WO2014102730A1 - Three‑dimensional culture perfusion chamber for tissue engineering - Google Patents

Three‑dimensional culture perfusion chamber for tissue engineering Download PDF

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WO2014102730A1
WO2014102730A1 PCT/IB2013/061341 IB2013061341W WO2014102730A1 WO 2014102730 A1 WO2014102730 A1 WO 2014102730A1 IB 2013061341 W IB2013061341 W IB 2013061341W WO 2014102730 A1 WO2014102730 A1 WO 2014102730A1
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perfusion
cells
culture
tissue
bioreactor
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PCT/IB2013/061341
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French (fr)
Portuguese (pt)
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Marivalda de Magalhães PEREIRA
Alexandra Rodrigues Pereira da SILVA
Alfredo Miranda de GÓES
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Universidade Federal De Minas Gerais - Ufmg
Fundação De Amparo À Pesquisa Do Estado De Minas Gerais- Fapemig
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture

Definitions

  • the present technology describes a tissue culture perfusion chamber whose fabrication material may involve different types of polymers having good thermal and electrical properties, moderate strength, easy machining and autoclaving, an essential factor when the proposal is to work. with biological material.
  • the camera can have different formats, provided that respecting the conditions of cultivation of the fabric of interest.
  • the developed perfusion chamber makes it possible to perform a three-dimensional culture of seeded cells in a matrix which, when properly adapted to the perfusion chamber, favors the flow of flow through this scaffold, ensuring that the medium can reach most if not all. thus favoring cell viability and proliferation.
  • Tissue engineering was defined as a research field only in the early 1980s and today progress in this field is related to the development of various study model approaches. Tissue engineering supports its scientific base in many areas of knowledge, such as cell biology, biochemistry, molecular biology, materials science, chemical engineering and bioengineering, which allow the rational application of engineering principles in living systems.
  • the scientific challenge of tissue engineering involves understanding cellular mechanisms and fabricating materials to provide matrices and models that guide tissue regeneration (VACANTI, J .; VACANTI, C.
  • VACANTI The History and Scope of Tissue Engineering. Principles of Tissue Engineering , 3rd ed., Elsevier, Inc, Chapter 1, pp. 3-6,).
  • Living stem cells are required for the manufacture of tissue substitutes. These cells create or recreate functional structures using preprogrammed information and signaling in their genetic code.
  • the acquisition of cells for the production of organic structures is a major challenge, because for the manufacture of large-scale structures, there is a need to obtain large numbers of cells that are immunologically compatible with the recipient individual (VACANTI, J.; VACANTI, C.
  • VACANTI J.
  • VACANTI C.
  • Bone marrow contains mesenchymal stem cells (MSC), but harvesting is an invasive procedure.
  • Adipose mesenchymal stem cells are able to differentiate into chondrogenic, adipogenic, myogenic and osteogenic lineages, suggesting their applicability in tissue reconstitution (ZUK, PA; ZHU, M .; MIZUNO, H .; HUANG, J .; FUTRELL, JW; KATZ, AJ; BENHAIM, P.; LORENZ, HP; HEDRICK, MH Multilineage Celis from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineering, 7, 211-228, 2001.).
  • Bioreactors are the main tools to mimic cellular microenvironments and provide cellular constructs with physiologically relevant stimuli that direct the conversion of a "set of cells" into a given tissue (FRESHNEY, RI; OBRADOVIC, B .; GRAYSON, W .; CANNIZZARO, C; VUNJAK-NOVAKOVIC, G. Principles of Tissue Culture and Bioreactor Design. Principies of Tissue Engineering, 3rd ed., Elsevier, Inc, Chapter 12, p. 155-183, 2007).
  • the bioreactor-induced hydrodynamic shear is believed to simulate interstitial flow in the canalicular spaces during natural bone formation associated with compression loading.
  • the resulting mechanical forces are an integral part of bone physiology that regulates homeostasis, bone remodeling and repair, however few studies apply mechanical deformation to explore their role in promoting cell growth and tissue organization (ALLORI, A. C; SAILON, AM; PAN).
  • JR WARREN SM Biological Basis of Bone Formation, Remodeling, and Repair — Part III: Biomechanical Forces, Tissue Engineering: Part B, v. 14, no. 3, pp. 285-293, 2008).
  • perfusion chambers in a bioreactor system are intended to increase the supply of nutritive material to cells and remove metabolites generated by cell activity, thereby enabling a favorable environment for cell growth (Dick, D. An Easily Made Tissue Culture Perfusion Chamber, Quarterly Journal of Microscopical Science (363, 1995). Since its invention in the mid-1907, as new cell and tissue culture techniques have emerged, new models of perfusion chambers have been developed (Wolf, D; Sluder, G. Methods in Celi Biology, v. 56, p. 256 , 1998). Currently, there is still no consensus in the literature on a standard perfusion chamber and bioreactor model for tissue engineering for cell engineering and several laboratories around the world are looking for this.
  • the present work has as main objective the construction of a perfusion chamber suitable for tissue culture, basing its efficiency on an analysis of the cellular differentiation of human adipose tissue stem cells, sown in a glass foam matrix. bioactive and grown in a bioreactor system for the production of a construct to be applied in tissue engineering.
  • bioactive glass foams produced by the sol-gel process (Hiratsuka, R. et al.) The process solgllma bibliographic review, p. 171 - 179, 1994).
  • COELHO MB Development of methodology for the production of porous three-dimensional bioactive glass structures for application in tissue engineering. Belo Horizonte: UFMG School of Engineering, 2003. 145p.
  • the development of the present technology proved to be very efficient for the cultivation of stem cells derived from human adipose tissue which, when sown in bioactive glass, were able to proliferate and differentiate into the phenotype of interest, producing typical osteogenic cell proteins and expressing genes. characteristic of osteoblasts. This efficiency can be expanded to other tissues as long as conditions, flow rate, culture matrix and perfusion chamber dimensions are adapted.
  • Figure 1 Illustration of bioreactor organization scheme: Greenhouse at 37 Q C (1), Biomaterial (2), Infusion Chamber (3), Culture Medium + O2 (4), Peristaltic Pump (5), Connection Tubes Inside the CO 2 Greenhouse (6).
  • Figure 2 Shows a non-limiting representation of the perfusion chamber.
  • Figure 3 Newly seeded hASC bioactive glass foam.
  • FIG. 4 Scanning electron microscopy (SEM) images at 21 days of cultivation in the bioreactor showing: (A) the surface of the bioactive glass sample with several attached cell clusters (indicated by arrows); (B) a pore of the sample with several adhered cells, (C) larger pore enlargement showing better cells and material roughness, and (D) greater increase in material-adhered hASC and secretion granules in the cell plasma membrane.
  • SEM scanning electron microscopy
  • Figure 5 Representative graph of the viability and proliferation of hASC grown in normal (static) medium and in bioactive (dynamic) glass matrices containing leibovitz (LEI) or leibovitz medium plus osteogenic factors (LEI O in the caption: static O and dynamic O). All times between static and dynamic and between static O and dynamic O are p ⁇ 0.001).
  • Figure 6 Representative graph of alkaline phosphatase activity of hASC grown in bioactive glass matrices containing leibovitz medium (LEI) or medium leibovitz plus osteogenic factors (LAW O in the caption: static O and dynamic O).
  • LAI leibovitz medium
  • LAW O medium leibovitz plus osteogenic factors
  • Figure 7 Immunofluorescence analysis for (A) osteocalcin, (B) osteopontin and (C) hASC type I collagen seeded in bioactive glass matrix and cultured in basal culture medium in perfusion bioreactor after 21 days. Cell nuclei have blue marking and specific proteins green.
  • Figure 8 Immunofluorescence analysis for hASC osteocalcin protein seeded in bioactive glass matrix and cultured in osteogenic culture medium in perfusion bioreactor after (A) 7, (B) 14 and (C) 21 days.
  • Figure 9 Immunofluorescence analysis for hASC osteopontin protein sown in bioactive glass matrix and cultured in osteogenic culture medium in perfusion bioreactor after (A) 7, (B) 14 and (C) 21 days.
  • Figure 10 Immunofluorescence analysis for hASC type I collagen protein sown in bioactive glass matrix and cultured in osteogenic culture medium in perfusion bioreactor after (A) 7, (B) 14 and (C) 21 days.
  • Figure 11 Detection of osteocalcin, osteopontin and alkaline phosphatase at 7, 14 and 21 days in hASC cultured on the bioactive glass matrix, LEI O medium and perfusion biorector. Human osteosarcoma (SOS) cells as positive control and C- is negative control.
  • SOS Human osteosarcoma
  • the present technology consists of a tissue culture perfusion chamber (Figure 2), the fabric of which may involve different types of polymers which have good thermal and electrical properties, moderate strength, easy machining and can be autoclaved, an essential factor. when the proposal is to work with biological material.
  • a tissue culture perfusion chamber (Figure 2), the fabric of which may involve different types of polymers which have good thermal and electrical properties, moderate strength, easy machining and can be autoclaved, an essential factor.
  • polypropylene is used, a low density resin that offers a good balance of thermal and electrical properties, as well as moderate strength, is nontoxic, easily machined and allows autoclaving.
  • the camera can have different formats and dimensions, as long as it respects the conditions of interest.
  • the matrix of choice for the cultivation of a given tissue must be correctly adapted to the perfusion chamber to favor the flow of the flow through the matrix, ensuring that the medium can reach most if not all cells, thus favoring the viability and cell proliferation.
  • the perfusion chamber was designed with one inlet and one outlet (7 and 9, respectively) to allow perfusion of the culture medium throughout its interior, where the construct (biomaterial and seeded cells) must be adequately adapted.
  • the matrix material (2) as well as the chamber shape vary according to the cell type of the tissue chosen for cultivation.
  • the perfusion chamber ( Figure 2) developed can be maintained in different environments, from a B.O.D (Biochemical Oxygen Demand) greenhouse to a water bath, provided the necessary conditions for cultivation are respected.
  • the perfusion chamber proposed to compose the bioreactor system (3) was able to efficiently promote bone tissue culture.
  • the developed perfusion bioreactor (1) is a closed, passive sterilization system where the cell culture medium is constantly pumped at a constant flow rate through a peristaltic pump (5) and through connected hoses (6) to one end of the infusion chamber.
  • the pumped medium passes through the construct and returns through the connecting hoses, connected at the other end of the chambers, to the culture medium reservoir as shown in Figure 1.
  • the tubes chosen to connect the bioreactor system are calibrated silicone peristaltic tubes with 2.6 mm internal diameter. These tubes were chosen because they are autoclavable, translucent and have biocompatibility and high gas permeability.
  • the reservoir for the culture media is screw-capped glass bottles. In the bottle caps, three holes were drilled, two of them for fitting the connection hoses - one for entering and one for leaving the culture medium - and one for fitting a breather filter to avoid the formation of vacuum inside the bottles during the tests and possibility of being another way of gas exchange.
  • the peristaltic pump of choice produces smooth, pulse-free flow, has 8 channels, allowing up to 8 perfusion chambers to work simultaneously, fed by a constant flow of culture medium from 0.05mL / min to 40mL / min per channel.
  • the bioreactor culture medium was partially changed every seven days during cell culture, and only half of the medium was removed in order not to eliminate the new components produced by cells, such as growth factors, hormones and enzymes, which are important. in the regulation of the cellular mechanisms themselves (GOLDSTEIN, AS; JUAREZ, TM; HELMKE, CD; GUSTIN, M. C; Mikos, AGE). Convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. Biomaterials, v. 22p. 1279 - 1288, 2001; MARTIN, Y; VERMETTE, P. Bioreactors for Tissue Mass Culture: Design, Characterization, and Recent Advances. Biomaterials, v. 26, pp. 7481-7503, 2005.).
  • the physiological conditions of bioreactor temperature and pH were controlled.
  • the temperature of 37 Q C was assured by maintaining the hose and reservoirs in the culture medium bath and monitored daily with the aid of a thermometer.
  • Already the pH was monitored with each change of culture medium with the aid of a pH meter.
  • the method chosen for sterilization of bioreactor accessories was autoclaving.
  • Adipose tissue stem cells were used for differentiation into bone tissue.
  • the chamber was made of polypropylene and in a cylindrical shape, obeying the following internal dimensions: 7mm in diameter and 10mm in length (8).
  • the chamber was made of polypropylene and in a cylindrical shape, obeying the following internal dimensions: 7mm in diameter and 10mm in length (8).
  • the chamber was made of polypropylene and in a cylindrical shape, obeying the following internal dimensions: 7mm in diameter and 10mm in length (8).
  • conduction tubes are connected which connect the chamber to the culture medium and a propellant pump (7 and 9, respectively).
  • the hASCs were grown on bioactive glass foam matrix, with dimensions adapted to the bioreactor dimensions, and were placed in individual perfusion chambers, each connected to a reservoir. containing Leibovitz medium plus osteogenic factors and kept in a water bath at 37 ° C for 7.14 and 21 days.
  • bone tissue we analyzed: cell viability and proliferation, alkaline phosphatase enzyme activity, osteogenic cell markers, as well as morphology and gene expression.
  • cell viability and proliferation we analyzed: cell viability and proliferation, alkaline phosphatase enzyme activity, osteogenic cell markers, as well as morphology and gene expression.
  • the biomaterial chosen for the cultivation of hASC was processed, in this case, the bioactive glass foam, and its characterization so that its chemical and structural characteristics could be verified.
