clinical medicine. Therefore, substantial effort has been devoted to the development of novel bone regenerative therapies. Due to their potent osteoinductive properties, Bone Morphogenetic Proteins (BMPs) have been the basis for the development of novel strategies for bone regeneration. The use of animal models is an indispensable part of the preclinical testing of novel therapeutic solutions. The rat subcutaneous assay became the initial screening procedure for the evalu- ation of promising BMP-based osteoinductive devices for bone regeneration because only osteogenic BMPs can induce new bone at any ectopic rodent site. Moreover, this model is used for research on the mechanisms of ectopic bone formation as well as for the evaluation of the inflammatory response to different materials. In this review, we provided an overview of the assay development and previously conducted studies with different methods (flow cytometry, histological and microCT analyses) for the study outcome evaluation. Moreover, we addressed essential issues in the experimental design such as the follow-up period and the sample size. The rat subcutaneous bone induction assay layed the founda- tion for isolation and identification of BMPs followed by testing of new osteogenic devices in higher animal species and humans

State-of-the-art of the Bone Morphogenetic Protein research field: 13th International BMP Conference, Dubrovnik 2022

The 13th International BMP Conference was held in October 2022 in Dubrovnik. The conference was attended by more than 240 participants from North America, Europe, Asia, and Australia who got an insight into the latest achievements in basic, translational, and clinical research of BMP mol- ecules through 75 lectures categorized into several scientific sections. This review paper provides the most important novel findings on the structure, function, and signaling of BMPs, the role of BMPs in patterning and organoids as well as the role of BMP in metabolism. Moreover, we discussed the role of BMPs in various diseases including cancer pathogenesis, pulmonary arterial hyperten- sion, and fibrodysplasia ossificans progressiva (FOP). Finally, we provided an overview of the new BMP-based therapies in regenerative medicine that are currently in different stages of preclinical and clinical trials

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

Comparison of the dynamics of ectopic bone formation using bone morphogenetic protein 2 on a collagen sponge carrier and bone morphogenetic protein 6 in an autologous blood coagulum

