BR102019011781A2

BR102019011781A2 - POLYMERIC HYBRID BIOCOMPOSITES OF POROUS AND AMORPLE HYDROXIAPATITE, FIBROUS COLLAGEN (TYPE I) EXTRACTED FROM BIOLOGICAL SOURCES, PREPARATION AND USES IN DENTAL APPLICATIONS

Info

Publication number: BR102019011781A2
Application number: BR102019011781-8A
Authority: BR
Inventors: Jose Jovanny Bermudez Sierra; Felipe Domingos de Sousa; Renato De Azevedo Moreira; Ana Cristina De Monteiro Moreira
Original assignee: Fundação Edson Queiroz
Priority date: 2019-06-11
Filing date: 2019-06-11
Publication date: 2020-12-15

Abstract

biocompósitos hibridos poliméricos de hidroxiapatita porosa e amorfa, colágeno fibroso (tipo i) extraído de fontes biológicas, preparação e usos em aplicações odontológicas a presente invenção refere-se ao desenvolvimento de um biocompósito hibrido-polimérico (hemiceluloses de origem vegetais) de hap/col, desenhado biomimeticamente a partir das escamas de tilápia do nilo (oreochromis sp), e descreve o processo de extração, purificação e tratamento dos biomateriais como a hidroxiapatita (hap) e colágeno (col), extraídos por hidrólises química e por precipitação seletiva (salting-out), o desenho de um biocompósito polimérico de tipo hemiceluloses de origem vegetais (galactomanana) associado com hap (nano-amorfa, micro e macro-porosa) e colágeno fibroso como biomateriais biocompatíveis, osteoindutores e osteocondutores com potencial aplicação na engenharia tecidual na regeneração óssea e tecidual guiada (rog e rtg), especialmente utilizando enxertos ósseos xenógenos tipo biocompósito poroso nos tratamentos de odontologia reconstrutiva.polymeric hybrid biocomposites of porous and amorphous hydroxyapatite, fibrous collagen (type i) extracted from biological sources, preparation and uses in dental applications the present invention relates to the development of a hybrid / polymeric biocomposite (plant-derived hemicelluloses) of hap / col , drawn biomimetically from the scales of Nile tilapia (oreochromis sp), and describes the process of extraction, purification and treatment of biomaterials such as hydroxyapatite (hap) and collagen (col), extracted by chemical hydrolysis and by selective precipitation (salting) -out), the design of a polymeric biocomposite of hemicellulose type of vegetable origin (galactomannan) associated with hap (nano-amorphous, micro and macro-porous) and fibrous collagen as biocompatible, osteoinductive and osteoconductive biomaterials with potential application in tissue engineering in guided bone and tissue regeneration (rog and rtg), especially using pore biocomposite xenogenous bone grafts only in reconstructive dentistry treatments.

Description

POLYMERIC HYBRID BIOCOMPOSITES OF POROUS AND AMORPLE HYDROXIAPATITE, FIBROUS COLLAGEN (TYPE I) EXTRACTED FROM BIOLOGICAL SOURCES, PREPARATION AND USES IN DENTAL APPLICATIONS

[01] O presente pedido de invenção descreve um processo de extração, purificação e tratamento de biomateriais como a hidroxiapatita (Hap) e colágeno (Col), extraídos por hidrólises química e por precipitação seletiva (Salting-out), respectivamente, a partir da escama de Tilápia do Nilo (Oreochromis Sp.). O desenho de um biocompósito hibrido-polimérico de tipo hemiceluloses de origem vegetais (galactomanana) associado com Hap (amorfa, micro e macro porosa) e colágeno fibroso (Tipo I) como biomateriais biocompatíveis, osteoindutores e osteocondutores com potencial aplicação na engenharia tecidual e regeneração óssea e tecidual guiada (ROG e RTG), especialmente utilizando enxerto ósseos xenógenos de tipo biocompósito porosos nos tratamentos de odontologia reconstrutiva.[01] The present invention application describes a process of extraction, purification and treatment of biomaterials such as hydroxyapatite (Hap) and collagen (Col), extracted by chemical hydrolysis and by selective precipitation (Salting-out), respectively, from the scale of Nile Tilapia (Oreochromis Sp.). The design of a hybrid-polymeric biocomposite of plant-type hemicelluloses (galactomannan) associated with Hap (amorphous, micro and macro porous) and fibrous collagen (Type I) as biocompatible, osteoinductive and osteoconductive biomaterials with potential application in tissue engineering and regeneration guided bone and tissue (ROG and RTG), especially using porous biocomposite xenogenous bone grafts in reconstructive dentistry treatments.

BACKGROUND OF THE INVENTION

[02] A reconstrução dos defeitos ósseos alveolares graves representa um grande desafio para os profissionais da odontologia na atualidade. Essas deformidades são comuns em pacientes que sofrem a perda de dentes, doença periodontal avançada ou malformações congênitas.[02] The reconstruction of severe alveolar bone defects represents a major challenge for dental professionals today. These deformities are common in patients suffering from tooth loss, advanced periodontal disease or congenital malformations.

[03] Portanto, os anteriores biocompósitos híbridos, formam uma matriz biomimética porosa, com propriedades osteoindutoras, osteocondutoras e osteogênicas, potencialmente replicando a funcionalidade de um autoenxerto. A matriz osteocondutora torna-se um sistema disponível para agentes bioativos, determinando menor quimiotaxia e migração de células progenitoras de osteoblastos para o local do enxerto. A inoculação direta de cultivos celulares pode levar a uma recuperação óssea mais rápida e consistente.[03] Therefore, the previous hybrid biocomposites, form a porous biomimetic matrix, with osteoinductive, osteoconductive and osteogenic properties, potentially replicating the functionality of an autograft. The osteoconductive matrix becomes a system available for bioactive agents, causing less chemotaxis and migration of osteoblast progenitor cells to the graft site. Direct inoculation of cell cultures can lead to faster and more consistent bone recovery.

