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WO2020208553A1 - Procédé de distribution de facteurs actifs dans un tissu artificiel, produits et utilisations de ceux-ci - Google Patents

Procédé de distribution de facteurs actifs dans un tissu artificiel, produits et utilisations de ceux-ci Download PDF

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WO2020208553A1
WO2020208553A1 PCT/IB2020/053366 IB2020053366W WO2020208553A1 WO 2020208553 A1 WO2020208553 A1 WO 2020208553A1 IB 2020053366 W IB2020053366 W IB 2020053366W WO 2020208553 A1 WO2020208553 A1 WO 2020208553A1
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previous
biomaterial
tissue
hap
gradient
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Raphael CANADAS
Joaquim Miguel OLIVEIRA
Alexandra MARQUES
Rui REIS
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Association for the Advancement of Tissue Engineering and Cell Based Technologies and Therapies A4TEC
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Association for the Advancement of Tissue Engineering and Cell Based Technologies and Therapies A4TEC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces

Definitions

  • the present disclosure relates to thermally-induced gradients in biomaterials for programmable spatial distribution of bioactive factors, preferably in an artificial tissue, in particular a graft.
  • the present disclosure further relates to a method for controlling hydroxyapatite microparticles distribution in gellan gum gel by induced convection, and also a method to distribute biomolecules, in particular growth factors; namely using a multi-chamber reactor.
  • OC unit The osteo- chondral unit
  • This phenotypic transition typically consists of precise spatial arrangements of multiple cell types, matrix components and mineralization in hierarchical structures. 5 Thus, precise functional roles are associated with these spatial patterns.
  • the present disclosure relates to thermally-induced gradients in biomaterials for programmable spatial distribution of bioactive factors.
  • the present disclosure further relates to a method for controlling microparticles distribution in hydrogels by induced convection.
  • Methylene blue was selected as a model drug allowing to modulate drug incorporation and investigate its spatial diffusion along the gradient structure. 27, 28 The dye was spatially confined to half hydrogel, meaning that a spatial control is achievable when small molecules are incorporated. This was confirmed when, GG modified with FITC was used to compare the location of the dyes with the mixing of the polymer itself, and similar profiles were observed even under the combination of solutions at different temperatures. Methylene blue was electrostatically bonded to GG, and therefore the electrostatic forces were more relevant than diffusion or convection. Furthermore, the mix of polymeric phases was possibly inhibited by the negative charges of GG, which was previously reported for other polymeric systems.
  • the HAp microparticles could cross the interface region from the bottom to the top layer.
  • Mass transport (of particles) in a fluid can either be diffusive or advective, in which matter or/and heat is transported by the larger-scale motion of the convective streams in the fluid.
  • thermal effects which trigger hydrodynamic instabilities.
  • well-known instabilities triggered by thermal effects such as Rayleigh-Bénard convection, are often found in systems with well-defined initial conditions.
  • 60°C on top and bottom layers, respectively, was selected considering the calcified gradient characteristic of the bone-to-cartilage OC tissue unit.
  • the gradient of HAp resulted in structures with bioactive-to-non-bioactive properties, interesting for bone- cartilage interface regeneration, as supported with fat pad hASCs (adipose-derived stem cells) cultures and the differences in ALP activity, an in vitro bone formation index.
  • the dependence of inflammatory response to the HAp morphology and size was previously reported, but here we showed that inflammation responds to a gradient of HAp in a subcutaneous model.
  • the present disclosure provides a simple way of controlling microparticles distribution in hydrogels by induced convection.
  • the performed experimental modulation showed how temperature, layer volumes, and particle distribution can be programmed for spatial control of bioactive molecules and particles, creating patterns in 3-D environments with potential utility in fundamental stem cell studies, drug testing, graft fabrication, and interfaces modelling and with potential interest for broader fields from architecture to energy infrastructure.
  • the present disclosure also combines guided crosslinking and freezing mechanisms as a top-down fabrication method to create anisotropy either as linear or radial porous organization in spongy-like hydrogels presenting microparticle gradients formed by convection in creaming emulsions.
  • the combination of such principles in hydrogels made of LAGG-GelMA polymeric blends allowed to combine a polysaccharide with a fibrous protein, as it is representative of the native extracellular matrix (ECM), generating patterned gradients of HAp with controlled porosity orientation.
  • ECM native extracellular matrix
  • the fabricated isotropic and anisotropic gradient structures showed, at the same polymeric concentration, not only differences in storage modulus but also in cell adhesion and proliferation. Further, GelMA and LAGG presented different degradation rates.
  • Using a single stem cell type to create a tissue interface required a spatial control over the 3D environment, which was achieved by the custom-made bioreactor for biochemical gradients.