U cilju razvoja boljih terapeutskih rješenja za regeneraciju kostiju, razvijen je novi osteoinduktivni sustav koji se sastoji od rhBMP6 i nosača u formi autolognog krvnog ugruška, koji je novo terapeutsko rješenje za regeneraciju kosti koje se trenutno testira u kliničkim ispitivanjima. U sklopu ovog rada istražena je biologija i vremenski tijek ektopičnog stvaranja koštanog tkiva pod utjecajem rhBMP6 u autolognom krvnom ugrušku (ABC) kao nosaču koji je uspoređen sa zlatnim standardom za cijeljenje kostiju, rhBMP2 na nosaču kolagenske spužvice (ACS), pri čemu su oba osteoinduktivna sustava ispitana na modelu potkožne ugradnje implantata u aksilarnu regiju štakora. Također, u seriji in vitro pokusa dinamike otpuštanja pokazano je kako apsorbirajuća kolagenska spužvica nema zadovoljavajući profil otpuštanja. U cilju ispitivanja staničnih populacija koje sudjeluju u ranoj osteogenezi (24h i 72h nakon potkožne ugradnje) koja je inducirana spomenutim osteoinduktivnim sustavima analiziran je udio limfoidnih, mijeloidnih i mezenhimalnih staničnih populacija. Adipoprogenitori su u najvećem broju bili prisutni u rhBMP2/ACS implantatima, dok su matične stanice u najvećem broju prisutne u rhBMP6/ABC implantatima. Nadalje, superiornost rhBMP6/ABC u odnosu na rhBMP2/ACS dokazana je u eseju potkožne ugradnje, gdje je u svim promatranim vremenskim točkama najveću količinu novostvorene kosti inducirao rhBMP6/ABC u dozi od 20 μg. Primijećeno je da se kolagenska spužvica kao nosač na ektopičnom mjestu sporije razgrađuje u odnosu na autologni krvni ugrušak. Posljedično, proces osifikacije je odgođen prilikom primjene rhBMP2/ACS zbog guste kolagenske mreže koja usporava ulazak progenitornih stanica, pa je samim time proces osifikacije odgođen u odnosu na krvni ugrušak. U cilju ispitivanja efikasnosti korištenih rekombinantnih proteina, rhBMP2 i rhBMP6, njihov osteoinduktivni potencijal ispitan je na jednakom nosaču koji se sastoji od autolognog krvnog ugruška i bifazične keramike u rangu veličine čestica od 500 do 1700 μm u tri različite doze (5, 20, 50 μg), pri čemu se rhBMP6 pokazao superiornijim u odnosu na rhBMP2 u dozi od 50 μg. Potencijalni razlog tomu je već dokazana superiornost rhBMP6 na inhibiciju nogginom. Osim toga, dodatkom biokompatibilne keramike u svojstvu matriksa otpornog na kompresiju poboljšavaju se biomehanička svojstva implantata, te je proces osifikacije osteoinduktivnih implantata s dodatkom keramičkih čestica drugačiji u odnosu na krvni ugrušak sam.In order to develop a better therapeutic solutions for bone regeneration, a new osteoinductive device has been developed consisting of rhBMP6 and autologous blood coagulum as a carrier, which is currently being tested in clinical trials as a new therapeutic solution for bone repair and regeneration. Part of this research was focused on the investigation of the biology and time course of ectopic bone formation induced by rhBMP6 in autologous blood coagulum (ABC) and compared with the gold standard for bone healing, rhBMP2 on absorbable collagen sponge (ACS), where both osteoinductive devices were evaluated in the rat subcutaneous assay. Also, in a series of in vitro experiments release dynamics of both osteoinductive devices was tested showing that absorbable collagen sponge does not have a satisfactory release profile. In order to examine the cell populations involved in early osteogenesis (24h and 72h after subcutaneous implantation) induced by the aforementioned osteoinductive devices, the number of lymphoid, myeloid and mesenchymal cell populations were analyzed. Adipoprogenitors were most abundant in rhBMP2/ACS implants, while mesenchimal stem cells were predominately present in rhBMP6/ABC implants. Furthermore, the superiority of rhBMP6/ABC over rhBMP2/ACS was demonstrated in a rat subcutaneous assay, where at all observed time points, the largest amount of newly formed bone was induced by rhBMP6 / ABC at a dose of 20 μg. It has been observed that the absorbable collagen sponge as a carrier at the ectopic site degrades more slowly compared to the autologous blood coagulum. Consequently, the ossification process is delayed when rhBMP2/ACS is used due to the dense collagen meshwork that slows down the entry of progenitor cells, and thus the ossification process is delayed in comparison to the blood coagulum. In order to test the efficacy of the recombinant proteins used, rhBMP2 and rhBMP6, their osteoinductive potential was tested on an equal scaffold consisting of autologous blood coagulum and biphasic ceramics in the particle size range of 500 to 1700 μm in three different doses (5, 20, 50 μg), where rhBMP6 was superior to rhBMP2 at a dose of 50 μg. A potential reason for this is already proven resistance of rhBMP6 to noggin inhibition. Also, the addition of biocompatible ceramics as a compression-resistant matrix improves the biomechanical properties of implants, and the process of ossification of osteoinductive implants with the addition of ceramic particles is different compared to the blood coagulum itself

Preparation of natural eutectic solvents and their application in cryoprotection of cell lines