[04] Alguns pesquisadores afirmam que a utilização desta biomassa residual, a partir de subprodutos de gado e suínos na extração de Hap e colágeno para a elaboração de biocompósitos (enxerto ósseo xenógeno), apresentam alguns riscos, já que estão frequentemente associados com alergias e zoonoses (encefalopatia espongiforme bovina, fevre aftosa, influenza aviar e porcina) e a proibição de sua utilização em produtos farmacêuticos, alimentares e cosméticos em algumas culturas religiosas provenientes do meio oriente.[04] Some researchers claim that the use of this residual biomass, from by-products of cattle and pigs in the extraction of Hap and collagen for the elaboration of biocomposites (xenogenous bone graft), presents some risks, since they are frequently associated with allergies and zoonoses (bovine spongiform encephalopathy, foot-and-mouth disease, avian and porcine influenza) and the ban on its use in pharmaceutical, food and cosmetic products in some religious cultures from the Middle East.

[05] Fazendo o levantamento do estado a técnica, com duas patentes que possuem algumas características em comum, mas são diferentes no processo e na fonte do material ou biomassa, são elas:[05] Making the status survey the technique, with two patents that have some characteristics in common, but are different in the process and in the source of the material or biomass, they are:

[06] A patente PI 1012502-7 PROCESSO DE OBTENÇÃO DE GEL, COMPLEXOS COM HIDROXIAPATITA E/OU CERÃMICA ÓSSEA, FILMES, ESPONJAS E MICROCRISTAIS RICOS EM FIBRAS DE COLÁGENO TIPO I PURO. A presente invenção trata-se de um processo de descelularização de uma matriz biológica para a obtenção de produtos ricos em fibras de colágeno tipo I puro, dentre eles um gel de diferentes viscosidades empregado em terapia de lesões dérmicas. Os outros produtos são derivados deste gel rico em fibras de colágeno tipo I puro, que poderá ser complexado com cerâmica óssea (CO) e/ou Hap, gerando complexos que podem ser utilizados em implantes ósseos, ou então ser tratadas para a obtenção de filmes, esponjas e microcristais de colágeno, com amplas aplicações terapêuticas.[06] The patent PI 1012502-7 PROCESS OF OBTAINING GEL, COMPLEXES WITH HYDROXIAPATITE AND / OR BONE CERAMIC, FILMS, SPONGES AND MICRO CRYSTALS RICH IN PURE COLLAGEN FIBERS TYPE I. The present invention is a process of decellularization of a biological matrix to obtain products rich in pure type I collagen fibers, among them a gel of different viscosities used in dermal injury therapy. The other products are derived from this gel rich in pure type I collagen fibers, which can be complexed with bone ceramics (CO) and / or Hap, generating complexes that can be used in bone implants, or be treated to obtain films , sponges and collagen microcrystals, with wide therapeutic applications.

[07] “E a patente BR 11 2016 001093 0 MATERIAL COMPÓSITO DE HIDROXIAPATITA-COLÁGENO BIOMIMÉTICO”. A invenção refere-se a: - um material compósito de hidroxiapatita-colágeno biomimético compreendendo um andaime de colágeno pelo menos parcialmente fibroso incluindo fibras de colágeno nativa maduras que possuem helicidade tripla como mostrado por Espectroscopia de Dicroísmo circular, em que aquelas fibras de colágeno nativo maduro são pelo menos parcialmente cobertas com cristais crescidos de forma epitáctica de hidroxiapatita nanocristalina, pelo que os nanocristais crescidos de forma epitáctica têm a mesma morfologia que mineral de osso humano e o mesmo tamanho que mineral de osso humano, a saber, um comprimento de 30 a 50 nm e uma largura de 14 a 25 nm, - um processo de preparar o material compósito de hidroxiapatita-colágeno biomimético acima compreendendo as etapas de: a) imergir um andaime de colágeno pelo menos parcialmente fibroso incluindo fibras de colágeno nativo maduras acima em uma solução aquosa saturada de Ca2+ saturado/HxPO4(3-x), para começar o processo de formação do material de implante compósito pelo que nanocristais de hidroxiapatita crescidos de forma epitáctica serão formadas nas fibras de colágeno nativo maduras, os nanocristais de hidroxiapatita crescidos de forma epitáctica tendo a mesma morfologia e mesmo tamanho que o mineral de osso humano, b) parar o processo de formação do material de implante compósito por separar material sólido a partir da solução aquosa, enxaguar com água e secar, e c) opcionalmente esterilizar o material separado vindo da etapa b), bem como o uso do material compósito de hidroxiapatita-colágeno biomimético acima como um implante ou prótese para formação de osso, regeneração de osso, reparo de osso e/ou substituição de osso em um local de defeito em um sujeito humano ou em um animal, ou como um implante para regeneração combinação de osso e cartilagem.[07] “And the patent BR 11 2016 001093 0 BIOMIMETIC HYDROXIAPATITE-COLLAGEN COMPOSITE MATERIAL”. The invention relates to: - a biomimetic hydroxyapatite-collagen composite material comprising at least partially fibrous collagen scaffold including mature native collagen fibers that have triple helicality as shown by Circular Dichroism Spectroscopy, in which those native collagen fibers mature are at least partially covered with epitatically grown crystals of nanocrystalline hydroxyapatite, so nanocrystals grown epitatically have the same morphology as human bone mineral and the same size as human bone mineral, namely a length of 30 at 50 nm and a width of 14 to 25 nm, - a process of preparing the above biomimetic hydroxyapatite-collagen composite material comprising the steps of: a) immersing a collagen scaffolding at least partially fibrous including mature native collagen fibers above a saturated aqueous solution of Ca2 + saturated / HxPO4 (3-x), to begin the process of formation of the composite implant material whereby epitatically grown hydroxyapatite nanocrystals will be formed on mature native collagen fibers, the epitactically grown hydroxyapatite nanocrystals having the same morphology and same size as the human bone mineral, b) stop the process of human formation of the composite implant material by separating solid material from the aqueous solution, rinsing with water and drying, and c) optionally sterilizing the separated material coming from step b), as well as the use of the above biomimetic hydroxyapatite-collagen composite material as a implant or prosthesis for bone formation, bone regeneration, bone repair and / or bone replacement at a defect site in a human subject or animal, or as an implant for combined bone and cartilage regeneration.