  • fat pad adipose-derived stem cells isolated from human Hoffa’s body usually removed during arthroscopic surgical interventions were used. These cells were stimulated to perform the codifferentiation, since this fat pad is located in the infrapatellar knee joint, the target tissue of the developed strategy.
  • the dual-chamber bioreactor allowed controlling the mixing of two different culture media creating a three-layered environment. Osteogenic, chondrogenic, and their mixed culture medium cocktail at the interface induced phenotypic changes observed in the ASCs through different regions of a single construct.
  • the codifferentiation of stem cells in a single environment was spatially controlled, which can potentially be applied for broader tissue interfaces.
  • the triggering of the ALP activity of ASCs from Hoffa’s body within the generated 3-D gradient structures was evaluated to determine stem cells ⁇ response toward the HAp gradient. These results were further validated by subcutaneous implantation in mice. The invasion of immune cells and host tissue infiltration in response to the HAp gradient were assessed.
  • a method for producing three-dimensional biomaterial with a plurality of layers comprises the following steps:
  • each biopolymer in a multi-chamber bioreactor, where the bioreactor chambers allow the contact of the biopolymeric gel solutions, for forming a single biomaterial comprising a plurality of layers; incubating the biomaterial with a suitable cell media.
  • the method for producing three-dimensional biomaterial further comprising preparing a further biopolymeric gel solution and applying different temperature to each biopolymeric gel solution for inducing different gradients of the bioactive factor into the three-dimensional biomaterial.
  • the method for producing three-dimensional biomaterial further comprising guided diffusion calcium solution into the biomaterial for vertical or horizontal ionic cross-linking to obtain isotropic or anisotropic porous structures; incubating the biomaterial with cell media or with an ionic solution.
  • the method for producing three-dimensional biomaterial further comprising directional freezing of the biomaterial to obtain linear or radial, or linear and radial porous structures. After directional freezing, the biomaterial is then subjected to freeze-drying.
  • the first and second biomaterial is the same.
  • the first and second biomaterial is different.
  • the biopolymeric gel solution is gellan gum, preferably a low acyl gellan gum gel.
  • the biopolymeric gel solution is gelatine, preferably a methacrylated gelatine.
  • the biopolymeric gel solution is gellan gum and gelatine, preferably a low acyl gellan gum gel and methacrylated gelatine.
  • the biopolymeric gel solution is gellan gum, gelatine, and laminin, preferably a low acyl gellan gum gel and methacrylated gelatine.
  • the temperature applied to each biopolymeric gel solutions varies between 30°C to 80°C, said temperatures have a difference of 5°C to 10°C.
  • the temperature applied to each biopolymeric gel solutions varies between 35°C to 40°C, preferably around 37 °C.
  • the bioactive factor is a growth factor, a growth factor precursor, a mineral, a cell, a biodegradable particle, a drug, a hormone, or mixtures thereof.
  • the bioactive factor is hydroxyapatite, preferably microparticle or nanoparticle.
  • the concentration of the bioactive factor is between 5- 50 % (w/v), more preferably between 10 to 30 % (w/v).
  • the concentration of the bioactive factor is preferably 20-25 % (w/v).
  • the biomaterial has a pore size lower than 500 ⁇ m, more preferable lower than 200 ⁇ m.
  • the cell medium is osteogenic, preferably osteogenic- endothelial media.
  • the cell medium is chondrogenic.
  • the cell medium is neural, preferably neural medium supplemented with fibroblast growth factor 2 (FGF2) or Reelin.
  • FGF2 fibroblast growth factor 2
  • Reelin fibroblast growth factor 2
  • the method may be used for producing a three-dimensional hierarchical synthetic living tissue.
  • the three-dimensional biomaterial comprising a plurality of layers comprises:
  • bioactive factor concentration gradient wherein the concentration of the bioactive factor varies along the three-dimensional biomaterial
  • a plurality of pores comprising a pore size lower than 200 ⁇ m wherein the pores are oriented in a suitable direction.
  • the three-dimensional biomaterial is linear or radial or random or combinations thereof.
  • At least two of the layers of the three-dimensional biomaterial have different pore orientation.
  • the three-dimensional biomaterial is an artificial tissue, a tissue graft, a hierarchical synthetical living tissue.
  • the three-dimensional biomaterial is an artificial tissue is osteochondral tissue, or vasculature formation, or osteochondral tissue with spatially controlled vasculature, or brain tissue, or skin tissue, or blood-brain barrier tissue, or lung epithelium, or intestinal epithelium.
  • the three-dimensional gradient gel may be used for drug testing, graft fabrication, interface modelling, bone-cartilage regeneration, osteochondral tissue.
  • Figure 1 shows the fabrication method for hierarchical hydrogels formation.
  • Figure 2 shows the convection-driven HAp dispersion patterned by HAp controlled dispersion in 3-D structures programmed by gels temperature.