Prirodna eutektična otapala (eng. Natural Deep Eutectic Solvents, NADES) su nova generacija ekološki prihvatljivih otapala sa mogućim primjenama u različitim industrijskim područjima. Definiraju se kao smjesa dviju ili više komponenata koje se međusobno mogu povezati vodikovim vezama te koje u određenom omjeru imaju niže talište nego pojedinačne komponente smjese. S obzirom na njihovu široku primjenu, u ovom radu ispitana je mogućnost njihove primjene kao krioprotektanata na staničnoj liniji HEK 293T. U ovom radu pripravljena su dva različita NADES-a u različitim molarnim odnosima (kolin klorid:glukoza, prolin:glukoza) te s različitim udjelima vode. Učinkovitost ovih NADES-a kao krioprotektanata uspoređena je sa standardnim krioprotektantima, glicerolom i dimetil sulfoksidom. Istraživanje je pokazalo da testirani spojevi nisu dostojna zamjena standardnim kriprotektantima.Natural deep eutectic solvents (NADES) are new generation of environmentally friendly solvents with potential application in various industrial fields. They are defined as a mixture of two or more components connected to each other by hydrogen bonds. At a certain ratio of their building components, NADES can have lower melting points than the individual components of the mixture. Considering their widespread application, NADES are tested as cryoprotectants on cell line HEK 293T. Two different NADES are made for this experiment; they are consisted of proline/glucose and choline chloride/glucose in different molar ratio and with different percentage of water content. Efficiency of those NADES are compared with standard cryoprotectants, glycerol and dimethyl sulfoxide. The results showed that NADES cannot serve as an appropriate alternative to the standard cryoprotectants

Comparison of the dynamics of ectopic bone formation using bone morphogenetic protein 2 on a collagen sponge carrier and bone morphogenetic protein 6 in an autologous blood coagulum