  • the structure of the bioactive glass foams was preliminarily evaluated by scanning electron microscopy (SEM) and it was observed that they had high porosity, with spherical pores, average pore sizes ranging from 100 to 500pm and an interconnected pore network. .
  • the analysis performed in various areas of bioactive glass foams detected the presence of the chemical elements silicon (Si), calcium (Ca), phosphorus (P) and gold (Au), the latter being due to the matrix metallization process.
  • the X-ray diffractogram analysis of the bioactive glass foam sample presents a characteristically amorphous material.
  • the perfusion bioreactor system was adapted for use in a water bath in order to maintain the temperature of 37 Q C, ideal for cell culture ..
  • To ensure the maintenance of the pH of the cell culture was Leibovitz tested Independent CO 2, which is able to maintain pH stability in the long term under atmospheric CO 2.
  • the hASCs isolated from the stromal vascular fraction resulting from the liposuction processing showed satisfactory results, presenting cells with fibroblast-like shape and adhering to the plastic surface, corroborating the data of Zuk (ZUK, PA; ZHU, M .; MIZUNO, H. ; HUANG, J .; FUTRELL, JW; KATZ, AJ; BENHAIM, P.; LORENZ, HP; HEDRICK, MH Multilineage Celis from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineering, 7, 21 1 -228, 2001).
  • Example 2 Since the samples used in the static tests (Example 2) were 3mm long and the samples grown in the perfusion bioreactor were 10mm, all samples were cut into three parts and subjected to subsequent biological analysis to allow comparison of the static culture results. and dynamic. After 7, 14 and 21 days, colonized matrices were prepared for SEM analysis to confirm the presence and adhesion of hASC to the surface and interior of colonized foams cultured in the perfusion bioreactor ( Figure 4).
  • Formazan crystals are solubilized and the optical density can be determined by spectrophotometer at 595 nm.
  • the number of viable cells is directly proportional to the amount of formazan crystals produced.
  • HASC-colonized bioactive glass foams cultured in the LEI and LEI O perfusion bioreactor were evaluated after each culture interval for hASC alkaline phosphatase activity by the BCIP / NBT assay ( Figure 6) based on chromogenic reaction resulting from the cleavage of a phosphate group BCIP (5-bromo 4-chloro 3-indolylphosphate p-toluidine) by the alkaline phosphatase produced by the cell.
  • hASC osteogenic lineage cells when cultured in bioactive glass matrices and perfusion bioreactor was evaluated by the expression of specific proteins produced by osteoblasts using the immunofluorescence technique.
  • cells were washed with PBS and fixed in 4% p-formaldehyde for 15 minutes at room temperature.
  • Cells were washed with PBS and plasma membrane permeabilization was performed using 0.1% Triton-100x in PBS for 10 minutes. After permeabilization, the cells were washed again with PBS. The reaction was then blocked by incubating the cells with 1% BSA (bovine serum albumin) and 5% goat serum in PBS for 1 hour at room temperature.
  • BSA bovine serum albumin
  • cells were incubated with primary antibody diluted in 1% BSA solution in PBS for 2 hours at room temperature and in a humid chamber. After incubation with primary antibody, the cells were again washed with PBS and then incubated with their secondary antibodies diluted in 1% BSA 1% solution in PBS for 1 hour in a humid chamber at no light and at room temperature. Negative controls were made using only the respective secondary antibody. Subsequently, cells were washed with PBS and incubated with the Hoechst probe (1 pg / ml) (Molecular Probes) for 30 minutes for nucleus labeling. Further washes were made with PBS and the coverslips were mounted with Hydromount on slides.
  • Hoechst probe (1 pg / ml) (Molecular Probes) for 30 minutes for nucleus labeling.
  • the slides were visualized and analyzed through the Confocal Microscope (Zeiss LSM 510 Meta) using the Carl Zeiss Laser Scanning Microscope LSM 510 ⁇ software.
  • the assay used to verify the expression of osteogenesis-related genes was the Polymerase Chain Reaction (PCR).
  • the constitutive gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered as a control for reaction amplification.
  • the hASCs sown in the bioactive glass were grown in the bioreactor in LEI O medium for 7, 14 and 21 days and, after each period, RNA extraction was performed.
  • RNA extraction from hASC was attempted by applying 1 ml Trizol (Gibco) on the bioactive glass matrices, incubating for 5 minutes at room temperature. The formed solution was processed following the RNA extraction steps usually used for cells grown in culture bottles, but when the absorbance reading at 260 / 280nm was taken, the results were close to zero.
  • aHASC-seeded bioactive glass samples taken from the bioreactor were cut into three parts and each into four parts and trypsinized for 5 minutes in a CO 2 oven so that the cells could be removed from inside the biomaterial. During trypsinization, the samples were gently shaken every 2 minutes. After trypsinization, the biomaterial was washed three times with LEI medium and the total solution was centrifuged for 5 minutes at 252 g. The pellet formed was resuspended in 1 ml Trizol and incubated for 5 minutes for cell lysis and homogenization at room temperature and for complete dissociation of nucleoprotein complexes.
  • the formed solution was transferred to a microtube 1, 5 mL and centrifuged for 15 minutes at 4 Q C and 12,000 g.
  • the upper phase was collected and transferred to a new microtube 1, 5mL which was added 0.2 ml of chloroform, followed by homogenization, two minutes incubation at room temperature and centrifugation at 12,000 g for 15 minutes at 4 Q C to three-phase separation where the superficial colorless phase contained the RNA.
  • the colorless phase was transferred to a new microtube and the RNA It was precipitated with 0.5 ml of isopropyl alcohol for 10 minutes at room temperature.
  • RNA was solubilized in DNAse and RNAse free distilled water and immediately stored at -80 Q C. RNA concentration was determined by reading the absorbance at 260 / 280nm in Nanodrop.
  • CDNAs were synthesized from total RNAs using the RevertAid TM H Minus First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's recommendations. Two micrograms of total RNA from each sample were incubated with 0.5 ug oligo (dT) 18 at 65 Q C for 5 minutes and then the samples were incubated on ice. Immediately after it was added to the samples: 5X reaction buffer, 20 units Ribolock TM Ribonuclease Inhibitor, 10mM dNTP mix and incubated at 37 Q C for 5 minutes. Then it was added 200 units of enzyme RevertAid TM H Minus M-MuLV RT (Fermentas) and the samples were incubated for 60 minutes at 42 Q C. The reaction was stopped by heating at 70 Q C for 10 minutes.
  • RevertAid TM H Minus First Strand cDNA Synthesis Kit Fermentas
  • the bioactive glass has had a osteoinductive action represented by the increased production of the enzyme in the period 14 days and decrease of this production in the 21 Q day, suggesting a probable compromise with the osteogenic phenotype.
  • the flux had a synergistic action to that of bioactive glass, which demonstrated an increase of alkaline phosphatase synthesis in the bioreactor constructs.
  • the immunofluorescence assay was performed to verify whether hASCs sown in bioactive glass foams and cultured in the perfusion bioreactor began to express osteogenic cell markers.
  • the specific antibodies used were osteocalcin, osteopontin and type I collagen and initially the test was performed on cells cultured in LEI medium after 21 days of culture. It can be observed in the confocal microscopy images that the hASC were labeled for the three proteins tested ( Figure 7). This result corroborates the results of MTT and alkaline phosphatase activity, where, from 14 Q day, there was a decrease in cell proliferation and an increase in the production of alkaline phosphatase enzyme, suggesting a compromise of hASC with osteogenic phenotype. .
  • confocal microscopy images also show a decrease in hASC length, also suggesting a change in morphology related to change in phenotype ( Figure 8).
  • osteogenic differentiation of HASC when cultured on bioactive glass matrix and means ACT in the perfusion bioreactor can be suggested that from 14 Q day of cultivation, the HASC seem to have committed to the osteogenic phenotype.
  • the evaluation of osteocalcin, osteopontin and collagen type I differentiation markers was performed in hASC grown on bioactive glass in LEI O medium for 7, 14 and 21 days in the perfusion bioreactor.
  • the confocal microscopy images show an increase in fluorescence labeling of from 7 to 21 Q day for three tested proteins: osteocalcin (Figure 8), osteopontin (Figure 9) and collagen type I ( Figure 10);
  • the cells move from the typical fusiform shape of a stem cell to the cuboidal shape, characteristic of osteoblastic cells.
  • hASC sown in the three-dimensional bioactive glass matrix and cultured in LEI O medium in the bioreactor showed a compromise with the osteogenic phenotype.

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Abstract

The present technology describes a perfusion chamber for tissue culture, the chamber being made of a material that can include different types of polymer that have good thermal and electrical properties and moderate strength, and that are easy to machine and capable of being autoclaved, which is an essential factor when the intention is to work with biological material. In terms of the physical aspect thereof, the chamber may have different formats, provided the conditions for culture of the tissue of interest are complied with. The perfusion chamber developed makes it possible to achieve three‑dimensional culture of cells seeded in a matrix that, when correctly adapted to the perfusion chamber, promotes the passage of the flow through this scaffold, guaranteeing that the medium is able to reach the majority of the cells, if not all the cells, thereby promoting cell viability and proliferation.

Description

CÂMARA DE PERFUSÃO DE CULTIVO TRIDIMENSIONAL PARA A  PERFUSION CHAMBER OF THREE-DIMENSIONAL CROP FOR
ENGENHARIA DE TECIDOS  FABRIC ENGINEERING
A presente tecnologia descreve uma câmara de perfusão para cultivo de tecidos, cujo material de confecção pode envolver diferentes tipos de polímeros que possuam boas propriedades térmicas e elétricas, moderada resistência, de fácil usinagem e passível de ser autoclavado, fator essencial quando a proposta é trabalhar com material biológico. Em seu aspecto físico, a câmara pode apresentar diferentes formatos, desde que respeitando as condições de cultivo do tecido de interesse. A câmara de perfusão desenvolvida torna possível a realização de um cultivo tridimensional de células semeadas em uma matriz que, quando corretamente adaptada à câmara de perfusão, favorece a passagem do fluxo através desse scaffold, garantindo que o meio possa chegar à maioria, senão à totalidade das células, favorecendo, desta forma, a viabilidade e proliferação celular. The present technology describes a tissue culture perfusion chamber whose fabrication material may involve different types of polymers having good thermal and electrical properties, moderate strength, easy machining and autoclaving, an essential factor when the proposal is to work. with biological material. In its physical aspect, the camera can have different formats, provided that respecting the conditions of cultivation of the fabric of interest. The developed perfusion chamber makes it possible to perform a three-dimensional culture of seeded cells in a matrix which, when properly adapted to the perfusion chamber, favors the flow of flow through this scaffold, ensuring that the medium can reach most if not all. thus favoring cell viability and proliferation.
A engenharia de tecidos foi definida como um campo de pesquisa apenas no início dos anos 80 e atualmente, o progresso neste campo está relacionado ao desenvolvimento de diversas abordagens de modelos de estudo. A engenharia de tecidos apoia sua base científica em diversas áreas do conhecimento, como biologia celular, bioquímica, biologia molecular, ciência dos materiais, engenharia química e bioengenharia, que permitem a aplicação racional dos princípios da engenharia nos sistemas vivos. O desafio científico da engenharia de tecidos envolve a compreensão dos mecanismos celulares e a fabricação de materiais para fornecer matrizes e modelos que guiem a regeneração tecidual (VACANTI, J.; VACANTI, C. The History and Scope of Tissue Engineering. Principies of Tissue Engineering, 3- ed., Elsevier, Inc, Capítulo 1 , p. 3-6,).  Tissue engineering was defined as a research field only in the early 1980s and today progress in this field is related to the development of various study model approaches. Tissue engineering supports its scientific base in many areas of knowledge, such as cell biology, biochemistry, molecular biology, materials science, chemical engineering and bioengineering, which allow the rational application of engineering principles in living systems. The scientific challenge of tissue engineering involves understanding cellular mechanisms and fabricating materials to provide matrices and models that guide tissue regeneration (VACANTI, J .; VACANTI, C. The History and Scope of Tissue Engineering. Principles of Tissue Engineering , 3rd ed., Elsevier, Inc, Chapter 1, pp. 3-6,).