  • Figure 3 shows the control over the 3-D gradient by tuning the volume of gels and the HAp concentration.
  • Figure 4 shows the control of the 3-D gradient by HAp concentration, viscosity, and temperature.
  • Figure 5 shows the transition from bioactive to non-bioactive and, the porous architecture within the 3-D structures.
  • Figure 6 shows the water absorption, the degradation and the mechanical properties of the HAp gradient within the 3-D structures.
  • Figure 7 shows that the stem cells respond towards HAp gradient within the 3-D structures.
  • Figure 8 shows the schematic representation of 3D porosity guided structure fabrication.
  • Figure 9 shows that the cells respond to the pore orientation being guided by the structure architecture.
  • Figure 10 shows the schematic for the control over particles gradient dispersion and pore orientation within the structures, as well as the biochemical gradient to promote hierarchical living tissue maturation.
  • Figure 11 shows the tomographic characterization of gradient structures in isotropic and anisotropic porous structure of random and guided orientation, respectively.
  • Figure 12 shows the cell orientation in 3D gradient structures with isotropic and anisotropic porosity.
  • Figure 13 shows the biochemical gradient for osteochondral differentiation in isotropic and anisotropic 3D structures.
  • Figure 14 shows the pre-vasculature spatial control induced by temporal alterations of biochemical gradient of pro- and anti-angiogenic conditions.
  • Figure 15 shows the phenotypic gradient for neural stem cells cultured and differentiated under biochemical growth factors gradient spatially controlled in the multi-chamber reactor for neuron differentiation and stem cell proliferation.
  • the present disclosure relates to thermally-induced gradients in gels for programmable spatial distribution of bioactive factors.
  • the present disclosure further relates to a method for controlling microparticles distribution in hydrogels by induced convection.
  • the present disclosure provides a simple way of controlling microparticles distribution in hydrogels by induced convection.
  • the performed experimental modulation showed how temperature, layer volumes, and particle distribution can be programmed for spatial control of bioactive molecules and particles, creating patterns in 3-D environments with potential utility in fundamental stem cell studies, drug testing, graft fabrication, and interfaces modelling and with potential interest for broader fields from architecture to energy infrastructure.
  • each gel solution was stained with red or blue dyes.
  • Alizarin red was used to stain the HAp concentrated solution, while methylene blue was used as a dye for the HAp-free solution.
  • Methylene blue was selected as a positively charged drug model that can interact with negatively charged GG.
  • the red layer was first injected into the mould, followed by the blue layer. The blue layer was injected on top of the red layer. Changing the temperature of both layers showed no considerable differences in the diffusion of the blue and red dyes (figure 2B). However, differences were observed in the top, interface and bottom layers. Methylene blue was confined in the top region, showing low mixing with the bottom one.
  • the fluorescence of GG-FITC (gel 1) and the autofluorescence of HAp (gel 2) was analysed and traced (figures 1 and 2).
  • the profile of GG-FITC corresponded to the methylene blue distribution profile.
  • the HAp diffused through the whole hydrogel independently of the mixing of the two gels creating different distribution profiles.
  • the temperature of the gel incorporating HAp was lower, the interface region increased in size.
  • the profile of HAp throughout the structures was quantified by fluorescence and a polynomial regression was adjusted to HAp obtained distribution (figure 2B).
  • a polynomial regression was adjusted to HAp obtained distribution (figure 2B).
  • both gels were at 45°C, which is the temperature around the sol-gel transition point of GG, a gradient with a high slope of the HAp profile at the interface was formed.
  • the first gel was at a higher temperature (60°C) than the second one (50°C)
  • the formed HAp gradient had an approximately linear profile.
  • the temperature of the two polymeric solutions was changed to assess the effect on the HAp gradient formation (figure 2).
  • the resultant HAp profiles were quantified.
  • Each combination of temperatures generated similar mixing profiles for each dye, but different HAp distribution profiles. Macroscopic observations showed that the mixing of both phases was larger at higher temperature.
  • the formation of a HAp gradient-shaped profile was predominantly achieved when applying different temperatures on top and bottom layers, as shown in the cases of 60°C
  • the convective thermal forces promoted by the difference between the applied temperatures had a direct influence on their distribution, creating different profiles, while GG mainly kept in separated layers.
  • 60°C was selected to demonstrate the control over the HAp gradient slope, either by changing the volumes of each gel or the concentration of HAp in one of the gels.
  • microCT was used (figure 3).
  • the volume of HAp-free solution was wider or narrower, respectively.
  • the HAp distribution profile was inverted.
  • the interconnectivity, porosity and wall thickness were not significantly affected.
  • the pore size decreased with the increasing amount of HAp from 671 ⁇ m up to 490 ⁇ m, and 469 ⁇ m.