U cilju razvoja boljih terapeutskih rješenja za regeneraciju kostiju, razvijen je novi osteoinduktivni sustav koji se sastoji od rhBMP6 i nosača u formi autolognog krvnog ugruška, koji je novo terapeutsko rješenje za regeneraciju kosti koje se trenutno testira u kliničkim ispitivanjima. U sklopu ovog rada istražena je biologija i vremenski tijek ektopičnog stvaranja koštanog tkiva pod utjecajem rhBMP6 u autolognom krvnom ugrušku (ABC) kao nosaču koji je uspoređen sa zlatnim standardom za cijeljenje kostiju, rhBMP2 na nosaču kolagenske spužvice (ACS), pri čemu su oba osteoinduktivna sustava ispitana na modelu potkožne ugradnje implantata u aksilarnu regiju štakora. Također, u seriji in vitro pokusa dinamike otpuštanja pokazano je kako apsorbirajuća kolagenska spužvica nema zadovoljavajući profil otpuštanja. U cilju ispitivanja staničnih populacija koje sudjeluju u ranoj osteogenezi (24h i 72h nakon potkožne ugradnje) koja je inducirana spomenutim osteoinduktivnim sustavima analiziran je udio limfoidnih, mijeloidnih i mezenhimalnih staničnih populacija. Adipoprogenitori su u najvećem broju bili prisutni u rhBMP2/ACS implantatima, dok su matične stanice u najvećem broju prisutne u rhBMP6/ABC implantatima. Nadalje, superiornost rhBMP6/ABC u odnosu na rhBMP2/ACS dokazana je u eseju potkožne ugradnje, gdje je u svim promatranim vremenskim točkama najveću količinu novostvorene kosti inducirao rhBMP6/ABC u dozi od 20 μg. Primijećeno je da se kolagenska spužvica kao nosač na ektopičnom mjestu sporije razgrađuje u odnosu na autologni krvni ugrušak. Posljedično, proces osifikacije je odgođen prilikom primjene rhBMP2/ACS zbog guste kolagenske mreže koja usporava ulazak progenitornih stanica, pa je samim time proces osifikacije odgođen u odnosu na krvni ugrušak. U cilju ispitivanja efikasnosti korištenih rekombinantnih proteina, rhBMP2 i rhBMP6, njihov osteoinduktivni potencijal ispitan je na jednakom nosaču koji se sastoji od autolognog krvnog ugruška i bifazične keramike u rangu veličine čestica od 500 do 1700 μm u tri različite doze (5, 20, 50 μg), pri čemu se rhBMP6 pokazao superiornijim u odnosu na rhBMP2 u dozi od 50 μg. Potencijalni razlog tomu je već dokazana superiornost rhBMP6 na inhibiciju nogginom. Osim toga, dodatkom biokompatibilne keramike u svojstvu matriksa otpornog na kompresiju poboljšavaju se biomehanička svojstva implantata, te je proces osifikacije osteoinduktivnih implantata s dodatkom keramičkih čestica drugačiji u odnosu na krvni ugrušak sam.In order to develop a better therapeutic solutions for bone regeneration, a new osteoinductive device has been developed consisting of rhBMP6 and autologous blood coagulum as a carrier, which is currently being tested in clinical trials as a new therapeutic solution for bone repair and regeneration. Part of this research was focused on the investigation of the biology and time course of ectopic bone formation induced by rhBMP6 in autologous blood coagulum (ABC) and compared with the gold standard for bone healing, rhBMP2 on absorbable collagen sponge (ACS), where both osteoinductive devices were evaluated in the rat subcutaneous assay. Also, in a series of in vitro experiments release dynamics of both osteoinductive devices was tested showing that absorbable collagen sponge does not have a satisfactory release profile. In order to examine the cell populations involved in early osteogenesis (24h and 72h after subcutaneous implantation) induced by the aforementioned osteoinductive devices, the number of lymphoid, myeloid and mesenchymal cell populations were analyzed. Adipoprogenitors were most abundant in rhBMP2/ACS implants, while mesenchimal stem cells were predominately present in rhBMP6/ABC implants. Furthermore, the superiority of rhBMP6/ABC over rhBMP2/ACS was demonstrated in a rat subcutaneous assay, where at all observed time points, the largest amount of newly formed bone was induced by rhBMP6 / ABC at a dose of 20 μg. It has been observed that the absorbable collagen sponge as a carrier at the ectopic site degrades more slowly compared to the autologous blood coagulum. Consequently, the ossification process is delayed when rhBMP2/ACS is used due to the dense collagen meshwork that slows down the entry of progenitor cells, and thus the ossification process is delayed in comparison to the blood coagulum. In order to test the efficacy of the recombinant proteins used, rhBMP2 and rhBMP6, their osteoinductive potential was tested on an equal scaffold consisting of autologous blood coagulum and biphasic ceramics in the particle size range of 500 to 1700 μm in three different doses (5, 20, 50 μg), where rhBMP6 was superior to rhBMP2 at a dose of 50 μg. A potential reason for this is already proven resistance of rhBMP6 to noggin inhibition. Also, the addition of biocompatible ceramics as a compression-resistant matrix improves the biomechanical properties of implants, and the process of ossification of osteoinductive implants with the addition of ceramic particles is different compared to the blood coagulum itself

Comparison of the dynamics of ectopic bone formation using bone morphogenetic protein 2 on a collagen sponge carrier and bone morphogenetic protein 6 in an autologous blood coagulum