As células-tronco vivas são requeridas para a fabricação de substitutos teciduais. Estas células criam ou recriam estruturas funcionais usando informações e sinalizações pré-programadas em seu código genético. A aquisição de células para a produção de estruturas orgânicas é um grande desafio, pois para a fabricação de estruturas em larga escala, há a necessidade da obtenção de grande quantidade de células que sejam imunologicamente compatíveis com o indivíduo receptor (VACANTI, J.; VACANTI, C. The History and Scope of Tissue Engineering. Principies of Tissue Engineering, 3- ed., Elsevier, Inc, Capítulo 1 , p. 3-6, 2007). A medula óssea contém células-tronco mesenquimais (MSC), mas a coleta é um procedimento invasivo. Portanto, é de relevante interesse avaliar a possibilidade de obtenção de células-tronco multipotentes de outras fontes teciduais, tais como periósteo ou tecido adiposo obtido por lipoaspiração (CANCEDDA, R.; BIANCHI, G.; DERUBEIS, A.; QUARTO, R. Celi therapy for bone disease: a review of current status. Stern Celis, v. 21 , p. 610-619, 2003). A coleta, a quantidade de células e a rápida expansão in vitro são vantagens das células-tronco derivadas do tecido adiposo em relação àquelas derivadas da medula óssea quando da contemplação de estratégias clínicas. As células- tronco mesenquimais adiposas são capazes de se diferenciar nas linhagens condrogênica, adipogênica, miogênica e osteogênica, sugerindo sua aplicabilidade na reconstituição de tecidos (ZUK, P. A.; ZHU, M.; MIZUNO, H.; HUANG, J.; FUTRELL, J. W.; KATZ, A. J.; BENHAIM, P.; LORENZ, H. P.; HEDRICK, M. H. Multilineage Celis from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineering, 7, 21 1 -228, 2001 .). Living stem cells are required for the manufacture of tissue substitutes. These cells create or recreate functional structures using preprogrammed information and signaling in their genetic code. The acquisition of cells for the production of organic structures is a major challenge, because for the manufacture of large-scale structures, there is a need to obtain large numbers of cells that are immunologically compatible with the recipient individual (VACANTI, J.; VACANTI, C. The History and Scope of Tissue Engineering. Principles of Tissue Engineering, 3rd ed., Elsevier, Inc, Chapter 1, pp. 3-6, 2007). Bone marrow contains mesenchymal stem cells (MSC), but harvesting is an invasive procedure. Therefore, it is of interest to evaluate the possibility of obtaining multipotent stem cells from other tissue sources, such as periosteum or adipose tissue obtained by liposuction (CANCEDDA, R .; BIANCHI, G .; DERUBEIS, A .; QUARTO, R. Celi therapy for bone disease: a review of current status (Stern Celis, v. 21, pp. 610-619, 2003). In vitro harvesting, cell numbers and rapid expansion are advantages of adipose-derived stem cells over bone marrow-derived stem cells when contemplating clinical strategies. Adipose mesenchymal stem cells are able to differentiate into chondrogenic, adipogenic, myogenic and osteogenic lineages, suggesting their applicability in tissue reconstitution (ZUK, PA; ZHU, M .; MIZUNO, H .; HUANG, J .; FUTRELL, JW; KATZ, AJ; BENHAIM, P.; LORENZ, HP; HEDRICK, MH Multilineage Celis from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineering, 7, 211-228, 2001.).
Para aplicação da engenharia de tecidos, grandes quantidades de células devem ser mantidas vivas, tanto in vitro, quanto in vivo. O desenvolvimento de sistemas para garantir esta sobrevivência celular inclui os sistemas de biorreatores, uma estratégia para suprir as limitações de transferência de massa que os tecidos vivos necessitam para adequada nutrição e eliminação de resíduos celulares, além de proporcionarem estímulos mecânicos específicos para o desenvolvimento tecidual através da pressão hidrodinâmica e tensões de cisalhamento (VACANTI, J.; VACANTI, C. The History and Scope of Tissue Engineering. Principies of Tissue Engineering, 3- ed., Elsevier, Inc, Capítulo 1 , p. 3-6, 2007).  For tissue engineering application, large amounts of cells must be kept alive, both in vitro and in vivo. Systems development to ensure cell survival includes bioreactor systems, a strategy to address the mass transfer limitations that living tissues need for proper nutrition and waste disposal, and to provide specific mechanical stimuli for tissue development through hydrodynamic pressure and shear stresses (VACANTI, J .; VACANTI, C. The History and Scope of Tissue Engineering. Principles of Tissue Engineering, 3rd ed., Elsevier, Inc, Chapter 1, p. 3-6, 2007) .
Os biorreatores são as principais ferramentas para mimetizar os microambientes celulares e fornecer construtos celulares com estímulos fisiologicamente relevantes que direcionem a conversão de um "conjunto de células" em um determinado tecido (FRESHNEY, R. I.; OBRADOVIC, B.; GRAYSON, W.; CANNIZZARO, C; VUNJAK-NOVAKOVIC, G. Principies of Tissue Culture and Bioreactor Design. Principies of Tissue Engineeríng, 3- ed., Elsevier, Inc, Capítulo 12, p. 155-183, 2007). Bioreactors are the main tools to mimic cellular microenvironments and provide cellular constructs with physiologically relevant stimuli that direct the conversion of a "set of cells" into a given tissue (FRESHNEY, RI; OBRADOVIC, B .; GRAYSON, W .; CANNIZZARO, C; VUNJAK-NOVAKOVIC, G. Principles of Tissue Culture and Bioreactor Design. Principies of Tissue Engineering, 3rd ed., Elsevier, Inc, Chapter 12, p. 155-183, 2007).
Acredita-se que o cisalhamento hidrodinâmico induzido pelo biorreator simula o fluxo intersticial nos espaços canaliculares durante a formação óssea natural associada à carga de compressão. As forças mecânicas resultantes são parte integral da fisiologia óssea que regula a homeostase, remodelagem e reparo ósseo, entretanto poucos estudos aplicam deformação mecânica para explorar seu papel na promoção do crescimento celular e organização tecidual (ALLORI, A. C; SAILON, A. M.; PAN, J. H.; WARREN, S. M. Biological Basis of Bone Formation, Remodeling, and Repair— Part III: Biomechanical Forces. Tissue Engineeríng: Part B, v. 14, n. 3, p. 285-293, 2008). Desse modo, o carregamento dinâmico é necessário para simular o correto microambiente para determinados tipos celulares (FRESHNEY, R. I.; OBRADOVIC, B.; GRAYSON, W.; CANNIZZARO, C; VUNJAK-NOVAKOVIC, G. Principies of Tissue Culture and Bioreactor Design. Principies of Tissue Engineeríng, 3- ed., Elsevier, Inc, Capítulo 12, p. 155-183, 2007).  The bioreactor-induced hydrodynamic shear is believed to simulate interstitial flow in the canalicular spaces during natural bone formation associated with compression loading. The resulting mechanical forces are an integral part of bone physiology that regulates homeostasis, bone remodeling and repair, however few studies apply mechanical deformation to explore their role in promoting cell growth and tissue organization (ALLORI, A. C; SAILON, AM; PAN). JR WARREN, SM Biological Basis of Bone Formation, Remodeling, and Repair — Part III: Biomechanical Forces, Tissue Engineering: Part B, v. 14, no. 3, pp. 285-293, 2008). Thus dynamic loading is required to simulate the correct microenvironment for certain cell types (FRESHNEY, RI; OBRADOVIC, B.; GRAYSON, W .; CANNIZZARO, C.; VUNJAK-NOVAKOVIC, G. Principles of Tissue Culture and Bioreactor Design. Principles of Tissue Engineering, 3rd ed., Elsevier, Inc, Chapter 12, pp. 155-183, 2007).
Os biorreatores ósseos atuais são projetados para direcionar o fluxo do meio de cultura através dos construtos, a fim de expor todas as células ao cisalhamento (BANCROFT, G.N.; SIKAVITSAS, V. I.; VAN DER DOLDER, J.; SHEFFIELD, T. L; AMBROSE, C. G.; JANSEN, J. A.; MIKOS, A. G. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose dependent manner. Proc. Natl. Acad. Sei. USA, v. 99, p. 12600-12605, 2002; SIKAVITSAS, V. I.; BANCROFT, G. N.; HOLTORF, H. L; JANSEN, J. A.; MIKOS, A. G. Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces. Proc Natl Acad Sei USA., v. 100, p. 14683-14688, 2003; BRACCINI, A.; WENDT, D.; JAQUIERY, C; JAKOB, M.; HEBERER, M.; KENINS, L; WODNAR-FILIPOWICZ, A.; QUARTO, R.; MARTIN, I. Three-Dimensional Perfusion Culture of Human Bone Marrow Celis and Generation of Osteoinductive Grafts. Stern Celis, v. 23, p. 1066-1072, 2005; HOLTORF, H. L; SHEFFIELD, T. L; AMBROSE, C. G.; JANSEN, J. A.; MIKOS, A. G. Flow perfusion culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics. Ann Biomed Eng 33, 1238-1248, 2005; ALLORI, A. C; SAILON, A. M.; PAN, J. H.; WARREN, S. M. Biological Basis of Bone Formation, Remodeling, and Repair— Part III: Biomechanical Forces. Tissue Engineeríng: Part B, v. 14, n. 3, p. 285-293, 2008; FROLICH, M.; GRAYSON, W. L; MAROLT, D.; GIMBLE, J. M.; KREGAR-VELIKONJA, N.; VUNJAK- NOVAKOVIC, G. Bone Grafts Engineered from Human Adipose-Derived Stern Cells in Perfusion Bioreactor Culture. Tissue Engineeríng. Part A, v. 16, n. 1 , p. 179-189 2010). Current bone bioreactors are designed to direct the flow of culture medium through the constructs to expose all cells to shear (BANCROFT, GN; SIKAVITSAS, VI; VAN DER DOLDER, J .; SHEFFIELD, T. L; AMBROSE. , CG; JANSEN, JA; MIKOS, AG Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose dependent manner Proc. Natl. Acad. Sci. USA, v. 99, pp. 12600-12605, 2002; SIKAVITSAS, VI; BANCROFT, GN; HOLTORF, H.L; JANSEN, JA; MIKOS, AG Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture with increasing fluid shear forces Proc Natl Acad Sci USA, v. 100, pp. 14683-14688, 2003; BRACCINI, A .; WENDT, D.; JAQUIERY, C.; JAKOB, M.; HEBERER, M .; KENINS, L.; WODNAR-FILIPOWICZ, A .; QUARTO, R .; MARTIN, I. Three-Dimensional Perfusion Culture of Human Bone Marrow Celis and Generation of Osteoinductive Grafts Stern Celis, v. 23, pp. 1066-1072, 2005; HOLTORF, H.L; SHEFFIELD, T. L. AMBROSE, CG; JANSEN, JA; MIKOS, AG Flow perfusion culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics. Ann Biomed Eng 33, 1238-1248, 2005; ALLORI, A. C; SAILON, AM; PAN, JH; WARREN, SM Biological Basis of Bone Formation, Remodeling, and Repair — Part III: Biomechanical Forces. Tissue Engineering: Part B, v. 14, no. 3, p. 285-293, 2008; FROLICH, M .; GRAYSON, W.L; MAROLT, D .; GIMBLE, JM; KREGAR-VELIKONJA, N .; VUNJAK- NOVAKOVIC, G. Bone Grafts Engineered from Human Adipose-Derived Stern Cells in Perfusion Bioreactor Culture. Tissue Engineering. Part A, v. 16, no. 1, p. 179-189 2010).
O uso de câmaras de perfusão em um sistema de biorreator tem por finalidade aumentar o fornecimento de material nutritivo para células e remover os metabólitos gerados pela atividade celular, viabilizando dessa forma, um ambiente favorável para crescimento das células (Dick, D. An Easily Made Tissue Culture Perfusion Chamber. Quarterly Journal of Microscopical Science p. 363, 1995). Desde sua invenção em meados de 1907, à medida que novas técnicas de cultivo de células e tecidos surgiam, novos modelos de câmaras de perfusão foram desenvolvidos (Wolf, D; Sluder, G. Methods in Celi Biology, v. 56, p. 256, 1998). Atualmente, ainda não há um consenso na literatura de um modelo padrão de câmara de perfusão e biorreator para o cultivo de células para a engenharia de tecidos e diversos laboratórios no mundo estão à procura disso. Dentro desse contexto, o presente trabalho tem como objetivo principal a construção de uma câmara de perfusão adequada para cultivo de tecidos, baseando sua eficiência em uma análise da diferenciação celular de células-tronco do tecido adiposo humano, semeadas em uma matriz de espuma de vidro bioativo e cultivadas em um sistema de biorreator para a produção de um construto a ser aplicado na engenharia de tecidos.  The use of perfusion chambers in a bioreactor system is intended to increase the supply of nutritive material to cells and remove metabolites generated by cell activity, thereby enabling a favorable environment for cell growth (Dick, D. An Easily Made Tissue Culture Perfusion Chamber, Quarterly Journal of Microscopical Science (363, 1995). Since its invention in the mid-1907, as new cell and tissue culture techniques have emerged, new models of perfusion chambers have been developed (Wolf, D; Sluder, G. Methods in Celi Biology, v. 56, p. 256 , 1998). Currently, there is still no consensus in the literature on a standard perfusion chamber and bioreactor model for tissue engineering for cell engineering and several laboratories around the world are looking for this. Within this context, the present work has as main objective the construction of a perfusion chamber suitable for tissue culture, basing its efficiency on an analysis of the cellular differentiation of human adipose tissue stem cells, sown in a glass foam matrix. bioactive and grown in a bioreactor system for the production of a construct to be applied in tissue engineering.