DESCRIPTION OF THE FIGURES

[08] Com o intuito de melhor definir e esclarecer o conteúdo do presente pedido de patente, as seguintes figuras são apresentadas:[08] In order to better define and clarify the content of this patent application, the following figures are presented:

[09] A figura 1 ilustra um diagrama geral das fazes de obtenção do material biológico.[09] Figure 1 illustrates a general diagram of the procedures for obtaining biological material.

[010] A figura 2 ilustra um diagrama de preparação do material biológico.[010] Figure 2 shows a diagram of the preparation of biological material.

[011] A figura 3 ilustra um diagrama de extração e purificação da Hap.[011] Figure 3 illustrates a diagram of Hap extraction and purification.

[012] A figura 4 ilustra um diagrama de extração e purificação do colágeno.[012] Figure 4 illustrates a diagram of collagen extraction and purification.

[013] A figura 5 ilustra um de diagrama sínteses da Hap micro e macroporosa (HapP).[013] Figure 5 illustrates a synthesis diagram of Hap micro and macroporous (HapP).

[014] A figura 6 ilustra um diagrama o desenho biocompósito polimérico HapP/Col.[014] Figure 6 illustrates a diagram of the HapP / Col polymeric biocomposite design.

[015] A figura 7 apresenta o espectro Infravermelho (FTIR) da Hap extraída da fonte biológica.[015] Figure 7 shows the Infrared spectrum (FTIR) of Hap extracted from the biological source.

[016] A figura 8 apresenta o espectro de Raios X (XRD) da Hap extraída da fonte biológica.[016] Figure 8 shows the X-ray (XRD) spectrum of Hap extracted from the biological source.

[017] A figura 9 apresenta o espectro de RAMAN da Hap extraída da fonte biológica.[017] Figure 9 shows the RAMAN spectrum of Hap extracted from the biological source.

[018] A figura 10 apresenta uma imagem de microscopia eletrônica de varredura (MEV) da Hap purificada, extraída da fonte biológica (10.000x).[018] Figure 10 presents a scanning electron microscopy (SEM) image of the purified Hap, extracted from the biological source (10,000x).

[019] A figura 11 apresenta uma imagem do espetro da analises por energia dispersiva de raios X (MEV-EDX) da amostra de Hap Purificada.[019] Figure 11 presents an image of the spectrum of X-ray dispersive energy analysis (SEM-EDX) of the Hap Purified sample.

[020] A figura 12 apresenta uma imagem MEV da HapP (micro e macroporosa), extraída da fonte biológica (50.000x).[020] Figure 12 presents an MEP image of HapP (micro and macroporous), extracted from the biological source (50,000x).

[021] A figura 13 apresenta uma imagem do espetro de analise por energia dispersiva de raios X (MEV-EDX) da amostra de HapP (micro-macro-porosa).[021] Figure 13 shows an image of the X-ray dispersive energy analysis (SEM-EDX) spectrum of the HapP sample (micro-macro-porous).

[022] A figura 14 apresenta o espectro infravermelho (FTIR) do Colágeno, extraída da fonte biológica.[022] Figure 14 shows the collagen infrared spectrum (FTIR), extracted from the biological source.

[023] A figura 15 apresenta o SDS-PAGE do colágeno, (padrão colágeno) extraída da fonte biológica.[023] Figure 15 shows the SDS-PAGE of collagen, (collagen pattern) extracted from the biological source.

[024] A figura 16 apresenta uma imagem MEV do Colágeno extraída da fonte biológica (1000x).[024] Figure 16 presents an SEM image of Collagen extracted from the biological source (1000x).

[025] A figura 17 apresenta uma imagem MEV de corte horizontal do biocompósito hibrido de HapP/Col (100x).[025] Figure 17 shows a horizontal cross-sectional SEM image of the HapP / Col hybrid biocomposite (100x).

SUMMARY OF THE INVENTION

[026] A aplicação para produtos a partir de fontes de biomassa residual da indústria do peixe é presumivelmente muito mais segura, por apresentar uma maior diferença evolutiva entre peixes e humanos, representando um baixo risco de aquirir doenças de transmissão. Diversas pesquisas, relacionadas com os produtos de origem marinhos e pesqueiros, demostram a obtenção de biomateriais potencialmente aplicáveis na fabricação de enxertos ósseos xenógenos para a indústria odontológica e biomédica, os quais sustentam características para a ROG: biodegradável, biocompatível, osteoindutora, baixo custo e alta disponibilidade de matéria prima. Adicionalmente, contribui-se à descontaminação ambiental, mitigando o descarte destes sub-produtos como resíduos de processamento (escamas; exoesqueletos, ossos, vísceras e peles).[026] The application for products from sources of residual biomass from the fish industry is presumably much safer, as it presents a greater evolutionary difference between fish and humans, representing a low risk of acquiring transmission diseases. Several researches, related to products of marine and fishery origin, demonstrate the obtaining of biomaterials potentially applicable in the manufacture of xenogenic bone grafts for the dental and biomedical industry, which support characteristics for ROG: biodegradable, biocompatible, osteoinductive, low cost and high availability of raw material. Additionally, it contributes to environmental decontamination, mitigating the disposal of these by-products as processing residues (scales; exoskeletons, bones, viscera and skins).