  • HAp distribution was affected by the amount of particles; when the concentration of HAp was increased, the slope of the profile was more pronounced.
  • a polynomial regression was adapted to each condition therefore the exact HAp concentration in each region of the gradient can be predicted.
  • the correlation between the mechanical features of each region and the concentration of Hap in the 3-D gradient structures was assessed after dynamic mechanical analysis (figure 6).
  • the values at 1 Hz were highlighted since this is a meaningful frequency for mechanical stimuli, e.g. the walking step frequency.
  • the loss moduli were approximately constant along the structure, the energy dissipation ability (tan d) decreased from 0.32 to 0.26 was registered for the low- (originally HAp- free) to the high HAp-containing regions, respectively, meaning that the first one has higher capability to disperse energy under mechanical compressive stress.
  • the effect of the HAp gradient over cell ALP activity was assessed. Constructs were cultured in osteogenic and chondrogenic (as a negative control) media (figure 6C). A significant increase in ALP activity was observed in relation to the control condition in both HAp-rich and -residual regions of the gradient. An increase of ALP activity was also detected in the HAp-rich region in relation to the HAp- residual one
  • mice to evaluate the level of the inflammatory response provoked by the 3-D gradient structures, a subcutaneous implantation was performed in mice. The histological analysis showed the response of inflammatory process to the gradient of HAp. Few host cells or tissue infiltrating was observed in the region of low HAp concentration, while a significantly higher number of immune cells and more percentage of tissue infiltration was observed in the increased concentration of microparticles of the 3-D structures.
  • HAp was synthesised.
  • HAp was prepared by precipitation reaction of calcium hydroxide and ortho-phosphoric acid 85% solutions in an aqueous system at room temperature. The mixing was gently performed at pH 11, adjusted by addition of concentrated ammonium hydroxide (1 M). The ceramic powder formed was sieved and particles with less than 263 ⁇ m were used.
  • Viscosity tests were performed using a Kinexus Pro+ rheometer. Samples were directly poured from the flask on a cone-plate geometry (40 mm) and the excess of material trimmed with a spoon and paper. A low viscosity oil solution was poured around the GG solution inside the cone- plate to avoid evaporation during the measurements at high temperatures. All data was obtained after an imposed shear rate of 100 s -1 to mimic the perfusion by injection and reset the deformation history of all samples. Viscosity data was obtained for single point measurement at shear rates of 0.01 s -1 to 0.1 s -1 .
  • ramps were performed ranging the shear rates from 0.01 s -1 to 0.1 s -1 . This range, being the typical range of levelling phenomena, was chosen to mimic the interface of gels mixing at a steady state. The process was sequentially repeated for 45°C, 50°C, 55°C, 60°C, and 80°C.
  • gels mixing at interface were experimentally modulated.
  • the mixing of two gel layers was continuously monitored and the corresponding graphs were traced by measuring the color mixing over 15s.
  • Pure GG or GG with dispersed HAp were dissolved in distilled water at 90°C for 30 minutes at 2% (w/v).
  • the gel phase made of GG and HAp was stained with alizarin red, and the pure GG phase was colored with methylene blue. After stabilization at the target temperature, e.g.
  • the first layer was injected in a transparent silicon mold (20 x 8 mm) at 0.44 mL/s, followed by the second one which was slowly injected at a flow rate of approximately 0.02 mL/s.
  • a camera was placed for the continuous recording of the mixing of the two layers. Images at 0, 1, 5, 10, and 15 seconds were selected for the analysis. Image J was used to quantitatively evaluate the mixing gels from the RGB histograms of red and blue phases, over time, and at the bottom, interface and top regions of the mold.
  • gradient 3-D structures were prepared for biological studies.
  • Low acyl GG was dissolved in two beakers at 90°C for 30 min in distilled water at 2% (w/v).
  • HAp was then added to one of the solutions at 10%, 20% or 30% (w/w) and dispersed under stirring.
  • 800 ⁇ L of this solution was added into a mold of 20x8 mm at 60oC and topped with a second 800 ⁇ L of solution at 50°C within 15 seconds after pipetting the first one.
  • hydrogels were obtained by immersing the mold in Ca 2+ at 0.25 M for 24h for total ionic-crosslinking of the GG molecular chains.
  • the hydrogels formed were frozen at -80°C and then freeze- dried. After 4 days, cylinders of 6x4 mm for in vitro trials or 5x5 mm for in vivo assays were punched from the obtained dried structures. The structures were submitted to ethylene oxide treatment for sterilization before in vitro or in vivo experimentations.