Dentre as possibilidades de matrizes específicas para cultivo de tecidos, a escolhida para ser aplicada nos experimentos foram espumas de vidro bioativo produzidas pelo processo sol-gel (Hiratsuka, R. et a\. O processo sol- gekllma revisão bibliográfica, p. 171 -179, 1994). Alguns estudos mostraram que suas características de bioatividade, degradabilidade e estrutura de poros são adequadas aos ensaios in vitro e in vivo (COELHO, M. B. Desenvolvimento de metodologia para produção de estruturas tridimensionais porosas de vidro bioativo para aplicação na engenharia de tecidos. Belo Horizonte: Escola de Engenharia da UFMG, 2003. 145p. (Tese, Doutorado em Engenharia Metalúrgica e de Materiais).; PEREIRA, M. M.; JONES, J. R..; HENCH, L. L. Bioactive glass and hybrid scaffolds prepared by sol-gel method for bone tissue engineering. Advances in Applied Ceramics, v. 104, n. 1 , p. 35-42, 2005; VALÉRIO, P.; GUIMARÃES, M. H. R.; PEREIRA, M. M.; LEITE, M. F.; GOES, A. M. Primary osteoblast cell response to sol-gel derived bioactive glass foams. Journal of Materials Science: Materials in Medicine, v. 16, p. 851 - 856, 2005; DUTRA, C. E. A.; PEREIRA, M. M.; SERAKIDES, R.; REZENDE, C. M. F. In vivo evaluation of bioactive glass foams associated with platelets rich plasma in bone defects. J Tissue Eng Regen Med, v. 2, p. 221 -227, 2008). Among the possibilities of specific matrices for tissue culture, the one chosen to be applied in the experiments were bioactive glass foams produced by the sol-gel process (Hiratsuka, R. et al.) The process solgllma bibliographic review, p. 171 - 179, 1994). Some studies have shown that its bioactivity, degradability and pore structure characteristics are suitable for in vitro and in vivo testing (COELHO, MB Development of methodology for the production of porous three-dimensional bioactive glass structures for application in tissue engineering. Belo Horizonte: UFMG School of Engineering, 2003. 145p. (Thesis, PhD in Metallurgical and Materials Engineering) .; PEREIRA, MM; JONES, JR.; HENCH, LL Bioactive glass and hybrid scaffolds prepared by sol-gel method for bone tissue engineering. Advances in Applied Ceramics, v. 104, paragraph 1, p 35-42, 2005; VALÉRIO, P .; GUIMARÃES, MHR; PEREIRA, MM; MILK, MF; GOES, AM Primary osteoblast cell response to soluble gel derived from bioactive glass foams Journal of Materials Science: Materials in Medicine, v 16, pp. 851 - 856, 2005; DUTRA, CEA; PEREIRA, MM; SERAKIDES, R.; REZENDE, CMF In vivo evaluation of bioactive glass foams associated with rich plasma platelets in bone defects J Tissue Eng Regen Med, v 2, pp 221-227, 2008).
O desenvolvimento da presente tecnologia se mostrou bastante eficiente para o cultivo de células-tronco derivadas do tecido adiposo humano que, quando semeadas no vidro bioativo, foram capazes de proliferar e se diferenciar no fenótipo de interesse, produzindo proteínas típicas de células osteogênicas e expressando genes característicos de osteoblastos. Essa eficiência pode ser expandida para outros tecidos, desde que adaptadas as condições, a velocidade do fluxo, a matriz de cultivo e as dimensões da câmara de perfusão.  The development of the present technology proved to be very efficient for the cultivation of stem cells derived from human adipose tissue which, when sown in bioactive glass, were able to proliferate and differentiate into the phenotype of interest, producing typical osteogenic cell proteins and expressing genes. characteristic of osteoblasts. This efficiency can be expanded to other tissues as long as conditions, flow rate, culture matrix and perfusion chamber dimensions are adapted.
É importante destacar que estudos semelhantes encontrados na literatura não demonstraram a diferenciação osteogênica de células cultivadas em matrizes e em biorreatores de perfusão, demonstrando que o fluxo do biorreator não foi suficiente para induzir a diferenciação das células-tronco derivadas do tecido adiposo, mesmo quando cultivadas por 5 semanas (FROLICH, M.; GRAYSON, W. L; MAROLT, D.; GIMBLE, J. M.; KREGAR- VELIKONJA, N.; VUNJAK-NOVAKOVIC, G. Bone Grafts Engineered from Human Adipose-Derived Stern Celis in Perfusion Bioreactor Culture. Tissue Engineering. Part A, 16 (1 ), 2010).  It is important to highlight that similar studies found in the literature did not demonstrate osteogenic differentiation of cells cultured in matrices and perfusion bioreactors, showing that bioreactor flow was not sufficient to induce differentiation of adipose-derived stem cells, even when cultured for 5 weeks (FROLICH, M .; GRAYSON, W.L; MAROLT, D .; GIMBLE, JM; KREGAR-VELIKONJA, N.; VUNJAK-NOVAKOVIC, G. Bone Grafts Engineered from Human Adipose-Derived Stern Celis in Perfusion Bioreactor Culture, Tissue Engineering, Part A, 16 (1), 2010).
Também, não foram encontrados, no estado da técnica, documentos que descrevam a elaboração de uma câmara de perfusão semelhante a presente invenção. O documento US20110229970, intitulado: Dual-Chamber Perfusion Bioreactor for Orthopedic Tissue Interfaces and Methods of Use, relata o uso de uma matriz tridimensional aplicada em múltiplas câmaras de perfusão. Este documento relata a produção de um biorreator, no entanto, as câmaras de perfusão produzidas, que podem ser alimentadas por um fluxo paralelo ou transversal, compartilham da mesma fonte de meio de cultura e inoculo, o que pode facilitar a comparação de várias condições de operação. Porém, este sistema é limitante em se tratando do controle individual do meio de cultura que alimenta cada câmara, pois aumenta o risco de contaminação entre as câmaras. Also, no prior art describing the design of a perfusion chamber similar to present invention. US20110229970, entitled Dual-Chamber Perfusion Bioreactor for Orthopedic Tissue Interfaces and Methods of Use, reports the use of a three-dimensional matrix applied in multiple perfusion chambers. This document reports the production of a bioreactor, however, the produced perfusion chambers, which may be fed by a parallel or transverse flow, share the same source of culture medium and inoculum, which may facilitate the comparison of various conditions of perfusion. operation. However, this system is limiting when it comes to the individual control of the culture medium that feeds each chamber, because it increases the risk of contamination between the chambers.
BREVE DESCRIÇÃO DAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figura 1 : Ilustração do esquema de organização do biorreator: Estufa a 37QC (1 ) , Biomaterial (2), Câmara de perfusão (3), Meio de Cultura + O2 (4), Bomba Peristáltica (5), Tubos de Conexão Dentro da Estufa de CO2 (6). Figure 1: Illustration of bioreactor organization scheme: Greenhouse at 37 Q C (1), Biomaterial (2), Infusion Chamber (3), Culture Medium + O2 (4), Peristaltic Pump (5), Connection Tubes Inside the CO 2 Greenhouse (6).
Figura 2: Mostra um a representação não limitante da câmara de perfusão. Onde: Entrada de Tubos Conectores (7), Parte Interna da Câmara para adaptação da matriz (8), Saída de Tubos Conectores (9) Figure 2: Shows a non-limiting representation of the perfusion chamber. Where: Connector Pipe Inlet (7), Inside Chamber for Matrix Adaptation (8), Connector Pipe Outlet (9)
Figura 3: Espuma de vidro bioativo recém-semeada com hASC. Figure 3: Newly seeded hASC bioactive glass foam.
Figura 4: Imagens por microscopia eletrônica de varredura (MEV) em 21 dias de cultivo no biorreator evidenciando: (A) a superfície da amostra de vidro bioativo com vários aglomerados de células aderidas (indicados pelas setas); (B) um poro da amostra com várias células aderidas, (C) maior aumento do poro evidenciando melhor as células e a rugosidade do material, e (D) maior aumento das hASC aderidas ao material e grânulos de secreção na membrana plasmática celular. Figure 4: Scanning electron microscopy (SEM) images at 21 days of cultivation in the bioreactor showing: (A) the surface of the bioactive glass sample with several attached cell clusters (indicated by arrows); (B) a pore of the sample with several adhered cells, (C) larger pore enlargement showing better cells and material roughness, and (D) greater increase in material-adhered hASC and secretion granules in the cell plasma membrane.
Figura 5: Gráfico representativo da viabilidade e proliferação das hASC cultivadas em meio normal (estático) e em matrizes de vidro bioativo (dinâmico), contendo meio leibovitz (LEI) ou meio leibovitz acrescido de fatores osteogênicos (LEI O, na legenda: estático O e dinâmico O). Todos os tempos entre estático e dinâmico e entre estático O e dinâmico O são p<0,001 ).  Figure 5: Representative graph of the viability and proliferation of hASC grown in normal (static) medium and in bioactive (dynamic) glass matrices containing leibovitz (LEI) or leibovitz medium plus osteogenic factors (LEI O in the caption: static O and dynamic O). All times between static and dynamic and between static O and dynamic O are p <0.001).
Figura 6: Gráfico representativo da atividade da fosfatase alcalina das hASC cultivadas em matrizes de vidro bioativo contendo meio leibovitz (LEI) ou meio leibovitz acrescido de fatores osteogênicos (LEI O, na legenda: estático O e dinâmico O). De maneira estática e dinâmica (todos os tempos entre estático e dinâmico e entre estático O e dinâmico O são ***p<0,001 ) Figure 6: Representative graph of alkaline phosphatase activity of hASC grown in bioactive glass matrices containing leibovitz medium (LEI) or medium leibovitz plus osteogenic factors (LAW O in the caption: static O and dynamic O). Statically and dynamically (all times between static and dynamic and between static O and dynamic O are *** p <0.001)
Figura 7: Análise de imunofluorescência para as proteínas (A) osteocalcina, (B) osteopontina e (C) colágeno tipo I de hASC semeadas em matriz de vidro bioativo e cultivadas em meio de cultura basal em biorreator de perfusão após 21 dias. Os núcleos celulares apresentam marcação em azul e as proteínas específicas em verde. Figure 7: Immunofluorescence analysis for (A) osteocalcin, (B) osteopontin and (C) hASC type I collagen seeded in bioactive glass matrix and cultured in basal culture medium in perfusion bioreactor after 21 days. Cell nuclei have blue marking and specific proteins green.
Figura 8: Análise de imunofluorescência para a proteína osteocalcina de hASC semeadas em matriz de vidro bioativo e cultivadas em meio de cultura osteogênico em biorreator de perfusão após (A) 7, (B) 14 e (C) 21 dias.  Figure 8: Immunofluorescence analysis for hASC osteocalcin protein seeded in bioactive glass matrix and cultured in osteogenic culture medium in perfusion bioreactor after (A) 7, (B) 14 and (C) 21 days.
Figura 9: Análise de imunofluorescência para a proteína osteopontina de hASC semeadas em matriz de vidro bioativo e cultivadas em meio de cultura osteogênico em biorreator de perfusão após (A) 7, (B) 14 e (C) 21 dias. Figure 9: Immunofluorescence analysis for hASC osteopontin protein sown in bioactive glass matrix and cultured in osteogenic culture medium in perfusion bioreactor after (A) 7, (B) 14 and (C) 21 days.
Figura 10: Análise de imunofluorescência para a proteína colágeno tipo I de hASC semeadas em matriz de vidro bioativo e cultivadas em meio de cultura osteogênico em biorreator de perfusão após (A) 7, (B) 14 e (C) 21 dias. Figure 10: Immunofluorescence analysis for hASC type I collagen protein sown in bioactive glass matrix and cultured in osteogenic culture medium in perfusion bioreactor after (A) 7, (B) 14 and (C) 21 days.
Figura 11 : Detecção de osteocalcina, osteopontina e fosfatase alcalina em 7, 14 e 21 dias em hASC cultivadas sobre a matriz de vidro bioativo, em meio LEI O e no biorretor de perfusão. Células de osteossarcoma humano (SÃOS) como controle positivo e C- é o controle negativo. Figure 11: Detection of osteocalcin, osteopontin and alkaline phosphatase at 7, 14 and 21 days in hASC cultured on the bioactive glass matrix, LEI O medium and perfusion biorector. Human osteosarcoma (SOS) cells as positive control and C- is negative control.
DESCRIÇÃO DETALHADA DA TECNOLOGIA DETAILED DESCRIPTION OF TECHNOLOGY
A presente tecnologia consiste em uma câmara de perfusão (Figura 2) para cultivo de tecidos, cujo material de confecção pode envolver diferentes tipos de polímeros que possuam boas propriedades térmicas e elétricas, moderada resistência, de fácil usinagem e passível de ser autoclavado, fator essencial quando a proposta é trabalhar com material biológico. Preferencialmente, utiliza-se o polipropileno, uma resina de baixa densidade que oferece bom equilíbrio de propriedades térmicas e elétricas, além de moderada resistência, ser atóxico, de fácil usinagem e permite autoclavagem. Em seu aspecto físico, a câmara pode apresentar diferentes formatos e dimensões, desde que respeitando as condições de cultivo do tecido de interesse. A matriz de escolha para o cultivo de determinado tecido deve ser corretamente adaptada à câmara de perfusão, para favorecer a passagem do fluxo através da matriz, garantindo que o meio possa chegar à maioria, senão à totalidade das células, favorecendo, desta forma, a viabilidade e proliferação celular. A câmara de perfusão foi projetada com uma entrada e uma saída (7 e 9, respectivamente) para permitir a perfusão do meio de cultivo por todo o seu interior, onde o construto (biomaterial e células semeadas) deve estar adequadamente adaptado. The present technology consists of a tissue culture perfusion chamber (Figure 2), the fabric of which may involve different types of polymers which have good thermal and electrical properties, moderate strength, easy machining and can be autoclaved, an essential factor. when the proposal is to work with biological material. Preferably, polypropylene is used, a low density resin that offers a good balance of thermal and electrical properties, as well as moderate strength, is nontoxic, easily machined and allows autoclaving. In its physical aspect, the camera can have different formats and dimensions, as long as it respects the conditions of interest. The matrix of choice for the cultivation of a given tissue must be correctly adapted to the perfusion chamber to favor the flow of the flow through the matrix, ensuring that the medium can reach most if not all cells, thus favoring the viability and cell proliferation. The perfusion chamber was designed with one inlet and one outlet (7 and 9, respectively) to allow perfusion of the culture medium throughout its interior, where the construct (biomaterial and seeded cells) must be adequately adapted.