[027] Diante do exposto, faz-se necessário utilizar tecnologias simples e de baixa inversão que permitem o aproveitamento de certas partes descartáveis do peixe especificamente e não obrigatoriamente a Tilápia e, desta forma, aumentar o valor agregado do produto e mitigar a contaminação ambiental. É importante mencionar que, no caso particular das escamas, elas têm baixo valor agregado que, normalmente, são utilizadas como peças de artesanato ou descartadas em ecossistemas por apresentar uma estrutura rígida e de baixo interesse na busca de possíveis usos. Porém, algumas pesquisas reportam diversas metodologias de extração de Hap e hidrolisados proteicos (Colágeno tipo I), extraídos das escamas da Tilápia do Nilo (Oreochromis Sp), que podem ser utilizados como matéria prima para o desenho de biocompósitos de natureza porosa que, em alguns casos, associados com polímeros naturais ou sintéticos não-tóxicos, acrescentam o potencial na ROG em tratamentos de odontologia estética.[027] Given the above, it is necessary to use simple and low inversion technologies that allow the use of certain disposable parts of the fish specifically and not necessarily the Tilapia and, in this way, increase the added value of the product and mitigate environmental contamination . It is important to mention that, in the particular case of scales, they have low added value, which are normally used as handicrafts or discarded in ecosystems because they have a rigid structure and are of low interest in the search for possible uses. However, some research reports several methodologies for the extraction of Hap and protein hydrolysates (Collagen type I), extracted from the scales of the Nile Tilapia (Oreochromis Sp), which can be used as raw material for the design of porous biocomposites that, in some cases, associated with natural or non-toxic synthetic polymers, add the potential in ROG in cosmetic dentistry treatments.

DETAILED DESCRIPTION OF THE INVENTION

[028] O pedido de patente apresenta o desenvolvimento de um biocompósito polimérico (hemiceluloses de origem vegetais) de Hap/colágeno, desenhado biomimeticamente a partir das escamas de Tilápia do Nilo (Oreochromis Sp), têm vantagens em comparação aos compostos mencionados acima. Pois observamos que esses produtos comerciais são sintetizados quimicamente ou extraídos a partir de biomassa residual do subproduto do gado (Hap) ou suíno (Col tipo I), especialmente dos ossos bovinos e peles de porcinos, que requerem uma série de operações unitárias para obtenção dos biomateriais, o que ocasiona um aumento no valor comercial dos subprodutos como matéria prima dos biocompósitos híbridos. Outros sim, a utilização destes produtos no desenho do biocompósito, ao serem utilizados como enxertos ósseos, pode apresentar um risco de zoonoses como problema de saúde coletiva. Em compensação, ao aproveitar as escamas de Tilápia, como subproduto do processamento na extração de Hap e Col, pode-se obter um material com maior biocompatibilidade, mais acessibilidade econômica e, além disso, com a perspectiva de contribuir para mitigar a contaminação ambiental pela indústria alimentar.[028] The patent application presents the development of a polymeric biocomposite (hemicelluloses of plant origin) of Hap / collagen, designed biomimetically from the scales of Nile Tilapia (Oreochromis Sp), have advantages in comparison to the compounds mentioned above. We observe that these commercial products are chemically synthesized or extracted from residual biomass from the by-product of cattle (Hap) or swine (Col type I), especially from bovine bones and porcine skins, which require a series of unitary operations to obtain the biomaterials, which causes an increase in the commercial value of by-products as raw material for hybrid biocomposites. Others, yes, the use of these products in the design of the biocomposite, when used as bone grafts, may present a risk of zoonoses as a collective health problem. On the other hand, by taking advantage of the scales of Tilápia, as a by-product of processing in the extraction of Hap and Col, one can obtain a material with greater biocompatibility, more economic accessibility and, in addition, with the perspective of contributing to mitigate environmental contamination by food industry.

[029] Apresente invenção é obtida por meio de um processo que compreende as seguintes etapas:[029] Present invention is obtained through a process that comprises the following steps:

[030] a. Preparação do material biológico (escamas):[030] a. Preparation of biological material (scales):

[031] As escamas de peixe especificamente não obrigatoriamente de Tilápia do Nilo (Oreochromis sp) serão lavadas inicialmente com água e detergentes (aniônico ou catiônicos) e deixadas por 6,0-12 h em solução desinfetante selecionados dentro de compostos, iodados, clorados, de amônio quaternário e aldeídos, preferencialmente o hipoclorito de sódio 3-5% (v/v), para reduzir a carga microbiológica e eliminar odores desagradáveis dos resíduos do processamento, posteriormente as escamas serão lavadas com água da torneira e levadas a estufa com recirculação de ar de 35 a 50 ± 2 °C entre 12 a 24 horas. As escamas secas serão reduzidas o tamanho de partícula, em que se obtiveram dois tipos de materiais, um em pó, que será utilizado para extrair a Hap crua, e o segundo, uma escama não triturada para obter o colágeno.[031] Fish scales specifically not required from Nile Tilapia (Oreochromis sp) will be washed initially with water and detergents (anionic or cationic) and left for 6.0-12 h in a disinfectant solution selected from compounds, iodized, chlorinated , quaternary ammonium and aldehydes, preferably sodium hypochlorite 3-5% (v / v), to reduce the microbiological load and eliminate unpleasant odors from processing residues, afterwards the scales will be washed with tap water and taken to the oven with air recirculation from 35 to 50 ± 2 ° C between 12 to 24 hours. The dry scales will be reduced to particle size, in which two types of materials were obtained, one in powder, which will be used to extract the raw Hap, and the second, an unground scale to obtain the collagen.

[032] b. Extração da Hidroxiapatita (Hap) crua por tratamento químico (TQ):[032] b. Extraction of raw Hydroxyapatite (Hap) by chemical treatment (TQ):

[033] O material em forma em pó será colocado em contato com uma solução de hidróxido de sódio (NaOH) entre 5,0 a 15,0 % p/v (razão de 1,0 gr-2,0 mL-1) por 12 a 24 horas a temperatura ambiente, depois levaram-se a ebulição entre 10 a 30 min. A solução a temperatura ambiente será filtrada com papel tipo qualitativo com posterior lavagem com agua destilada até atingir o pH neutro, logo o material e secado na estufa entre 35 a 50 ±2 °C por 12 a 24 horas. Depois o material seco é colocado em contato com uma solução acida de ácido clorídrico (HCl) de 0,5-2,5 % por 12 a 24 horas (razão de 1,0 gr·2,0 mL-1) logo será filtrado com agua destilada até atingir o pH neutro e levada a estufa a 35 a 50 ± 2 °C por 6,0 a 12 horas.[033] The material in powder form will be placed in contact with a solution of sodium hydroxide (NaOH) between 5.0 to 15.0% w / v (ratio of 1.0 gr-2.0 mL-1) for 12 to 24 hours at room temperature, then boiling for 10 to 30 min. The solution at room temperature will be filtered with qualitative paper and then washed with distilled water until it reaches neutral pH, then the material and dried in the oven at 35 to 50 ± 2 ° C for 12 to 24 hours. Then the dry material is placed in contact with an acidic solution of hydrochloric acid (HCl) of 0.5-2.5% for 12 to 24 hours (ratio of 1.0 gr · 2.0 mL-1) will soon be filtered with distilled water until it reaches neutral pH and brought to the oven at 35 to 50 ± 2 ° C for 6.0 to 12 hours.