  • computed X-ray micro-tomographic analysis was conducted. Structures were analysed by micro-CT after freeze-drying. Images were acquired by X- ray diffraction, and then used for a 3-D reconstruction. HAp profile was traced, and mean pores size and interconnectivity were quantified.3-D projections of the specimens were performed. The 3-D gradient structures were acquired with a SkyScan 1272 scanner (v1.1.3), with a pixel size of 14 ⁇ m. Approximately 400 to 550 projections were acquired over a rotation angle of 180° or 360°, with a rotation step of 0.45° or 0.68°. Data sets were reconstructed using a standardized cone-beam reconstruction software (NRecon 1.6.10.2).
  • the output format for each sample was bitmap images. Representative data set of the slices was segmented into binary images with a dynamic threshold of approximately 100–255 for the analysis of the ceramic phase, and 30-255 for soft polymeric phase (grey scale values– optimized per sample and analysis). Then, the binary images were used for morphometric analysis (CT Analyser, v1.15.4.0) and to build the 3-D models (CTvox, v 3.0.0). When needed, samples were vertically oriented in DataViewer (v1.5.2.3) before proceeding to CT Analyser and CTvox.
  • CT Analyser v1.15.4.0
  • CTvox 3-D models
  • bioactivity assay with simulated body fluid was done.
  • the GG-HAp structures were soaked in a solution of SBF, as described by Kokubo et al.
  • the SBF solution Na + 142.0, K + 5.0, Ca 2+ 2.5, Mg 2+ 1.5, Cl- 147.8, HCO 3 - 4.2, HPO 4 2- 1.0, SO 4 2- 0.5 mM
  • the SBF solution was made by dissolving reagent-grade NaCl, NaSO 4 , NaHCO 3 , KCL, K2PO 4 .3H 2 O, CaCl 2 and MgCl 2 .6H 2 O in distilled water at 36.5°C.
  • the solution pH was settled to 7.4 by addition of trishydroxymethylaminomethane and hydrochloric acid.
  • Each structure was immersed in 10 mL of SBF solution using 50 mL polystyrene flasks and kept for periods of 12 h, 1day, 3days, 7days, and 14 days. At each time point, the structures were removed and rinsed in distilled water, frozen at -80°C, and then freeze-dried for 4 days before SEM/EDS analysis. All measurements were performed in triplicate.
  • Scanning Electron Microscopy analysis was conducted.
  • the 3-D gradient structures were analyzed by EDS (SEM/EDS) to assess the bioactivity of the structures shown by apatite formation.
  • SEM scanning electron microscopy
  • specimens were coated with gold using a Fisons Instruments Coater with a current set at 18 mA, for a coating time of 120 s. SEM was performed to evaluate architecture morphology.
  • DMA Dynamic Mechanical Analysis
  • PBS phosphate buffer solution
  • fat pad ASCs were isolated and cultured. Fat pad-ASCs were isolated from human tissues obtained from Hoffa’s body removed during arthroscopic surgeries on male and female donors.
  • hASCs were isolated following an enzymatic digestion-based method with type II collagenase.
  • the extracted tissue was placed in PBS solution and washed several times with PBS containing 1% (v/v) antibiotic (AB) mixture, until total removal of blood and cut into small pieces.
  • Tissue digestion was performed by incubation at 37°C in a humidified atmosphere of 5% CO 2 for 10 h in a 10-20 mL 1:1 mixture of Minimum Essential Media alpha (MEM alpha) supplemented with 10% fetal bovine serum (FBS) and 1% AB mixture, with type II collagenase 0.15% (w/v) in PBS.
  • MEM alpha Minimum Essential Media alpha
  • FBS fetal bovine serum
  • AB fetal bovine serum
  • fat pad ASCs were seeded in a concentration of 1x10 6 cells per scaffold and cultured for 3 days before starting cell differentiation in a 24-well suspension cell culture plate.
  • Cells were suspended in 50 ⁇ L culture medium and seeded in both ends of each structure. After 1h, 500 ⁇ L of culture medium was added. On day 3, metabolic activity was measured before and after dividing each structure in two halves to assess the cell distribution in each layer versus the whole scaffold. Then, each layer of the construct was cultured in osteogenic differentiation medium over 14 days.
  • Osteogenic induction medium was MEM alpha supplemented with dexamethasone (100 nM), bglicerophosphate (10 mM), ascorbic acid 2-phosphate (50 mg/mL), 1% AB, and 10% FBS.
  • cytoskeleton staining was conducted. Samples from the in vitro assays were fixed with 10% formalin for 20 min at RT and washed with PBS. Red fluorescent phalloidin was used to label F-actin for cytoskeleton staining. The blue fluorescent 40,6-diamidino-2-phynylindole (DAPI) was used as a nuclear counter staining. After washing, 3-D structures were visualized with a transmitted and reflected light microscope.
  • DAPI blue fluorescent 40,6-diamidino-2-phynylindole
  • DNA quantification was done.