O material da matriz (2), bem como o formato da câmara, variam de acordo como tipo celular do tecido escolhido para cultivo. A câmara de perfusão (Figura 2) desenvolvida pode ser mantida em diferentes ambientes, desde uma estufa tipo B.O.D {Biochemical Oxygen Demand) até um banho- maria, desde que respeitadas as condições necessárias para o cultivo. A câmara de perfusão proposta para compor o sistema biorreator (3) foi capaz de promover, de forma eficiente, o cultivo de tecido ósseo.  The matrix material (2) as well as the chamber shape vary according to the cell type of the tissue chosen for cultivation. The perfusion chamber (Figure 2) developed can be maintained in different environments, from a B.O.D (Biochemical Oxygen Demand) greenhouse to a water bath, provided the necessary conditions for cultivation are respected. The perfusion chamber proposed to compose the bioreactor system (3) was able to efficiently promote bone tissue culture.
O biorreator de perfusão desenvolvido (1 ) é um sistema fechado, passivo de sofrer esterilização, onde o meio de cultura celular é constantemente bombeado a uma taxa de fluxo constante por meio de uma bomba peristáltica (5) e através de mangueiras conectadas (6) a uma das extremidades da câmara de perfusão. Assim, o meio bombeado passa através do construto e retorna através das mangueiras de conexão, ligadas à outra extremidade das câmaras, ao reservatório de meio de cultura, conforme esquematizado na Figura 1 .  The developed perfusion bioreactor (1) is a closed, passive sterilization system where the cell culture medium is constantly pumped at a constant flow rate through a peristaltic pump (5) and through connected hoses (6) to one end of the infusion chamber. Thus, the pumped medium passes through the construct and returns through the connecting hoses, connected at the other end of the chambers, to the culture medium reservoir as shown in Figure 1.
Os tubos escolhidos para conectar o sistema do biorreator são tubos peristálticos calibrados de silicone com 2,6 mm de diâmetro interno. Estes tubos foram escolhidos por serem autoclaváveis, translúcidos e apresentarem biocompatibilidade e elevada permeabilidade a gases. O reservatório para os meios de cultura são garrafas de vidro com tampa rosqueada. Nas tampas das garrafas, foram confeccionados três furos, dois deles para a adaptação das mangueiras de conexão - um para entrada e outro para saída do meio de cultura - e um deles para a adaptação de um filtro de respiro, com o intuito de evitar a formação de vácuo no interior das garrafas durante os testes e possibilidade de ser mais uma via de troca de gases. The tubes chosen to connect the bioreactor system are calibrated silicone peristaltic tubes with 2.6 mm internal diameter. These tubes were chosen because they are autoclavable, translucent and have biocompatibility and high gas permeability. The reservoir for the culture media is screw-capped glass bottles. In the bottle caps, three holes were drilled, two of them for fitting the connection hoses - one for entering and one for leaving the culture medium - and one for fitting a breather filter to avoid the formation of vacuum inside the bottles during the tests and possibility of being another way of gas exchange.
A bomba peristáltica de escolha produz um fluxo suave e livre de pulsos, possui 8 canais, ou seja, permite o trabalho de até 8 câmaras de perfusão simultaneamente, alimentadas por um fluxo constante de meio de cultura, de 0,05mL/min até 40mL/min por canal. O meio de cultura do biorreator foi parcialmente trocado a cada sete dias durante o cultivo celular, sendo removido apenas metade do meio com o objetivo de não eliminar os novos componentes produzidos pelas células, como os fatores de crescimento, hormônios e enzimas, que são importantes na regulação dos próprios mecanismos celulares (GOLDSTEIN, A. S.; JUAREZ, T. M.; HELMKE, C.D.; GUSTIN, M. C; Mikos, A. G.Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. Biomaterials, v. 22p. 1279- 1288, 2001 ; MARTIN, Y.; VERMETTE, P. Bioreactors for tissue mass culture: Design, characterization, and recent advances. Biomaterials, v. 26, p. 7481- 7503, 2005.).  The peristaltic pump of choice produces smooth, pulse-free flow, has 8 channels, allowing up to 8 perfusion chambers to work simultaneously, fed by a constant flow of culture medium from 0.05mL / min to 40mL / min per channel. The bioreactor culture medium was partially changed every seven days during cell culture, and only half of the medium was removed in order not to eliminate the new components produced by cells, such as growth factors, hormones and enzymes, which are important. in the regulation of the cellular mechanisms themselves (GOLDSTEIN, AS; JUAREZ, TM; HELMKE, CD; GUSTIN, M. C; Mikos, AGE). Convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. Biomaterials, v. 22p. 1279 - 1288, 2001; MARTIN, Y; VERMETTE, P. Bioreactors for Tissue Mass Culture: Design, Characterization, and Recent Advances. Biomaterials, v. 26, pp. 7481-7503, 2005.).
As condições fisiológicas de temperatura e pH do biorreator foram controladas. A temperatura de 37QC foi garantida pela manutenção das mangueiras e reservatórios de meio de cultura em banho e monitorada diariamente com o auxílio de um termómetro. Já o pH foi monitorado a cada troca de meio de cultura com o auxílio de um pHmetro. O método escolhido para esterilização dos acessórios do biorreator foi a autoclavagem. The physiological conditions of bioreactor temperature and pH were controlled. The temperature of 37 Q C was assured by maintaining the hose and reservoirs in the culture medium bath and monitored daily with the aid of a thermometer. Already the pH was monitored with each change of culture medium with the aid of a pH meter. The method chosen for sterilization of bioreactor accessories was autoclaving.
Foram utilizadas células-tronco do tecido adiposo (hASC) para diferenciação em tecido ósseo. Para tanto, a câmara foi confeccionada com polipropileno e em formato cilíndrico, obedecendo as seguintes dimensões internas: 7mm de diâmetro e 10mm de comprimento (8). Em cada extremidade da câmara existem orifícios por onde são conectados tubos de condução que ligam a câmara ao meio de cultura e a uma bomba propulsora (7 e 9, respectivamente).  Adipose tissue stem cells (hASC) were used for differentiation into bone tissue. To this end, the chamber was made of polypropylene and in a cylindrical shape, obeying the following internal dimensions: 7mm in diameter and 10mm in length (8). At each end of the chamber are holes through which conduction tubes are connected which connect the chamber to the culture medium and a propellant pump (7 and 9, respectively).
As hASC foram cultivadas em matriz de espuma de vidro bioativo, com dimensões adaptadas às dimensões do biorreator, e foram colocadas em câmaras individuais de perfusão conectadas, cada uma, a um reservatório individual contendo meio Leibovitz, acrescido de fatores osteogênicos, e mantidas em banho-maria a 37°C durante 7,14 e 21 dias. The hASCs were grown on bioactive glass foam matrix, with dimensions adapted to the bioreactor dimensions, and were placed in individual perfusion chambers, each connected to a reservoir. containing Leibovitz medium plus osteogenic factors and kept in a water bath at 37 ° C for 7.14 and 21 days.
Para análise da eficiência da câmara de perfusão no cultivo de tecidos, no caso o tecido ósseo, foram analisados: viabilidade e proliferação celular, atividade da enzima fosfatase alcalina, marcadores de células osteogênicas, bem como morfologia e expressão gênica. Para melhor compreensão, a presente tecnologia será descrita a seguir, de forma não limitante.  To analyze the efficiency of the perfusion chamber in tissue culture, in this case bone tissue, we analyzed: cell viability and proliferation, alkaline phosphatase enzyme activity, osteogenic cell markers, as well as morphology and gene expression. For a better understanding, the present technology will be described below without limitation.
Exemplo 1- Produção da matriz para a câmara de perfusão Example 1- Matrix Production for the Infusion Chamber
Inicialmente, realizou-se o processamento do biomaterial escolhido para cultivo das hASC, no caso, a espuma de vidro bioativo, e sua caracterização para que fossem verificadas as suas características químicas e estruturais.  Initially, the biomaterial chosen for the cultivation of hASC was processed, in this case, the bioactive glass foam, and its characterization so that its chemical and structural characteristics could be verified.
A síntese de espumas de vidro bioativo contendo 60%-SiO2-36%CaO- 4%P2O5 pelo método sol-gel - rota alcoóxido - levou à obtenção de matrizes tridimensionais com aspecto macroscópico esponjoso, de cor branca e medindo 6 mm de diâmetro e 20 mm de comprimento no molde pequeno. A estrutura das espumas de vidro bioativo foi avaliada, preliminarmente, através de microscopia eletronica de varredura (MEV) e observou-se que estas apresentavam elevada porosidade, com poros esféricos, tamanhos médios de poros variando entre 100 e 500pm e uma rede interconectada de poros. A espectroscopia de raios X por dispersão de energia, realizada em conjunto com a MEV, foi utilizada para analisar a composição química qualitativa das espumas de vidro bioativo produzidas. A análise realizada em diversas áreas das espumas de vidro bioativo detectou a presença dos elementos químicos silício (Si), cálcio (Ca), fósforo (P) e ouro (Au), sendo este último, devido ao processo de metalização das matrizes. A análise do difratograma de raios X da amostra de espuma de vidro bioativo apresenta um material caracteristicamente amorfo. The synthesis of bioactive glass foams containing 60% -SiO 2 -36% CaO- 4% P 2 O5 by the sol-gel method - alkoxide route - led to the obtaining of three-dimensional white spongy macroscopic matrices measuring 6 mm in diameter and 20 mm in length in the small mold. The structure of the bioactive glass foams was preliminarily evaluated by scanning electron microscopy (SEM) and it was observed that they had high porosity, with spherical pores, average pore sizes ranging from 100 to 500pm and an interconnected pore network. . Energy dispersion X-ray spectroscopy, performed in conjunction with SEM, was used to analyze the qualitative chemical composition of the bioactive glass foams produced. The analysis performed in various areas of bioactive glass foams detected the presence of the chemical elements silicon (Si), calcium (Ca), phosphorus (P) and gold (Au), the latter being due to the matrix metallization process. The X-ray diffractogram analysis of the bioactive glass foam sample presents a characteristically amorphous material.
Exemplo 2- Montagem da Câmara e do Biorreator  Example 2- Mounting the Camera and Bioreactor
O sistema do biorreator de perfusão foi adaptado para o uso dentro de um banho-maria com o objetivo de manter a temperatura de 37QC, ideal para o cultivo celular.. Para garantir a manutenção do pH das culturas celulares, foi testado o meio Leibovitz Independente de CO2, que é capaz de manter a estabilidade do pH a longo prazo sob CO2 atmosférico. The perfusion bioreactor system was adapted for use in a water bath in order to maintain the temperature of 37 Q C, ideal for cell culture .. To ensure the maintenance of the pH of the cell culture was Leibovitz tested Independent CO 2, which is able to maintain pH stability in the long term under atmospheric CO 2.
Exemplo 3- Isolamento e Cultivo das Células-Tronco a Partir do Tecido Adiposo Humano  Example 3- Isolation and Cultivation of Stem Cells from Human Adipose Tissue
O isolamento e o cultivo das células-tronco a partir do tecido adiposo humano (hASC) foram realizados conforme a descrição de Zuk e colaboradores (ZUK, P. A.; ZHU, M.; MIZUNO, H.; HUANG, J.; FUTRELL, J. W.; KATZ, A. J.; BENHAIM, P.; LORENZ, H. P.; HEDRICK, M. H. Multilineage Celis from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineeríng, 7, 21 1 -228, 2001 ). A partir da extração das hASC, alguns testes estáticos foram delineados para adaptar o cultivo ao meio independente de CO2 (LEI) e, em seguida, avaliar se esse meio seria capaz de manter as características fenotípicas, morfológicas e de proliferação e diferenciação celular com a mesma qualidade que o meio DMEM padronizado pela comunidade científica. O meio de cultura basal para cultivo das hASC no biorreator de perfusão passou a ser o meio LEI com adaptação mais gradual desde a primeira passagem. Para induzir osteogênese, foram adicionados ao meio LEI: 50 pg/mL de ácido ascórbico, 10 mM de β-glicerofosfato e 0, 1 μΜ dexametasona, constituindo assim o meio LEI O. Isolation and cultivation of stem cells from human adipose tissue (hASC) was performed as described by Zuk and colleagues (ZUK, PA; ZHU, M .; MIZUNO, H .; HUANG, J .; FUTRELL, JW. ; KATZ, AJ; BENHAIM, P.; LORENZ, HP; HEDRICK, MH Multilineage Celis from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineering, 7, 211-228, 2001). From the extraction of hASC, some static tests were designed to adapt the culture to CO 2 independent medium (LEI) and then to evaluate if this medium would be able to maintain the phenotypic, morphological and proliferation and differentiation characteristics with same quality as the DMEM medium standardized by the scientific community. The basal culture medium for culturing hASC in the perfusion bioreactor became the LEI medium with more gradual adaptation since the first pass. To induce osteogenesis, 50 µg / mL ascorbic acid, 10 mM β-glycerophosphate and 0.1 µm dexamethasone were added to the LEI medium, thus constituting the LEI O medium.