U cilju razvoja boljih terapeutskih rješenja za regeneraciju kostiju, razvijen je novi osteoinduktivni sustav koji se sastoji od rhBMP6 i nosača u formi autolognog krvnog ugruška, koji je novo terapeutsko rješenje za regeneraciju kosti koje se trenutno testira u kliničkim ispitivanjima. U sklopu ovog rada istražena je biologija i vremenski tijek ektopičnog stvaranja koštanog tkiva pod utjecajem rhBMP6 u autolognom krvnom ugrušku (ABC) kao nosaču koji je uspoređen sa zlatnim standardom za cijeljenje kostiju, rhBMP2 na nosaču kolagenske spužvice (ACS), pri čemu su oba osteoinduktivna sustava ispitana na modelu potkožne ugradnje implantata u aksilarnu regiju štakora. Također, u seriji in vitro pokusa dinamike otpuštanja pokazano je kako apsorbirajuća kolagenska spužvica nema zadovoljavajući profil otpuštanja. U cilju ispitivanja staničnih populacija koje sudjeluju u ranoj osteogenezi (24h i 72h nakon potkožne ugradnje) koja je inducirana spomenutim osteoinduktivnim sustavima analiziran je udio limfoidnih, mijeloidnih i mezenhimalnih staničnih populacija. Adipoprogenitori su u najvećem broju bili prisutni u rhBMP2/ACS implantatima, dok su matične stanice u najvećem broju prisutne u rhBMP6/ABC implantatima. Nadalje, superiornost rhBMP6/ABC u odnosu na rhBMP2/ACS dokazana je u eseju potkožne ugradnje, gdje je u svim promatranim vremenskim točkama najveću količinu novostvorene kosti inducirao rhBMP6/ABC u dozi od 20 μg. Primijećeno je da se kolagenska spužvica kao nosač na ektopičnom mjestu sporije razgrađuje u odnosu na autologni krvni ugrušak. Posljedično, proces osifikacije je odgođen prilikom primjene rhBMP2/ACS zbog guste kolagenske mreže koja usporava ulazak progenitornih stanica, pa je samim time proces osifikacije odgođen u odnosu na krvni ugrušak. U cilju ispitivanja efikasnosti korištenih rekombinantnih proteina, rhBMP2 i rhBMP6, njihov osteoinduktivni potencijal ispitan je na jednakom nosaču koji se sastoji od autolognog krvnog ugruška i bifazične keramike u rangu veličine čestica od 500 do 1700 μm u tri različite doze (5, 20, 50 μg), pri čemu se rhBMP6 pokazao superiornijim u odnosu na rhBMP2 u dozi od 50 μg. Potencijalni razlog tomu je već dokazana superiornost rhBMP6 na inhibiciju nogginom. Osim toga, dodatkom biokompatibilne keramike u svojstvu matriksa otpornog na kompresiju poboljšavaju se biomehanička svojstva implantata, te je proces osifikacije osteoinduktivnih implantata s dodatkom keramičkih čestica drugačiji u odnosu na krvni ugrušak sam.In order to develop a better therapeutic solutions for bone regeneration, a new osteoinductive device has been developed consisting of rhBMP6 and autologous blood coagulum as a carrier, which is currently being tested in clinical trials as a new therapeutic solution for bone repair and regeneration. Part of this research was focused on the investigation of the biology and time course of ectopic bone formation induced by rhBMP6 in autologous blood coagulum (ABC) and compared with the gold standard for bone healing, rhBMP2 on absorbable collagen sponge (ACS), where both osteoinductive devices were evaluated in the rat subcutaneous assay. Also, in a series of in vitro experiments release dynamics of both osteoinductive devices was tested showing that absorbable collagen sponge does not have a satisfactory release profile. In order to examine the cell populations involved in early osteogenesis (24h and 72h after subcutaneous implantation) induced by the aforementioned osteoinductive devices, the number of lymphoid, myeloid and mesenchymal cell populations were analyzed. Adipoprogenitors were most abundant in rhBMP2/ACS implants, while mesenchimal stem cells were predominately present in rhBMP6/ABC implants. Furthermore, the superiority of rhBMP6/ABC over rhBMP2/ACS was demonstrated in a rat subcutaneous assay, where at all observed time points, the largest amount of newly formed bone was induced by rhBMP6 / ABC at a dose of 20 μg. It has been observed that the absorbable collagen sponge as a carrier at the ectopic site degrades more slowly compared to the autologous blood coagulum. Consequently, the ossification process is delayed when rhBMP2/ACS is used due to the dense collagen meshwork that slows down the entry of progenitor cells, and thus the ossification process is delayed in comparison to the blood coagulum. In order to test the efficacy of the recombinant proteins used, rhBMP2 and rhBMP6, their osteoinductive potential was tested on an equal scaffold consisting of autologous blood coagulum and biphasic ceramics in the particle size range of 500 to 1700 μm in three different doses (5, 20, 50 μg), where rhBMP6 was superior to rhBMP2 at a dose of 50 μg. A potential reason for this is already proven resistance of rhBMP6 to noggin inhibition. Also, the addition of biocompatible ceramics as a compression-resistant matrix improves the biomechanical properties of implants, and the process of ossification of osteoinductive implants with the addition of ceramic particles is different compared to the blood coagulum itself