  • the amount of double strained DNA (dsDNA), that is directly proportional with the cell number, was determined using a fluorimetric dsDNA quantification kit (PicoGreen) according to the manufacturer’s instructions.
  • dsDNA double strained DNA
  • PicoGreen fluorimetric dsDNA quantification kit
  • a complete mechanical disruption of the materials was performed, followed by a thermal shock cycle by incubating the lysates at 75°C for 1h and freezing them at 80°C until further analysis.
  • Supernatant fluorescence was measured at an excitation wavelength of 485/20 nm and at an emission wavelength of 528/20 nm, in a microplate reader.
  • the quantification of DNA was calculated according to a standard curve prepared with concentrations ranging between 0 mg/mL and 2 mg/mL. Quantity of DNA relative to fluorescence intensity was read off the standard curve. Samples without cells were used as control.
  • Alkaline phosphatase (ALP) activity was quantified.
  • the alkaline phosphatase (ALP) activity at day 21 of cell culture was determined on the same samples collected for dsDNA quantification as a marker of osteogenic differentiation.
  • ALP activity was quantified by the specific conversion of pnitrophenyl phosphate (pNPP) into p-nitrophenol (pNP).
  • pNPP pnitrophenyl phosphate
  • pNPP p-nitrophenol
  • a buffer solution containing 0.2% (w/v) pNPP was added to the sample in a 96- well plate.
  • the enzyme reaction was carried out at 37°C for 45 min and then stopped by the addition of a solution containing 2 M NaOH and 0.2 mM EDTA in distilled water.
  • the absorbance of pNP formed was read at 405 nm in a microplate reader and concentrations were read off from a standard curve made using pNP values ranging from 0 to 0.2 mmol/mL. The obtained pNP concentrations were normalized against the DNA concentrations of the same samples and converted into enzyme activity per hour. Samples without cells were used as control. In an embodiment, subcutaneous implantation was conducted.
  • the 3-D structures were subcutaneously implanted in the back of 5 weeks old mice Hsd:ICR (CD- 1) and with an average weight of 27-32 g at the time of implantation .
  • Each mouse was anesthetized by intraperitoneal injection of Medetomidine 1 mg/Kg and Ketamine 75 mg/Kg. The hair of the mice was shaved at the implantation area, followed by disinfection with iodine.
  • two skin incisions (1 cm length) were made in the dorsal midline.
  • mice were euthanized 2 weeks’ post-surgery with an injection of Eutasil 200 mg/mL (pentobarbital sodium) and the implanted materials were retrieved along with the surrounding tissue.
  • Eutasil 200 mg/mL pentobarbital sodium
  • immunohistochemical analysis was conducted. Immunohistochemical staining of the explants from in vivo subcutaneous assay was performed after explanting the specimen. The explants were fixed with 10% (v/v) formalin for at least 5 days at RT and transferred to histological cassettes for paraffin embedding. Samples were then serially sectioned using a microtome (3.5 ⁇ m thick) and stained with hematoxylin & eosin (H&E) and Masson's trichrome. The histological sections were then observed using a light microscope. Immune cells invasion and host tissue infiltration were quantified using QuPath18.
  • LAGG 1000000
  • gelatine from porcine skin, gel strength 300, Type A
  • methacrylic anhydride were used
  • HAp was produced by precipitation reaction of calcium hydroxide and orthophosphoric acid 85% solutions in an aqueous system at RT. The mixing was gently performed at pH 11, adjusted by addition of concentrated ammonium hydroxide (1 m), with a continuous flow rate ranging from 8 to 15 mL min-1. The formed ceramic powder was sieved to obtain particles smaller than 250 ⁇ m.
  • Gelatin was modified by methacrylation (GelMA).
  • a solution of gelatin (from porcine skin, Type A, 300 mw) at 10% (w/v) was prepared by dissolving 2 g in 20 mL of phosphate-buffered saline (PBS), at 50 °C for 1 h and covered to avoid evaporation.0.8 mL per gram of gelatin of methacrylic anhydride were added to the solution at a rate of 0.1 mL min-1, counting 16 min down. The beaker was covered with aluminum to avoid light, and the temperature was kept between 42°C and 48°C during the addition. Methacrylic anhydride was directly added to gelatin solution under stirring of »160 rpm.
  • the solution was left to react for 2 h while temperature was kept between 40°C and 45°C at a slow stirring of »120 rpm. To finalize the reaction, the solution was added to a prewarmed dialysis bag (12–14 kDA cutoff) without precipitation of the solute.
  • dialysis was performed against distilled water in a 5 L beaker for one week keeping the temperature around 45°C to avoid gelation. Distilled water was changed three times on the preparation day and once a day afterward. After 1 week, GelMA solution was removed from dialysis membrane and transferred to 50 mL falcon tube. The solution was frozen in liquid nitrogen or at -80°C (this second options requires waiting for 1 d of freezing before drying). Freeze drying was performed in a freeze drier for 5 d. Lyophilized GelMA was frozen at -80°C until further use.