As hASC isoladas a partir da fração vascular estromal resultante do processamento dos lipoaspirados demonstrou resultados satisfatórios, apresentando células com formato tipo fibroblastóide e aderentes a superfície plástica, corroborando com os dados de Zuk (ZUK, P. A.; ZHU, M.; MIZUNO, H.; HUANG, J.; FUTRELL, J. W.; KATZ, A. J.; BENHAIM, P.; LORENZ, H. P.; HEDRICK, M. H. Multilineage Celis from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineeríng, 7, 21 1 -228, 2001 ).  The hASCs isolated from the stromal vascular fraction resulting from the liposuction processing showed satisfactory results, presenting cells with fibroblast-like shape and adhering to the plastic surface, corroborating the data of Zuk (ZUK, PA; ZHU, M .; MIZUNO, H. ; HUANG, J .; FUTRELL, JW; KATZ, AJ; BENHAIM, P.; LORENZ, HP; HEDRICK, MH Multilineage Celis from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineering, 7, 21 1 -228, 2001).
Exemplo 4- Efeito do Cultivo Dinâmico em Biorreator de Perfusão Sobre a Diferenciação Osteogênica das hASC Example 4- Effect of Dynamic Cultivation on Perfusion Bioreactor on hASC Osteogenic Differentiation
Sobre as amostras de espumas de vidro bioativo (Figura 3) descritas no Exemplo 1 , foram semeadas 3x105 células/matriz. O número de células utilizado neste ensaio foi três vezes maior que o número de células dos ensaios estáticos, devido à diferença no comprimento das amostras, que corresponde a uma área proporcionalmente três vezes maior. As amostras foram mantidas em atmosfera úmida com 5% CO2 por 4 horas para a adesão celular. Foram então colocadas em câmaras individuais de perfusão e cultivadas por 7, 14 e 21 dias em banho-maria a 37QC. A taxa de perfusão utilizada para todos os testes foi de 0,1 mL/min, valor estimado como uma média utilizada na literatura para a diferenciação de células-tronco em células ósseas. Cada câmara de perfusão foi conectada a um reservatório individual contendo 150ml_ de meio LEI ou LEI O, onde metade do volume do meio foi trocado a cada 7 dias. On the bioactive glass foam samples (Figure 3) described in Example 1, 3x10 5 cells / matrix were seeded. The number of cells used in this assay was three times greater than the number of static assay cells due to the difference in sample length, which corresponds to an area proportionally three times larger. The samples were kept in a humid atmosphere with 5% CO 2 for 4 hours for cell adhesion. They were then placed in individual perfusion chambers and cultured for 7, 14 and 21 days in a water bath at 37 Q C. The perfusion rate used for all tests was 0.1 ml / min, estimated as an average used in the literature for the differentiation of stem cells into bone cells. Each perfusion chamber was connected to an individual reservoir containing 150 ml of LEI or LEI O medium, where half of the volume of medium was changed every 7 days.
Como as amostras utilizadas nos testes estáticos (Exemplo 2) apresentavam 3mm de comprimento e as amostras cultivadas no biorreator de perfusão apresentavam 10mm, todas as amostras foram cortadas em três partes e submetidas às análises biológicas subsequentes, para possibilitar a comparação dos resultados do cultivo estático e dinâmico. Após 7, 14 e 21 dias, as matrizes colonizadas foram preparadas para análise por MEV para confirmar a presença e adesão das hASC à superfície e interior das espumas colonizadas cultivadas no biorreator de perfusão (Figura 4).  Since the samples used in the static tests (Example 2) were 3mm long and the samples grown in the perfusion bioreactor were 10mm, all samples were cut into three parts and subjected to subsequent biological analysis to allow comparison of the static culture results. and dynamic. After 7, 14 and 21 days, colonized matrices were prepared for SEM analysis to confirm the presence and adhesion of hASC to the surface and interior of colonized foams cultured in the perfusion bioreactor (Figure 4).
A proliferação e viabilidade das hASC semeadas nas espumas de vidro bioativo e cultivadas no biorreator de perfusão com os meios LEI e LEI O foram avaliadas pelo ensaio de MTT (Figura 5), baseado em um método colorimétrico que avalia a capacidade de enzimas desidrogenases, presentes em células viáveis em converter o sal de brometo de 3-(4,5-dimetiltiazol-2-il)-2,5- difeniltetrazolium, solúvel em água, em cristais de formazan, produto insolúvel em água (MOSMANN, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. Journal of Immunological Methods, v. 65, p. 55-63, 1983). Os cristais de formazan são solubilizados e a densidade ótica pode ser determinada pelo espectrofotômetro a 595 nm. O número de células viáveis é diretamente proporcional à quantidade de cristais de formazan produzidos. As espumas de vidro bioativo colonizadas por hASC e cultivadas no biorreator de perfusão com meio LEI e LEI O foram avaliadas, após cada intervalo de cultivo, em relação a atividade da fosfatase alcalina das hASC pelo ensaio de BCIP/NBT (Figura 6), baseado na reação cromogênica decorrente da clivagem de um grupamento fosfato do BCIP (5-bromo 4-cloro 3-indolilfosfato p-toluidina) pela fosfatase alcalina produzida pela célula. Essa reação produz um próton que reduz o NBT (nitroblue tetrazólio clorídrico), formando um precipitado insolúvel de cor púrpura (SMEJKAL, G. B.; KAUL, CA. Stability of nitroblue tetrazolium-based alkaline phosphatase substrates. J. Histochem. Cytochem., 49 (9), 1 189-1 190, 2001 ). The proliferation and viability of hASC sown in bioactive glass foams and cultured in the perfusion bioreactor with LEI and LEI O media were evaluated by MTT assay (Figure 5), based on a colorimetric method that assesses the capacity of enzymes dehydrogenases present. in cells viable in converting the water-soluble 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide salt to formazan crystals, water-insoluble product (MOSMANN, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays, Journal of Immunological Methods, v. 65, pp. 55-63, 1983). Formazan crystals are solubilized and the optical density can be determined by spectrophotometer at 595 nm. The number of viable cells is directly proportional to the amount of formazan crystals produced. HASC-colonized bioactive glass foams cultured in the LEI and LEI O perfusion bioreactor were evaluated after each culture interval for hASC alkaline phosphatase activity by the BCIP / NBT assay (Figure 6) based on chromogenic reaction resulting from the cleavage of a phosphate group BCIP (5-bromo 4-chloro 3-indolylphosphate p-toluidine) by the alkaline phosphatase produced by the cell. This reaction produces a proton that reduces nitroblue hydrochloric tetrazolium (NBT), forming an insoluble purple precipitate (SMEJKAL, GB; KAUL, CA. Stability of nitroblue tetrazolium-based alkaline phosphatase substrates. J. Histochem. Cytochem., 49 ( 9), 1189-1190, 2001).
A diferenciação das hASC em células da linhagem osteogênica quando cultivadas em matrizes de vidro bioativo e em biorreator de perfusão foi avaliada pela expressão de proteínas específicas produzidas por osteoblastos utilizando a técnica de imunofluorescência. Ao término de cada intervalo de indução, as células foram lavadas com PBS e fixadas em p-formaldeído a 4% por 15 minutos, à temperatura ambiente. As células foram lavadas com PBS e foi feita a permeabilização da membrana plasmática utilizando-se Triton-100x a 0,1 % em PBS por 10 minutos. Após a permeabilização, as células foram lavadas novamente com PBS. E em seguida, foi feito o bloqueio da reação, incubando as células com 1 % de BSA (albumina de soro bovino) e 5% de soro de cabra em PBS por 1 hora, à temperatura ambiente. Posteriormente, as células foram incubadas com o anticorpo primário diluído em solução de BSA a 1 % em PBS por 2 horas, à temperatura ambiente e em câmara úmida. Após incubação com anticorpo primário, as células foram novamente lavadas com PBS e, em seguida, foram incubadas com os respectivos anticorpos secundários diluídos em solução de 1 % de BSA em PBS na proporção de 1 :500, por 1 hora em câmara úmida, na ausência de luminosidade e à temperatura ambiente. Os controles negativos foram feitos utilizando-se apenas o respectivo anticorpo secundário. Posteriormente, as células foram lavadas com PBS e incubadas com a sonda Hoechst (1 pg/mL) (Molecular Probes), por 30 minutos para marcação do núcleo. Em seguida, foram feitas novas lavagens com PBS e as lamínulas foram montadas com Hydromount sobre lâminas. As lâminas foram visualizadas e analisadas através do Microscópio Confocal (Zeiss LSM 510 Meta), utilizando-se o programa Carl Zeiss Laser Scanning Microscope LSM 510©. O ensaio utilizado para a verificação de expressão dos genes relacionados à osteogênese foi a Reação em Cadeia da Polimerase (PCR). Foi considerado o gene constitutivo gliceraldeído-3-fosfato desidrogenase (GAPDH) como controle de amplificação das reações. As hASC semeadas no vidro bioativo foram cultivadas no biorreator em meio LEI O por 7, 14 e 21 dias e, após cada período, foi realizada a extração do RNA. The differentiation of hASC in osteogenic lineage cells when cultured in bioactive glass matrices and perfusion bioreactor was evaluated by the expression of specific proteins produced by osteoblasts using the immunofluorescence technique. At the end of each induction interval, cells were washed with PBS and fixed in 4% p-formaldehyde for 15 minutes at room temperature. Cells were washed with PBS and plasma membrane permeabilization was performed using 0.1% Triton-100x in PBS for 10 minutes. After permeabilization, the cells were washed again with PBS. The reaction was then blocked by incubating the cells with 1% BSA (bovine serum albumin) and 5% goat serum in PBS for 1 hour at room temperature. Subsequently, cells were incubated with primary antibody diluted in 1% BSA solution in PBS for 2 hours at room temperature and in a humid chamber. After incubation with primary antibody, the cells were again washed with PBS and then incubated with their secondary antibodies diluted in 1% BSA 1% solution in PBS for 1 hour in a humid chamber at no light and at room temperature. Negative controls were made using only the respective secondary antibody. Subsequently, cells were washed with PBS and incubated with the Hoechst probe (1 pg / ml) (Molecular Probes) for 30 minutes for nucleus labeling. Further washes were made with PBS and the coverslips were mounted with Hydromount on slides. The slides were visualized and analyzed through the Confocal Microscope (Zeiss LSM 510 Meta) using the Carl Zeiss Laser Scanning Microscope LSM 510 © software. The assay used to verify the expression of osteogenesis-related genes was the Polymerase Chain Reaction (PCR). The constitutive gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered as a control for reaction amplification. The hASCs sown in the bioactive glass were grown in the bioreactor in LEI O medium for 7, 14 and 21 days and, after each period, RNA extraction was performed.
Não foi encontrado na literatura um protocolo detalhado de extração de RNA de células cultivadas em amostras semelhantes ao vidro bioativo. Portanto, houve a necessidade de adaptar a extração de RNA de células cultivadas nesse tipo de amostra porosa e inorgânica. Inicialmente foi tentada a extração de RNA das hASC através da aplicação de 1 ml_ de Trizol (Gibco) sobre as matrizes de vidro bioativo, incubação por 5 minutos em temperatura ambiente. A solução formada foi processada seguindo os passos da extração do RNA usualmente utilizados para células cultivadas em garrafas de cultivo, mas quando realizada a leitura da absorbância a 260/280nm, os resultados foram próximos a zero. Após outras tentativas sem sucesso, a nova metodologia pode ser assim descrita: aAs amostras de vidro bioativo semeadas com hASC retiradas do biorreator foram cortadas em três partes e cada uma em quatro partes e tripsinizadas por 5 minutos em estufa de CO2 para que as células pudessem ser removidas do interior do biomaterial. Durante a tripsinização, as amostras foram agitadas suavemente a cada 2 minutos. Após a tripsinização, o biomaterial foi lavado três vezes com meio LEI e a solução total foi centrifugada por 5 minutos a 252 g. O pellet formado foi resuspendido em 1 mL de Trizol e mantido em incubação por 5 minutos para a lise e homogenização das células à temperatura ambiente e para completa dissociação dos complexos nucleoprotéicos. A solução formada foi transferida para um microtubo de 1 ,5mL e centrifugada por 15 minutos a 4QC e 12.000 g. A fase superior foi coletada e transferida para um novo microtubo de 1 ,5mL onde foram adicionados 0,2 mL de clorofórmio, seguido de homogeneização, dois minutos de incubação à temperatura ambiente e centrifugação a 12.000 g por 15 minutos à 4QC, para separação em três fases onde a fase incolor superficial continha o RNA. A fase incolor foi transferida para um novo microtubo e o RNA foi precipitado com 0,5ml_ de álcool isopropílico por 10 minutos a temperatura ambiente. Em seguida, foi feita uma nova centrifugação a 4°C e 12.000 g por 10 minutos e lavagem com 1 mL de etanol 75% (Merck), seguida de centrifugação de 10 minutos a 4°C e 7.500 g. O RNA foi solubilizado em água destilada DNAse e RNAse free e imediatamente estocado a -80QC. A concentração do RNA foi determinada pela leitura da absorbância a 260/280nm em Nanodrop. No detailed protocol for RNA extraction from cells grown on bioactive glass samples was found in the literature. Therefore, there was a need to adapt RNA extraction from cultured cells in this type of porous and inorganic sample. Initially, RNA extraction from hASC was attempted by applying 1 ml Trizol (Gibco) on the bioactive glass matrices, incubating for 5 minutes at room temperature. The formed solution was processed following the RNA extraction steps usually used for cells grown in culture bottles, but when the absorbance reading at 260 / 280nm was taken, the results were close to zero. Following other unsuccessful attempts, the new methodology can be described as follows: aHASC-seeded bioactive glass samples taken from the bioreactor were cut into three parts and each into four parts and trypsinized for 5 minutes in a CO 2 oven so that the cells could be removed from inside the biomaterial. During trypsinization, the samples were gently shaken every 2 minutes. After trypsinization, the biomaterial was washed three times with LEI medium and the total solution was centrifuged for 5 minutes at 252 g. The pellet formed was resuspended in 1 ml Trizol and incubated for 5 minutes for cell lysis and homogenization at room temperature and for complete dissociation of nucleoprotein complexes. The formed solution was transferred to a microtube 1, 5 mL and centrifuged for 15 minutes at 4 Q C and 12,000 g. The upper phase was collected and transferred to a new microtube 1, 5mL which was added 0.2 ml of chloroform, followed by homogenization, two minutes incubation at room temperature and centrifugation at 12,000 g for 15 minutes at 4 Q C to three-phase separation where the superficial colorless phase contained the RNA. The colorless phase was transferred to a new microtube and the RNA It was precipitated with 0.5 ml of isopropyl alcohol for 10 minutes at room temperature. Then, a fresh centrifugation was performed at 4 ° C and 12,000 g for 10 minutes and washed with 1 mL of 75% ethanol (Merck), followed by a 10 minute centrifugation at 4 ° C and 7,500 g. RNA was solubilized in DNAse and RNAse free distilled water and immediately stored at -80 Q C. RNA concentration was determined by reading the absorbance at 260 / 280nm in Nanodrop.