[034] iv) Pré-Purificação da Hap crua por tratamento termo alcalino (TA):[034] iv) Pre-purification of raw Hap by thermo alkaline treatment (TA):

[035] A Hap anteriormente extraída será colocada em contato com numa solução básica de hidróxido de sódio entre 5,0 a 15.0 % (w/v) razão de 1,0 gr·4,0 mL-1 por 12 a 24 horas, levara-se a digestão por micro-ondas entre 350 a 500 watts por 10 a 25 minutos, com repetição do processo novamente de digestão (3x). Depois de alcançar a temperatura ambiente esta será lavada com agua destilada até atingir o pH neutro e finalmente com metanol. Finalizando, a Hap é colocada em estufa com recirculação de ar entre 35 a 50 ± 2 °C por 12 a 24 horas.[035] The previously extracted Hap will be placed in contact with a basic solution of sodium hydroxide between 5.0 to 15.0% (w / v) ratio of 1.0 gr · 4.0 mL-1 for 12 to 24 hours, microwave digestion took between 350 to 500 watts for 10 to 25 minutes, with a repetition of the digestion process again (3x). After reaching room temperature, it will be washed with distilled water until it reaches neutral pH and finally with methanol. Finally, Hap is placed in an oven with air recirculation between 35 to 50 ± 2 ° C for 12 to 24 hours.

[036] v) Purificação da Hap por solubilidade acida e precipitação alcalina.[036] v) Purification of Hap by acid solubility and alkaline precipitation.

[037] A Hap pré-purificada será solubilizada numa solução ácida de tipo fraco. Os ácidos para essa solubilização podem ser: fórmico, propiônico, láctico e acético. Preferencialmente, utiliza-se o ácido acético, entre 1,0 a 5,0 % (v/v), razão de 1,0 gr· 100 mL-1 de solução. Estes compostos são colocados em agitação constante, entre 200 a 500 RPM, por 6 a 12 horas. Em seguida, filtra-se em bomba de vácuo, utilizando papel filtro de 0,45 μm de diâmetro de poro. À solução microfiltrada, novamente em agitação constante (200 a 500 RPM), será adicionada uma solução alcalina de NaOH entre 0,5 a 3,0 Mol·mL-1 (3,0 a 5,0 mL*min-1) até atingir o pH básico (8,5-10,0 ± 2). O precipitado obtido será lavado várias vezes, com água deionizada milli-Q, e, quando atingir o pH neutro, este biomaterial deverá ser filtrado, em papel filtro qualitativo e colocado em estufa até eliminar a umidade (35 a 50 ± 2 °C, por 12 a 24 horas).[037] The pre-purified Hap will be solubilized in a weak acidic solution. The acids for this solubilization can be: formic, propionic, lactic and acetic. Preferably, acetic acid is used, between 1.0 to 5.0% (v / v), with a ratio of 1.0 gr · 100 mL-1 of solution. These compounds are placed under constant agitation, between 200 to 500 RPM, for 6 to 12 hours. Then, filter in a vacuum pump, using 0.45 μm pore diameter filter paper. To the microfiltered solution, again under constant agitation (200 to 500 RPM), an alkaline solution of NaOH between 0.5 to 3.0 Mol · mL-1 (3.0 to 5.0 mL * min-1) will be added until reach basic pH (8.5-10.0 ± 2). The precipitate obtained will be washed several times, with milli-Q deionized water, and, when it reaches neutral pH, this biomaterial should be filtered, using qualitative filter paper and placed in an oven until the moisture is eliminated (35 to 50 ± 2 ° C, for 12 to 24 hours).

[038] c. Extração do colágeno por precipitação salina (PS):[038] c. Collagen extraction by saline precipitation (PS):

[039] As escamas não trituradas serão colocadas em solução de HCl 5,0-10,0 % (razão de 1,0 gr·5,0 mL-1) por 1,0 a 3.0 horas, logo serão colocadas a ebulição entre 10-20 min e deixadas por 6,0-12 horas em contato, depois a solução é liquidificado por 1,0 a 2,0 min por 3x. Posteriormente, será filtrado com agua aquecida (40 a 60 °C) em filtro de tela nylon (poro 20 um) com diversos lavagem de agua aquecida até obter a fração liquida descartando a parte solida. A solução filtrada a temperatura ambiente será adicionada em porções de NaCl (0,125 - 0,250 gr / mL-1) em agitação constante (200 a 500 RPM) até obter um sobrenadante (colágeno). Este concentrado será filtrado e transferido para um pano de nylon para ser lavado com porções de agua da torneira e ao final com destilada (5,0-10 °C) até retirar a sal remanente, depois se deixara em contato com agua destilada por 6,0 a 12 horas a 2,0 a 5,0 °C, para logo ser dialisado em um saco com poro de 15 kD em agua destilada e deionizada. Por último o concentrado será levado a freezer (-50 a -85 °C) para ser liofilizado por 24 a 48 horas, secado em estufa de circulação de ar a temperatura ambiente até desidratação completa. Ao final o biomaterial será reduzido o tamanho de partícula e armazenado em dessecador para posterior desenho dos biocompósitos.[039] The non-crushed scales will be placed in a 5.0-10.0% HCl solution (1.0 gr · 5.0 mL-1 ratio) for 1.0 to 3.0 hours, then they will be boiled between 10-20 min and left for 6.0-12 hours in contact, then the solution is liquefied for 1.0 to 2.0 min for 3x. Subsequently, it will be filtered with heated water (40 to 60 ° C) in a nylon screen filter (pore 20 um) with several heated water rinses until obtaining the liquid fraction discarding the solid part. The filtered solution at room temperature will be added in portions of NaCl (0.125 - 0.250 gr / mL-1) with constant stirring (200 to 500 RPM) until a supernatant (collagen) is obtained. This concentrate will be filtered and transferred to a nylon cloth to be washed with portions of tap water and at the end with distilled (5.0-10 ° C) until the remaining salt is removed, then left in contact with distilled water for 6 , 0 to 12 hours at 2.0 to 5.0 ° C, to be dialyzed in a 15 kD pore bag in distilled and deionized water. Finally, the concentrate will be taken to a freezer (-50 to -85 ° C) to be lyophilized for 24 to 48 hours, dried in an air circulation oven at room temperature until complete dehydration. At the end, the biomaterial will be reduced in particle size and stored in a desiccator for later design of the biocomposites.