  • TBSA 2,4,6-trinitrobenzene sulfonic acid
  • GelMA and LAGG 3D Gradient Structures were prepared LAGG and GelMA were dissolved at a total polymer concentration of 2% (w/v) in two separated beakers. First, LAGG was dissolved at 80 °C for 1 h in distilled water. Then, the solution was cooled down to 50°C, and 0.5% (w/v) 2-hydroxy-1-[4-(2- hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure)) was added to the polymeric solution and dissolved for 1 h. GelMA was then added for dissolution for 1 h at 45°C.
  • HAp was added at 30% (w/w) and dispersed under stirring in one of the two beakers with the LAGG/GelMA solution. After complete HAp dispersion, 800 ⁇ L of this solution was added into a mold of 10 ⁇ 7 mm at 55°C. A second injection of 800 ⁇ L was added on top of the first half at 45 °C within 30 s after pipetting the first gel. Afterward, hydrogels were obtained by immersing the gradients in a-minimum essential medium (MEM) medium without fetal bovine serum (FBS) for 24 h for total ionic-crosslinking of LAGG molecular chains. For the isotropic structure fabrication, the silicon molds were removed after 1 h in medium solution.
  • MEM a-minimum essential medium
  • FBS fetal bovine serum
  • a guided ionic crosslinking was performed.
  • cylindrical molds were covered on the bottom part with Parafilm and immersed in culture medium solution. After 24 h at 37°C, the ionic-crosslinked hydrogels were exposed to UV light (320–500 nm, 7.14 mW cm -2 ) for 60 s each side of the mold. After photo-crosslinking, a gradient temperature, reaching the -80°C, was applied using metallic plates previously frozen in dry ice.
  • a custom-made Styrofoam mold involving the silicone mold was used covering the polymeric solution all around except the top, where the frozen plate at -80 °C was directly placed for 2 h.
  • the fabricated structures were punched and sliced in cylinders of 4 mm ⁇ 3 mm.
  • the structures were submitted to three cycles of oxygen plasma treatment, 5 min each, for sterilization and surface hydrophilicity cure before cell seeding. 44 [00109]
  • the viscoelastic measurements were performed using a TRITEC8000B dynamic mechanical analyzer in the compressive mode. The measurements were carried out at 37°C. Samples were cut into cylindrical shapes of »8 mm diameter and 6 mm thickness (measured each sample accurately with a micrometer).
  • the scaffolds were always analyzed whilst immersed in a liquid bath placed in a Teflon reservoir.
  • the scaffolds had previously been immersed in phosphate buffer solution (PBS) until equilibrium was reached (37°C overnight).
  • PBS phosphate buffer solution
  • the geometry of the samples was then measured and the samples were clamped in the DMA apparatus. After equilibration at 37°C, the DMA spectra were obtained during a frequency scan between 0.1 Hz and 10 Hz.
  • the experiments were performed under a constant strain amplitude (50 ⁇ m). A small preload was applied to each sample to ensure that the entire scaffold surface was in contact with the compression plates before testing, and the distance between plates was equal for all scaffolds being tested. A minimum of three samples were used for each condition.
  • FTIR Analysis was conducted. Infrared spectra of the HAp were obtained by Fourier transform infrared spectrometry. For this purpose, HAp particles were mixed with KBr in the proportion of 1/150 (by weight) and pressed into a pellet. To assess the amorphous state of HAp in the structures, FTIR-attenuated total reflection (ATR) was performed instead. Each infrared spectrum was on the average between 32 scans and 40 scans over a range of 800–4400 cm -1 collected with a resolution of 4 cm -1 at room temperature.
  • ATR FTIR-attenuated total reflection
  • X-Ray Diffraction Analysis was conducted.
  • the qualitative and quantitative analyses of crystalline phases presented on the powders and scaffolds were obtained by XRD using a conventional Bragg–Brentano diffractometer equipped with CuKa radiation, produced at 40 kV and 40 mA. Data sets were collected in the 2q range of 10°–70° with a step size of 0.04° and 2 s for each step.
  • the phase composition of the powders was calculated on the basis of XRD patterns by means of Rietveld analysis with TOPAS 5.0 software. Refined parameters were scale factor, sample displacement, background as Chebyshev polynomial of fifth order, crystallite size, and lattice parameters.
  • the degradation rate of GelMA and LAGG polymeric scaffolds were assessed.
  • GelMA and LAGG polymeric scaffolds were assessed for degradation over 14 days and 30 days, respectively.