Os cDNAs foram sintetizados a partir dos RNAs totais, utilizando-se o RevertAid TM H Minus First Strand cDNA Synthesis Kit (Fermentas), de acordo com as recomendações do fabricante. Dois microgramas de cada amostra de RNA total foram incubados com 0,5 μg de oligo(dT)18, a 65QC por 5 minutos e, em seguida, as amostras foram incubadas no gelo. Logo após, foi adicionado às amostras: 5X reaction buffer, 20 unidades Ribolock TM Ribonuclease inhibitor, 10mM dNTP mix, e foram incubadas a 37QC por 5 minutos. Em seguida, foram adicionadas 200 unidades da enzima RevertAid TM H Minus M- MuLV RT (Fermentas) e as amostras foram incubadas por 60 minutos a 42QC. A reação foi interrompida pelo aquecimento a 70QC por 10 minutos. CDNAs were synthesized from total RNAs using the RevertAid TM H Minus First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's recommendations. Two micrograms of total RNA from each sample were incubated with 0.5 ug oligo (dT) 18 at 65 Q C for 5 minutes and then the samples were incubated on ice. Immediately after it was added to the samples: 5X reaction buffer, 20 units Ribolock TM Ribonuclease Inhibitor, 10mM dNTP mix and incubated at 37 Q C for 5 minutes. Then it was added 200 units of enzyme RevertAid TM H Minus M-MuLV RT (Fermentas) and the samples were incubated for 60 minutes at 42 Q C. The reaction was stopped by heating at 70 Q C for 10 minutes.
A quantificação relativa da expressão de osteocalcina, fosfatase alcalina e osteopontina foram avaliadas pela técnica de PCR (Figura 1 1 ). Como controle positivo da reação foi amplificado o segmento gênico que codifica a gliceraldeído fosfato desidrogenase (GAPDH), enzima expressa constitutivamente por todas as células. Ao final de 7, 14 e 21 dias de cultivo no biorreator de perfusão, as amostras foram removidas de maneira estéril e preparadas para as seguintes análises que se seguem.  Relative quantification of osteocalcin, alkaline phosphatase and osteopontin expression were assessed by PCR (Figure 11). As a positive reaction control, the gene segment encoding glyceraldehyde phosphate dehydrogenase (GAPDH), an enzyme constitutively expressed by all cells, was amplified. At the end of 7, 14 and 21 days of culture in the perfusion bioreactor, samples were sterile removed and prepared for the following analyzes.
Para avaliar se as hASC realmente aderiram ao vidro bioativo, realizou-se To assess whether hASC actually adhered to bioactive glass, a
MEV, cujos resultados demonstraram que as hASC foram capazes de aderir à matriz e estabelecerem conexões entre si e foi possível observar a presença de pequenas vesículas nas superfícies das membranas celulares, que indicam atividade de síntese e secreção proteica após 14 e 21 dias (Figura 4). SEM, whose results demonstrated that hASC were able to adhere to the matrix and establish connections between them and it was possible to observe the presence of small vesicles on the cell membrane surfaces, which indicate protein synthesis and secretion activity after 14 and 21 days (Figure 4 ).
Quando se comparam os resultados obtidos com as hASC semeadas na matriz de vidro bioativo e cultivadas em biorreator de perfusão aos resultados do cultivo estático sob as mesmas condições, pode-se observar uma semelhança no perfil de proliferação celular, onde inicialmente, as células cultivadas em meio LEI apresentaram uma proliferação celular maior, sendo esta proliferação significativamente maior entre as células cultivadas no biorreator de perfusão, porém, a partir do 14Q dia, a proliferação celular aumentou consideravelmente entre as células cultivadas em meio LEI O, independente do cultivo ser estático ou dinâmico (Figura 5). When comparing the results obtained with the hASC sown in the bioactive glass matrix and cultured in perfusion bioreactor to the results of static cultivation under the same conditions, one can observe a similarity in cell proliferation profile, where initially, cells cultured in LEI medium showed higher cell proliferation, this proliferation being significantly higher among cells cultured in perfusion bioreactor, but from 14 Q day, cell proliferation increased considerably. among cells grown in LEI O medium, regardless of whether the culture is static or dynamic (Figure 5).
Os valores de absorbância obtidos no ensaio de MTT foram superiores quando o cultivo foi realizado no biorreator de perfusão, demonstrando a viabilidade e proliferação celular favorecidas e, quando este dispositivo foi conjugado aos fatores de indução osteogênica, aumentou significativamente a proliferação celular (Figura 5). Esse resultado demonstrou que o cultivo tridimensional em biorreator de perfusão exerce um efeito positivo na proliferação e viabilidade das hASC.  The absorbance values obtained in the MTT assay were higher when the culture was performed in the perfusion bioreactor, demonstrating favored cell viability and proliferation, and when this device was combined with osteogenic induction factors, cell proliferation significantly increased (Figure 5). . This result demonstrated that three-dimensional perfusion bioreactor culture has a positive effect on hASC proliferation and viability.
Quando realizada a análise do gráfico representativo da produção de fosfatase alcalina, pôde-se observar que houve uma maior produção da fosfatase alcalina pelas células cultivadas em meio LEI entre 14 e 21 dias, quando comparadas às células cultivadas em meio LEI O, ambas no biorreator de perfusão (Figura 6).  When analyzing the graph representing alkaline phosphatase production, it was observed that there was a higher production of alkaline phosphatase by cells cultured in LEI medium between 14 and 21 days, when compared to cells cultured in LEI O medium, both in the bioreactor. infusion (Figure 6).
Considerando o aumento da atividade da fosfatase alcalina, um marcador precoce das células em diferenciação osteogênica (JAISWAL, N.; HAYNESWORTH, S. E.; CAPLAN, A. I.; BRUDER, S. P. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J. Celi Biochem., v. 64, p. 295-312, 1997; AUBIN, J. E. Bone stem cells. J Celi Biochem Suppl. V. 72, p. 73-82, 1998), o pico da atividade dessa enzima no 14Q dia sem indução das hASC semeadas sobre a matriz tridimensional e cultivadas no biorreator, corrobora com o ensaio de proliferação, onde evidencia a ação do fluxo sobre as células e indica o início da diferenciação em um fenótipo osteogênico sem a adição de fatores de indução. Considering the increased activity of alkaline phosphatase, an early marker of cells in osteogenic differentiation (JAISWAL, N .; HAYNESWORTH, SE; CAPLAN, AI; BRUDER, SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells. Celi Biochem, v. 64, pp. 295-312, 1997; AUBIN, JE Bone stem cells (Celi Biochem Suppl. V. 72, pp. 73-82, 1998), the peak of the activity of this enzyme in 14. The day without induction of hASC sown on the three-dimensional matrix and cultured in the bioreactor, corroborates the proliferation assay, which shows the action of flow on cells and indicates the onset of differentiation into an osteogenic phenotype without the addition of induction factors.
A partir do 14Q dia de cultivo, houve uma maior produção da enzima quando as amostras foram cultivadas em meio LEI, independente do cultivo ser estático ou dinâmico, e, comparando ambos os cultivos, a produção da fosfatase alcalina foi maior no cultivo dinâmico com meio LEI. Neste momento, o vidro bioativo exerceu uma ação decisiva em relação à atividade da fosfatase alcalina e, somada à ação do fluxo, conferiu ao grupo dinâmico LEI o melhor resultado. No 21 Q dia de cultivo, houve uma diminuição dessa atividade nas células cultivadas em meio LEI e manutenção da atividade nas células cultivadas em meio LEI O. Esse resultado sugere que o vidro bioativo exerceu uma ação osteoindutora representada pela maior produção da enzima no período de 14 dias e diminuição dessa produção no 21 Q dia, sugerindo um provável comprometimento com o fenótipo osteogênico. Neste caso, o fluxo teve uma ação sinérgica à do vidro bioativo, a qual demonstrou um aumento da síntese de fosfatase alcalina nos construtos cultivados no biorreator. From the 14 Q day of cultivation, there was a increased production of the enzyme when the samples were cultured in medium ACT, regardless of being static or dynamic culture, and comparing both cultures, the production of alkaline phosphatase was higher in the dynamic cultivation with kinda LAW. At this time, The bioactive glass exerted a decisive action in relation to the activity of alkaline phosphatase and, added to the flow action, gave the dynamic group LEI the best result. On day 21 of culture Q, there was a decrease in this activity in cells grown in ACT and maintaining activity in cells grown in ACT O. This result suggests that the bioactive glass has had a osteoinductive action represented by the increased production of the enzyme in the period 14 days and decrease of this production in the 21 Q day, suggesting a probable compromise with the osteogenic phenotype. In this case, the flux had a synergistic action to that of bioactive glass, which demonstrated an increase of alkaline phosphatase synthesis in the bioreactor constructs.
Portanto, esses resultados sugerem que a ação do fluxo do biorreator de perfusão teve um papel importante quando da proliferação celular aos 7 dias e aumento da atividade da fosfatase alcalina aos 14 dias de cultivo na ausência dos fatores de indução osteogênica.  Therefore, these results suggest that the action of perfusion bioreactor flow played an important role during cell proliferation at 7 days and increased alkaline phosphatase activity at 14 days of culture in the absence of osteogenic induction factors.
O ensaio de imunofluorescência foi realizado para verificar se as hASC semeadas em espumas de vidro bioativo e cultivadas no biorreator de perfusão passaram a expressar marcadores de células osteogênicas. Os anticorpos específicos utilizados foram osteocalcina, osteopontina e colágeno tipo I e inicialmente o teste foi realizado em células cultivadas em meio LEI após 21 dias de cultivo. Pode-se observar nas imagens de microscopia confocal que as hASC apresentaram marcação para as três proteínas testadas (Figura 7). Este resultado corrobora com os resultados do MTT e da atividade da fosfatase alcalina, nos quais, a partir do 14Q dia, houve uma diminuição da proliferação celular e um aumento na produção da enzima fosfatase alcalina, sugerindo um comprometimento das hASC com o fenótipo osteogênico. Além disso, as imagens de microscopia confocal também mostram uma diminuição no comprimento das hASC, sugerindo também uma alteração na morfologia relacionada à alteração no fenótipo (Figura 8). The immunofluorescence assay was performed to verify whether hASCs sown in bioactive glass foams and cultured in the perfusion bioreactor began to express osteogenic cell markers. The specific antibodies used were osteocalcin, osteopontin and type I collagen and initially the test was performed on cells cultured in LEI medium after 21 days of culture. It can be observed in the confocal microscopy images that the hASC were labeled for the three proteins tested (Figure 7). This result corroborates the results of MTT and alkaline phosphatase activity, where, from 14 Q day, there was a decrease in cell proliferation and an increase in the production of alkaline phosphatase enzyme, suggesting a compromise of hASC with osteogenic phenotype. . In addition, confocal microscopy images also show a decrease in hASC length, also suggesting a change in morphology related to change in phenotype (Figure 8).
Portanto, de acordo com os resultados expostos e em relação à hipótese da diferenciação osteogênica das hASC quando cultivadas em matriz de vidro bioativo e em meio LEI no biorreator de perfusão, pode-se sugerir que a partir do 14Q dia de cultivo, as hASC parecem ter se comprometido com o fenótipo osteogênico. A avaliação dos marcadores de diferenciação osteocalcina, osteopontina e colágeno tipo I foi realizada em hASC cultivadas no vidro bioativo em meio LEI O por 7, 14 e 21 dias no biorreator de perfusão. As imagens de microscopia confocal evidenciam uma evolução na marcação das fluorescências a partir do 7- até o 21 Q dia para as três proteínas testadas: osteocalcina (Figura 8), osteopontina (Figura 9) e colágeno tipo I (Figura 10); além de uma evolução na morfologia, na qual, as células passam de um formato fusiforme típico de uma célula-tronco para o formato cuboidal, característico das células osteoblásticas. Estes resultados, associados aos resultados de MTT e da atividade da fosfatase alcalina indicam uma alteração do fenótipo osteogênico das hASC. Therefore, according to the results above and in relation to the case of the osteogenic differentiation of HASC when cultured on bioactive glass matrix and means ACT in the perfusion bioreactor can be suggested that from 14 Q day of cultivation, the HASC seem to have committed to the osteogenic phenotype. The evaluation of osteocalcin, osteopontin and collagen type I differentiation markers was performed in hASC grown on bioactive glass in LEI O medium for 7, 14 and 21 days in the perfusion bioreactor. The confocal microscopy images show an increase in fluorescence labeling of from 7 to 21 Q day for three tested proteins: osteocalcin (Figure 8), osteopontin (Figure 9) and collagen type I (Figure 10); In addition to an evolution in morphology, the cells move from the typical fusiform shape of a stem cell to the cuboidal shape, characteristic of osteoblastic cells. These results, associated with the MTT and alkaline phosphatase activity results indicate a change in the osteogenic phenotype of hASC.