[040] d. Sínteses da Hap granulada micro e macro-porosa (HapP):[040] d. Syntheses of Hap granulated micro and macro-porous (HapP):

[041] Preparara-se uma solução de Hap purificada (0,2 a 1,0%) e galactomanana (0,2 a 1,0%) dissolvida em solução de HCl 0,2 a 2,0 Mol·mL-1, com agitação por 2,0 a 6,0 horas entre 700-1000 RPM, depois será adicionada gota a gota (3,0 a 5,0 mL·min-1) uma solução de NaOH 0,5-1,0 Mol·mL-1 até atingir alcalino (8,5-10 ± 2) com agitação constante até obter uma solução coloidal, esta será deixada em repouso entre 6 a 12 horas até uma precipitação total. Posteriormente, esta mistura é concentrada manualmente retirando o sobrenadante por decantação e posteriormente por centrifugação (2500-5000xg, 4,0 °C, 15-30 min.) depois, o concentrado será reidratado com água milli-Q até formar uma solução saturada-viscosa num béquer com agitação constante (200 a 500 RPM) por 30 a 60 min., em seguida a blenda será colocada em moldes cilíndricos de 2,0 ml (3,0 cm x 0,5 cm) e levada a freezer (-50 a -85 °C) para assim ser colocados por 24 a 48 horas no liofilizador. Depois da desidratação as blendas serão impregnadas com solventes de tipo orgânico selecionados entre éter de petróleo, etílico, hexano e acetona, preferivelmente com acetona e éter etílico (0,1-0,5 ml) para a expansão volumétrica do compósito, serão levados à desidratação em estufa entre 50 a 80 °C por 30 a 60 min e logo a digestão por micro-ondas entre 350 a 500 watts entre 10 a 25 minutos, depois de digeridas estes são sinterizada entre 300-700 °C por 4,0 a 8,0 horas com taxas de aquecimento de 2,0 °C·min-1, depois de atingir a temperatura ambiente estes será levemente trituradas e peneiradas com tamanho de poro entre 0,5 e 1,0 mm para ser armazenadas em dessecador.[041] A purified Hap solution (0.2 to 1.0%) and galactomannan (0.2 to 1.0%) dissolved in HCl solution 0.2 to 2.0 Mol · mL-1 , with stirring for 2.0 to 6.0 hours between 700-1000 RPM, then a 0.5-1.0 Mol NaOH solution will be added dropwise (3.0 to 5.0 mL · min-1) · ML-1 until alkaline (8.5-10 ± 2) is reached with constant agitation until a colloidal solution is obtained, which will be left to stand for 6 to 12 hours until total precipitation. Subsequently, this mixture is concentrated manually by removing the supernatant by decanting and later by centrifugation (2500-5000xg, 4.0 ° C, 15-30 min.) Then, the concentrate will be rehydrated with milli-Q water until it forms a saturated solution. viscous in a beaker with constant agitation (200 to 500 RPM) for 30 to 60 min., then the blend will be placed in 2.0 ml (3.0 cm x 0.5 cm) cylindrical molds and taken to the freezer (- 50 to -85 ° C) to be placed in the lyophilizer for 24 to 48 hours. After dehydration, the blends will be impregnated with organic type solvents selected from petroleum ether, ethyl, hexane and acetone, preferably with acetone and ethyl ether (0.1-0.5 ml) for the volumetric expansion of the composite, will be taken to oven dehydration at 50 to 80 ° C for 30 to 60 min and then microwave digestion between 350 to 500 watts in 10 to 25 minutes, after being digested these are sintered between 300-700 ° C for 4.0 to 8.0 hours with heating rates of 2.0 ° C · min-1, after reaching room temperature these will be lightly crushed and sieved with a pore size between 0.5 and 1.0 mm to be stored in a desiccator.

[042] e. Desenho do biocompósito hibrido-polimérico (HapP/Col):[042] e. Hybrid-polymeric biocomposite design (HapP / Col):