Application of deep eutectic solvents in extraction of phenolic compounds from grape skin

Razvoj zelenih otapala je važan zadatak za industriju, pa se stoga se u posljednje vrijeme intenzivno istražuju eutektična otapala zbog njihovih poželjnih karakteristika kao što su niska toksičnost i biorazgradivost. Eutektična otapala zbog mogućnost prilagodbe njihovih svojstava odabirom početnih sirovina, privukla su pozornost u sintetskoj kemiji i biokatalizi, separacijskim postupcima ali i kao medij za ekstrakciju prirodnih spojeva. Prilikom ekstrakcije fenolnih spojeva iz pokožice grožđa, od pet testiranih eutektičnih otapala, najučinkovitije se pokazalo ono sastavljeno od kolin klorida i oksalne kiseline s udjelom vode od 25 %. Navedeno eutektično otapalo pokazalo se učinkovitije i od klasične metode primjenom zakiseljenog metanola. Rezultati istraživanja utvrdili su mogućnost zamjene klasičnih organskih otapala eutektičnim otapalima kod ekstrakcije antocijana iz pokožice grožđa.Developing new green solvents is one of the most important tasks for industry. Lately, deep eutectic solvents have been researched due to their desirable characteristics like low toxcity and biodegrability. Deep Eutectic Solevents (DES), because of their adjustability, are widely used in synthetic chemistry and biocathalisis but also as extraction media for phenolic compounds. Among tested, DES made of choline chloride and oxal acid (with 25% of water in their content) showed the best performance in extraction of phenolic compounds from grape skin. It also showed better performance in comparison to conventional solvents. Results of this experiment showed that conventional solvents for extraction of phenolic compounds can be replaced with DESs

Application of deep eutectic solvents in extraction of phenolic compounds from grape skin

Razvoj zelenih otapala je važan zadatak za industriju, pa se stoga se u posljednje vrijeme intenzivno istražuju eutektična otapala zbog njihovih poželjnih karakteristika kao što su niska toksičnost i biorazgradivost. Eutektična otapala zbog mogućnost prilagodbe njihovih svojstava odabirom početnih sirovina, privukla su pozornost u sintetskoj kemiji i biokatalizi, separacijskim postupcima ali i kao medij za ekstrakciju prirodnih spojeva. Prilikom ekstrakcije fenolnih spojeva iz pokožice grožđa, od pet testiranih eutektičnih otapala, najučinkovitije se pokazalo ono sastavljeno od kolin klorida i oksalne kiseline s udjelom vode od 25 %. Navedeno eutektično otapalo pokazalo se učinkovitije i od klasične metode primjenom zakiseljenog metanola. Rezultati istraživanja utvrdili su mogućnost zamjene klasičnih organskih otapala eutektičnim otapalima kod ekstrakcije antocijana iz pokožice grožđa.Developing new green solvents is one of the most important tasks for industry. Lately, deep eutectic solvents have been researched due to their desirable characteristics like low toxcity and biodegrability. Deep Eutectic Solevents (DES), because of their adjustability, are widely used in synthetic chemistry and biocathalisis but also as extraction media for phenolic compounds. Among tested, DES made of choline chloride and oxal acid (with 25% of water in their content) showed the best performance in extraction of phenolic compounds from grape skin. It also showed better performance in comparison to conventional solvents. Results of this experiment showed that conventional solvents for extraction of phenolic compounds can be replaced with DESs