Portanto, pode-se sugerir que as hASC semeadas na matriz tridimensional de vidro bioativo e cultivadas em meio LEI O no biorreator demonstraram um comprometimento com o fenótipo osteogênico.  Therefore, it can be suggested that hASC sown in the three-dimensional bioactive glass matrix and cultured in LEI O medium in the bioreactor showed a compromise with the osteogenic phenotype.
Da mesma forma como foi observado nas imagens de imunofluorescência, pôde-se observar uma alteração progressiva da morfologia celular, especialmente nos estágios mais tardios, nos quais além das células cultivadas em meio LEI O dinâmico apresentarem uma maior expressão dos marcadores, ainda apresentaram uma morfologia cuboidal, morfologia típica de osteoblastos.  As observed in immunofluorescence images, a progressive change in cell morphology could be observed, especially in later stages, in which, besides the cells cultivated in dynamic LEI medium, presenting a higher expression of the markers, they also presented a morphology. cuboidal, typical morphology of osteoblasts.
Quando comparados o ensaio estático ao dinâmico, pôde-se concluir que a proliferação e viabilidade celular foi significativamente maior nas hASC cultivadas no biorreator de perfusão em 7 dias de cultivo. Em relação à atividade da fosfatase alcalina, que é um marcador da diferenciação celular e seu pico está relacionado ao 14Q dia de cultivo, o resultado foi mais expressivo nas hASC cultivadas em meio LEI e de maneira dinâmica, sugerindo novamente uma ação positiva do fluxo do biorreator de perfusão na diferenciação das hASC, como demonstrado em diversos estudos na literatura para MSC (BANCROFT, G.N.; SIKAVITSAS, V. I.; VAN DER DOLDER, J.; SHEFFIELD, T. L; AMBROSE, C. G.; JANSEN, J. A.; MIKOS, A. G. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose dependent manner. Proc. Natl. Acad. Sei. USA, v. 99, p. 12600-12605, 2002; SIKAVITSAS, V. I.; BANCROFT, G. N.; LEMOINE, J. J.; LIEBSCHNER,M. A. K.; DAUNER, M.; MIKOS, A. G. Flow Perfusion Enhances the Calcified Matrix Deposition of Marrow Stromal Cells in Biodegradable Nonwoven Fiber Mesh Scaffolds. Annals of Biomedical Engineering, v. 33, n. 1 , p. 63-70, 2005; BRACCINI, A.; WENDT, D.; JAQUIERY, C; JAKOB, M.; HEBERER, M.; KENINS, L; WODNAR- FILIPOWICZ, A.; QUARTO, R.; MARTIN, I. Three-Dimensional Perfusion Culture of Human Bone Marrow Cells and Generation of Osteoinductive Grafts. Stern Cells, v. 23, p. 1066-1072, 2005; HOLTORF, H. L; SHEFFIELD, T. L; AMBROSE, C. G.; JANSEN, J. A.; MIKOS, A. G. Flow perfusion culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics. Ann Biomed Eng 33, 1238-1248, 2005). Comparing the static to the dynamic assay, it was concluded that proliferation and cell viability were significantly higher in hASC cultured in the perfusion bioreactor at 7 days of cultivation. Regarding the activity of alkaline phosphatase, a marker of cell differentiation and its peak is related to the 14 Q day of culture, the result was more pronounced in cultured HASC among ACT and dynamically suggesting again a positive action of the flow of perfusion bioreactor in the differentiation of hASC, as demonstrated in several studies in the literature for MSC (BANCROFT, GN; SIKAVITSAS, VI; VAN DER DOLDER, J .; SHEFFIELD, T. L; AMBROSE, CG; JANSEN, JA; MIKOS, AG Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose dependent manner Proc. Natl. Acad. Sci. USA, v. 99, p. 12600-12605, 2002; SIKAVITSAS, VI; BANCROFT, GN; LEMOINE, JJ; LIEBSCHNER, MAK; DAUNER, M .; MIKOS, AG Flow Perfusion Enhancements Calcified Matrix Deposition of Marrow Stromal Cells in Biodegradable Nonwoven Fiber Mesh Scaffolds. Annals of Biomedical Engineering, v. 33, no. 1, p. 63-70, 2005; BRACCINI, A .; WENDT, D .; JAQUIERY, C; JAKOB, M .; HEBERER, M .; KENINS, L; WODNAR-FILIPOWICZ, A .; FOURTH, R .; MARTIN, I. Three-Dimensional Perfusion Culture of Human Bone Marrow Cells and Generation of Osteoinductive Grafts. Stern Cells, v. 23, p. 1066-1072, 2005; HOLTORF, H.L; SHEFFIELD, T. L; AMBROSE, CG; JANSEN, JA; MIKOS, AG Flow perfusion culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics. Ann Biomed Eng 33, 1238-1248, 2005).

Claims

REIVINDICAÇÕES
CÂMARA DE PERFUSÃO DE CULTIVO TRIDIMENSIONAL THREE-DIMENSIONAL CULTURE PERFUSION CHAMBER
caracterizada por possuir em suas extremidades, dois orifícios de passagem, um de entrada (7) e outro de saída (9) para o meio de cultivo permitindo a perfusão por todo construto e podendo ser conectadas a um meio de cultura isoladamente e possuir em sua parte interna (8) uma região para adaptação adequada da matriz tridimensional de cultivo. characterized in that it has at its ends two passage holes, one inlet (7) and one outlet (9) for the culture medium allowing perfusion throughout the construct and can be connected to a culture medium alone and have in its inner part (8) a region for proper adaptation of the three-dimensional crop matrix.
CÂMARA DE PERFUSÃO DE CULTIVO TRIDIMENSIONAL, de acordo com reivindicação 1 , caracterizada por, ter a possibilidade de ser produzida em diferentes formatos e dimensões, de acordo com o objetivo do cultivo.  TRIMIMENSIONAL CULTIVATION PERFUSION CHAMBER according to claim 1, characterized in that it has the possibility of being produced in different formats and dimensions, according to the purpose of the crop.
CÂMARA DE PERFUSÃO DE CULTIVO TRIDIMENSIONAL, de acordo com reivindicação 1 , caracterizada por, poder ser adaptado a diferentes ambientes, desde que respeitadas as condições para o cultivo tecido-específico.  Three-dimensional culturing perfusion chamber according to claim 1, characterized in that it can be adapted to different environments, provided that the conditions for tissue-specific cultivation are respected.
METODOLOGIA DE CULTIVO TRIDIMENSIONAL caracterizada por conectar as câmaras de perfusão de modo isolado a cada meio de cultivo.  THREE-DIMENSIONAL CROP METHODOLOGY characterized by connecting the perfusion chambers in isolation to each culture medium.
METODOLOGIA DE AVALIAÇÃO DE DIFERENCIAÇÃO DE CÉLULAS CULTIVADAS EM MATRIZ DE VIDRO BIOATIVO  BIOACTIVE GLASS CELL DIFFERENTIATION EVALUATION METHODOLOGY
caracterizada por, apresentar técnica de extração de RNA onde, em uma etapa anterior a extração convencional, as amostras de vidro bioativo semeadas com hASC retiradas do biorreator foram cortadas em partes tripsinizadas, preferencialmente por 5 minutos, em estufa CO2 e agitadas suavemente a cada 2 minutos; após, o biomaterial é lavado por preferencialmente or três vezes com meio Leibovitz e centrifugado preferencialmente por 5 minutos a 252g; o pellet formado é resuspendido em Trizol, preferencialmente 1 mL, e mantido em incubação por preferencialmente por 5 minutos à temperatura ambiente. characterized by its RNA extraction technique where, in a previous step to conventional extraction, bioactive glass samples sown with hASC from the bioreactor were cut into trypsinized parts, preferably for 5 minutes, in a CO 2 oven and gently shaken every 2 minutes; afterwards, the biomaterial is preferably washed three times with Leibovitz medium and centrifuged preferably for 5 minutes at 252g; The pellet formed is resuspended in Trizol, preferably 1 mL, and incubated for preferably 5 minutes at room temperature.
PCT/IB2013/061341 2012-12-28 2013-12-26 Three‑dimensional culture perfusion chamber for tissue engineering WO2014102730A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019051486A1 (en) * 2017-09-11 2019-03-14 3D Biotek, Llc Large-scale bioreactor
WO2022068029A1 (en) * 2020-09-29 2022-04-07 中国肉类食品综合研究中心 Special culture apparatus for 3d biological tissue, and method for preparing block-shaped cultured meat
US11566215B2 (en) 2016-08-27 2023-01-31 3D Biotek Llc Bioreactor with scaffolds

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6943008B1 (en) * 2002-08-21 2005-09-13 Florida State University Research Foundation, Inc. Bioreactor for cell culture
US20080311650A1 (en) * 2004-05-06 2008-12-18 University Hospital Of Basel Bioreactor for Tissue Engineering
PT104155A (en) * 2008-08-06 2010-02-08 Ass For The Advancement Of Tis MULTI-CAMERA BIOREACTOR WITH BIDIRECTIONAL PERFUSION INTEGRATED IN A CULTURE SYSTEM FOR APPLICATION IN STRATEGIES OF TISSUE ENGINEERING
US20110229970A1 (en) * 2010-03-05 2011-09-22 Florida State University Research Foundation Dual-chamber perfusion bioreactor for orthopedic tissue interfaces and methods of use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6943008B1 (en) * 2002-08-21 2005-09-13 Florida State University Research Foundation, Inc. Bioreactor for cell culture
US20080311650A1 (en) * 2004-05-06 2008-12-18 University Hospital Of Basel Bioreactor for Tissue Engineering
PT104155A (en) * 2008-08-06 2010-02-08 Ass For The Advancement Of Tis MULTI-CAMERA BIOREACTOR WITH BIDIRECTIONAL PERFUSION INTEGRATED IN A CULTURE SYSTEM FOR APPLICATION IN STRATEGIES OF TISSUE ENGINEERING
US20110229970A1 (en) * 2010-03-05 2011-09-22 Florida State University Research Foundation Dual-chamber perfusion bioreactor for orthopedic tissue interfaces and methods of use

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BANCROFT GN ET AL.: "Design of a flow perfusion bioreactor system for bone tissue-engineering applications.", TISSUE ENG., vol. 9, no. 3, 2003, pages 549 - 554 *
BJERRE L ET AL.: "Flow perfusion culture of human mesenchymal stem cells on silicate-substituted tricalcium phosphate scaffolds.", BIOMATERIALS, vol. 29, no. 17, 2008, pages 2616 - 2627 *
BRACCINI A ET AL.: "Three-dimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts.", STEM CELLS.., vol. 23, no. 8, 2005, pages 1066 - 1072 *
CARTMELL SH ET AL.: "Effects of medium perfusion rate on cell -seeded. three-dimensional bone constructs in vitro.", TISSUE ENG., vol. 9, no. 6, 2003, pages 1197 - 1203 *
GRAYSON WL ET AL.: "Optimizing the' medium perfusion rate in bone tissue engineering bioreactors.", BIOTECHNOL BIOENG., vol. 108, no. 5, 2011, pages 1159 - 1170 *
JONES JR ET AL.: "Optimising bioactive glass scaffolds for bone tissue engineering.", BIOMATERIALS, vol. 27, no. 7, 2006, pages 964 - 973 *
MARTIN I ET AL.: "The role of bioreactors in tissue engineering.", TRENDS BIOTECHNOL., vol. 22, no. 2, 2004, pages 80 - 86 *
MARTIN Y ET AL.: "Bioreactors for tissue mass culture: design, characterization, and recent advances.", BIOMATERIALS, vol. 26, no. 35, 2005, pages 7481 - 7503 *
SILVA, A R.P: "Avatiagao da Diferenciagao Osteogenica de celulas-tronco do tecido adiposo humane cultivadas em espuma de vidro bioativo e.biorreator de-perfusao para enganharia de tecidb osseo.", TESE DE DOUTORADO, March 2012 (2012-03-01), BELO HORIZONTE, pages 148 *
VALERIO P ET AL.: "Primary osteoblast cell response to sol-gel derived bioactive glass foams.", J MATER SCI MATER MED., vol. 16, no. 9, 2005, pages 851 - 856 *
WENDT D ET AL.: "Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity.", BIOTECHNOLOGY AND BIOENGINEERING, vol. 84, no. 2, 2003, pages 205 - 214 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11566215B2 (en) 2016-08-27 2023-01-31 3D Biotek Llc Bioreactor with scaffolds
US11926810B2 (en) 2016-08-27 2024-03-12 3D Biotek, Llc Bioreactor with scaffolds
WO2019051486A1 (en) * 2017-09-11 2019-03-14 3D Biotek, Llc Large-scale bioreactor
WO2022068029A1 (en) * 2020-09-29 2022-04-07 中国肉类食品综合研究中心 Special culture apparatus for 3d biological tissue, and method for preparing block-shaped cultured meat

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