[043] Preparara-se, inicialmente, uma solução aquosa, com a proporção de 0,1-1,0%, do polímero de tipo hemiceluloses de origem vegetal como a galactomanana. Posteriormente, acrescenta-se, à solução anterior (que deve estar em agitação constante entre 500-1000 RPM), o colágeno na concentração de 1,0 a 50% (w/w) até solubilização completa. Depois, será adicionada a Hap (granulada micro e macroporosa), na faixa porcentual de 50 a 99%, completando os 100% da blenda dos biomateriais que foram extraídos a partir da escama de Tilápia (Oreochromis sp). Ao estarem os componentes solubilizados na mistura, este serão aquecidos entre 35 a 50 °C ± 2 por 60 a 180 min. Em outro momento, a solução do biocompósito é resfriada e levada a pH alcalino, na faixa de 8,5 a 12.0 (NaOH de 0,5-5,0 Mol·mL-1), com adição do agente reticulante Trimetilfosfato de Trissodíco (95%) entre 1,0 a 5,0 % segundo o peso total dos biomateriais. Essa solução será aquecida novamente (40-60 °C) entre 15-30 minutos e resfriada até temperatura de 5,0-10 °C com agitação constante por 15 a 30 min. Em outra etapa, a blenda a temperatura ambiente será colocado em moldes cilíndricos de 2,0 ml (3,0 cm x 0,5 cm), e levada ao freezer (-50 a -85 °C) para, por fim, serem liofilizadas durante 24 a 48 horas. Após o período indicado, as amostras serão desidratadas em estufa (50-80 °C), por 6,0 a 12 horas, armazenadas em dessecador e, por último, embaladas a vácuo.[043] An aqueous solution, with a proportion of 0.1-1.0%, of the plant-type hemicellulosic polymer such as galactomannan was initially prepared. Subsequently, to the previous solution (which must be in constant agitation between 500-1000 RPM), collagen in the concentration of 1.0 to 50% (w / w) until complete solubilization. Then, Hap (micro and macroporous granules) will be added, in the percentage range of 50 to 99%, completing the 100% of the blend of biomaterials that were extracted from the scale of Tilapia (Oreochromis sp). When the components are solubilized in the mixture, it will be heated between 35 to 50 ° C ± 2 for 60 to 180 min. At another time, the biocomposite solution is cooled and brought to alkaline pH, in the range of 8.5 to 12.0 (0.5-5.0 Mol · mL-1 NaOH), with the addition of the trisodium trimethylphosphate crosslinking agent ( 95%) between 1.0 to 5.0% depending on the total weight of the biomaterials. This solution will be heated again (40-60 ° C) between 15-30 minutes and cooled to a temperature of 5.0-10 ° C with constant agitation for 15 to 30 min. In another stage, the blend at room temperature will be placed in cylindrical molds of 2.0 ml (3.0 cm x 0.5 cm), and taken to the freezer (-50 to -85 ° C) to finally be lyophilized for 24 to 48 hours. After the indicated period, the samples will be dehydrated in an oven (50-80 ° C), for 6.0 to 12 hours, stored in a desiccator and, finally, vacuum-packed.

Claims

POLYMERIC HYBRID BIOCOMPOSITES OF POROUS AND AMORPLE HYDROXIAPATITE, FIBROUS COLLAGEN (TYPE I) EXTRACTED FROM BIOLOGICAL SOURCES, PREPARATION AND USES IN DENTAL APPLICATIONS, characterized by a polymeric biocomposite / hemolytic compound (from polymeric to polymeric) fish.

POLYMERIC HYBRID BIOCOMPOSITES OF POROUS AND AMORPLE HYDROXIAPATITE, FIBROUS COLLAGEN (TYPE I) EXTRACTED FROM BIOLOGICAL SOURCES, PREPARATION AND USES IN DENTAL APPLICATIONS, according to claim 1 characterized by the silica of the silica of the silica .

POLYMERIC HYBRID BIOCOMPOSITES OF POROUS AND AMORPLE HYDROXIAPATITE, FIBROUS COLLAGEN (TYPE I) EXTRACTED FROM BIOLOGICAL SOURCES, PREPARATION AND USES IN DENTAL APPLICATIONS, according to claim 1 characterized by a water-based solution with a %, of the plant-type hemicellulose polymer such as galactomannan.

POLYMERIC HYBRID BIOCOMPOSITES OF POROUS AND AMORPLE HYDROXIAPATITE, FIBROSOUS COLLAGEN (TYPE I) EXTRACTED FROM BIOLOGICAL SOURCES, PREPARATION AND USES IN DENTAL APPLICATIONS, according to claim 1 and 3 in the concentration of 1.0% to 10% characterized by collagen / w) and addition of Hap (micro and macroporous granules), in the percentage range of 50 to 99%.

POLYMERIC HYBRID BIOCOMPOSITES OF POROUS AND AMORPLE HYDROXIAPATITE, FIBROUS COLLAGEN (TYPE I) EXTRACTED FROM BIOLOGICAL SOURCES, PREPARATION AND USES IN DENTAL APPLICATIONS, according to claim 1, 3 and 4 characterized by:
The. Preparation of biological material (scales)

The. i) the biological material is prepared for grinding in which two types of materials are obtained, one in powder, used to extract the raw hydroxyapatite (Hap), and the second, an unground scale to obtain the collagen;

B. Extraction of raw Hydroxyapatite (Hap) by chemical treatment (TQ)

B. i) the powdered material comes in contact with a solution of sodium hydroxide (NaOH) between 5.0 to 15% w / v (ratio of 1.0 gr · 2.0 mL-1) for 12 to 24 hours at room temperature, then boiling for 10 to 30 min;
b.ii) the solution obtained is filtered at room temperature with qualitative paper and then washed with distilled water until it reaches neutral pH, then the material is dried in the oven at 35 to 50 ± 2 ° C for 12 to 24 hours;
b.iii) the dry material is placed in contact with an acidic solution of hydrochloric acid (HCl) of 0.5-2.5% for 12 to 24 hours (ratio of 1.0 gr · 2.0 mL-1) , being then filtered with distilled water until it reaches neutral pH and taken to the oven at 35 to 50 ± 2 ° C for 12 to 24 hours;
b.iv) the pre-purification of raw Hap by thermo-alkaline treatment (TA) the previously extracted Hap is placed in contact with a basic sodium hydroxide solution between 5.0 to 15% (w / v) ratio of 1 , 0 gr · 4.0 mL-1 for 12 to 24 hours, microwave digestion took between 350 to 500 watts for 10 to 25 minutes, with repetition of the digestion process again (3x);
bv) after reaching room temperature it will be washed with distilled water until it reaches neutral pH and finally with methanol, finally Hap is placed in an oven with air recirculation between 35 to 50 ± 2 ° C for 12 to 24 hours;
b.vi) purification of Hap by acid solubility and alkaline precipitation, the pre-purified Hap is solubilized in a weak type acid solution, preferably acetic acid, between 1.0 to 5.0% (v / v), ratio of 1.0 gr · 100 mL-1 of solution and placed under constant agitation, between 200 to 500 RPM, for 6.0 to 12 hours;
b.vii) the material obtained is filtered in a vacuum pump, using filter paper of 0.45 μm pore diameter and the microfiltered solution is placed again under constant agitation (200 to 500 RPM), with an alkaline solution of NaOH being added between 0.5 to 3.0 Mol · mL-1 (3.0 to 5.0 mL · min-1) until the basic pH is reached (8.5-10 ± 2);
B. viii) the precipitate obtained is washed several times with deionized water and, when it reaches neutral pH, it is filtered on qualitative filter paper and placed in an oven until the moisture is eliminated (35 to 50 ± 2 ° C, for 12 to 24 hours);

ç. Collagen extraction by saline precipitation (PS)

ç. i) the non-crushed scales will be placed in 5.0-10.0% HCl solution (1.0 gr · 5.0 mL-1 ratio) for 1.0 to 3.0 hours, then they will be boiled between 10 -20 min and left for 6.0-12 hours in contact, then the solution is liquefied for 1.0 to 2.0 min for 3x;
c.ii) afterwards, it will be filtered with heated water (40 to 60 ° C) in a nylon screen filter (pore 20 um) with several heated water washing until obtaining the liquid fraction discarding the solid part;
c.iii) the filtered solution at room temperature will be added in portions of NaCl (0.125 - 0.250 gr / mL-1) under constant stirring (200 to 500 RPM) until a supernatant (collagen) is obtained;
c.iv) the concentrate obtained will be filtered and transferred to a nylon cloth to be washed with portions of tap water and at the end with distilled water (5.010 ° C) until the remaining salt is removed, then it will be left in contact with distilled water for 6.0 to 12 hours at 2.0 to 5.0 ° C, to then be dialyzed in a 15 kD pore bag in distilled and deionized water;
cv) the concentrate will be taken to a freezer (-50 to -85 ° C) to be lyophilized for 24 to 48 hours, dried in an air circulation oven at room temperature until complete dehydration;
ç. vi) the biomaterial will be reduced to particle size and stored in a desiccator for the design of biocomposites;

d. Syntheses of micro and macro-porous granules (HapP)

d. i) it is prepared from a solution of purified Hap (0.2 to 1.0%) and galactomannan (0.2 to 1.0%) dissolved in HCl solution 0.2 to 2.0 Mol · mL-1 , with agitation for 2.0 to 6.0 hours between 700-1000 RPM;
d.ii) a 0.5-1.0 Mol · mL-1 NaOH solution is added dropwise (3.0 to 5.0 mL · min-1) until alkaline (8.5-10 ± 2) is reached ) with constant stirring until a colloidal solution is obtained, it will be left to stand between 6.0 to 12 hours until total precipitation;
d.iii) the mixture is manually concentrated by removing the supernatant by decanting and then by centrifugation (2500-5000xg, 4.0 ° C, 15-30 min.) then, the concentrate will be rehydrated with milli-Q water until a solution is formed viscous saturated with constant agitation (200 to 500 RPM) for 30 to 60 min;
d.iv) the blend will be placed in 2.0 mL (3.0 cm x 0.5 cm) cylindrical molds and taken to a freezer (-50 to -85 ° C) to be placed in the oven for 24 to 48 hours. lyophilizer;
dv) after dehydration the blends will be impregnated with organic type solvents selected from petroleum ether, ethyl, hexane and acetone, preferably with acetone and ethyl ether (0.1-0.5 mL) for the volumetric expansion of the composite, taken to dehydration in an oven at 40 to 80 ° C for 30 to 60 min and then the digestion by microwave between 350 to 500 watts in 10 to 25 minutes;
d. vi) after drying, they are synthesized at 500-700 ° C for 3.0 to 6.0 hours with heating rates of 2.0 ° C · min-1, after reaching room temperature they will be crushed and sieved to size pore size between 0.5 and 1.0 mm to be stored in a desiccator;

and. Polymeric biocomposite design (HapP / Col)

and. i) an aqueous solution, with a proportion of 0.1-1.0%, of the plant-type hemicellulose polymer such as galactomannan was prepared;
e.ii) to the previous solution (which must be in constant agitation between 500-1000 RPM), the collagen in the concentration of 3.0 to 50% (w / w) until complete solubilization;
e.iii) Hap (micro and macroporous granules) is added, in the percentage range of 50 to 99%, completing the 100% of the blend of biomaterials that were extracted from the fish scale;
e.iv) when the components are solubilized in the mixture, it will be heated between 35 to 50 ° C ± 2 for 60 to 180 min;
and v) the biocomposite solution is cooled and brought to alkaline pH, in the range of 8.5 to 12.0 (0.5-5.0 Mol · mL-1 NaOH), with the addition of the trisodium trimethylphosphate crosslinking agent (95% ) between 1.0 to 5.0% according to the total weight of biomaterials;
e.vi) the solution will be heated again (40-60 ° C) between 15-30 minutes and cooled to a temperature of 5.0-10 ° C with constant agitation for 15 to 30 min;
e.vii) the blend will be placed at room temperature in cylindrical molds of 2.0 mL (3.0 cm x 0.5 cm), and taken to the freezer (-50 to -85 ° C) to finally be lyophilized for 24 to 48 hours;
e.viii) the samples will be dehydrated in an oven (50-80 ° C), for 6.0 to 12 hours, stored in a desiccator and, finally, vacuum-packed.

POLYMERIC HYBRID BIOCOMPOSITES OF POROUS AND AMORPLE HYDROXIAPATITE, FIBROUS COLLAGEN (TYPE I) EXTRACTED FROM BIOLOGICAL SOURCES, PREPARATION AND USES IN DENTAL APPLICATIONS, according to claim 4 (aorganic, characterized by a ) biomimetic, with the weight / weight percentage composition of fibrous collagen type I associated with Hap micro and macroporous being between 50-97 / 3-50, preferably at the ratios of 70/30 and 50/50.

POLYMERIC HYBRID BIOCOMPOSITES OF POROUS AND AMORPLE HYDROXIAPATITE, FIBROUS COLLAGEN (TYPE I) EXTRACTED FROM BIOLOGICAL SOURCES, PREPARATION AND USES IN DENTAL APPLICATIONS, according to claim 1, 2, 3, 4, your, for your use Organic-Inorganic hybrid biocomposite, like a bone graft as a scaffolded implant in neoformation and guided alveolar bone repair in dental procedures.