  • 3D Architecture Characterization of 3D Porous Structures and HAp Profile were conducted. Freeze-dried structures were analyzed by micro-CT. The gradient structures were acquired by X-ray diffraction and then reconstructed for qualitative and quantitative analyses. HAp profile, porosity, mean pores size, and anisotropic degree were quantified. 3D cross-sections of isotropic and anisotropic structures were designed. Samples were acquired with a SkyScan 1272 scanner (v1.1.3), with a pixel size of 10 ⁇ m. Approximately 300 projections were acquired over a rotation range of 180°, with a rotation step of 0.6°. Data sets were reconstructed using standardized cone-beam reconstruction software (NRecon 1.6.10.2).
  • the output format for each sample was bitmap images. Representative data set of the slices was segmented into binary images with a dynamic threshold of 30–255 for soft polymer analysis and 120–2255 for ceramic phase analysis (gray scale values). Then, the binary images were used for morphometric analysis (CT Analyser, v1.13) and to build the 3D models.
  • CT Analyser v1.13
  • the obtained supernatant was transferred to a 96-well plate and the fluorescence measured in a microplate reader. Then, the medium was removed by aspiration and the constructs were washed with fresh medium three times and kept in culture until the end of the assay. Scaffolds without cells were used as a negative control for fluorescent intensity correction.
  • hASCs were enzymatically isolated from Hoffa’s body.
  • hASCs were isolated following an enzymatic digestion-based method with type II collagenase from Hoffa’s body human tissue. The extracted tissue was placed in PBS solution and washed several times with 1% PBS/AA (v/v) solution, until blood or other bodily contaminants were totally removed, and cut into small pieces.
  • Tissue digestion was performed by incubating, at 37°C in a humidified atmosphere of 5% CO2 for 1–2 h, the small pieces with 10–20 mL of a mixture composed by cell medium MEM alpha medium, supplemented with 10% FBS and 1% A/A mixture, and type II collagenase (1:1).
  • the digested tissue was filtered and cell suspension centrifuged at 1220 rpm for 5 min.
  • the isolated cell populations were expanded in MEM alpha medium supplemented with 10% FBS and 1% of A/A mixture until reaching confluence.
  • fat pad ASCs were cultured in bilayered structures for 3 d, before starting cell differentiation.
  • the cells were seeded in a concentration of 1 ⁇ 10 6 cells per scaffold, having these scaffolds 6 mm ⁇ 4 mm high and diameter, respectively.
  • the two layers of each structure were divided and measured the metabolic activity of each layer versus the whole scaffold, in order to assess cell distribution in each layer.
  • hASCs were cultured for 21 d in osteogenic differentiation medium.
  • MEM alpha was supplemented with dexamethasone (100 ⁇ 10-9 m), b-glicerophosphate (10 ⁇ 10-3 m), ascorbic acid 2-phosphate (50 mg mL-1), 1% AB, and 10% FBS.
  • Chondrogenic medium was prepared supplementing Dulbecco’s modified Eagle’s medium high-glucose with dexamethasone (100 ⁇ 10-9 m), ascorbic acid 2-phosphate (50 ⁇ g mL-1), L-proline (40 ⁇ g mL-1), transforming growth factor-b3 (10 ng mL-1), 1% AB, and 10% FBS.
  • hAMECs Human Adipose Microvascular Endothelial Cells
  • Dual-Chamber Bioreactor hAMECs were stained with DiI live staining and then mixed with ASCs in a ratio of 4:1, respectively.
  • the mixed population was seeded in the top and bottom parts of the isotropic porous 3D structures placed in the dual-chamber bioreactor.
  • Basal MEM alpha supplemented with 10% FBS and 1% A/A was mixed with endothelium growth medium (Lonza) containing 2% FBS and VEGF in a ratio of 1:4, respectively, and equally perfused in both compartments of the dual-chamber.
  • the bioreactor chamber was perfused at 5 ⁇ L min-1 and placed at 37 °C in a humidified 5% CO2 atmosphere. After 5 d of perfusion, the cell culture medium in the bottom chamber was changed by mixing osteogenic differentiation medium with endothelial growth medium (1:4). Constructs were perfused for a further 2 days.
  • constructs were imaged on top and bottom regions at day 5 and day 7 by fluorescent microscopy.
  • immunofluorescence staining was done.
  • Cells in constructs were fixed with 4% paraformaldehyde for 20 min at RT. Fixed cells were washed with PBS, nonspecific binding was blocked with 1% bovine serum albumin (BSA) and permeabilized with 0.3% Triton-X 100 for 2 h at RT. Cells were incubated with rabbit anti-osteocalcin (Millipore), mouse anti-SOX9 (Santa Cruz Biotechnology), and goat anti- RUNX2 (Novus Biologicals) overnight at 4 °C.
  • BSA bovine serum albumin
  • the porous architecture of the gels was guided by ice- templating (figure 8).
  • the anisotropic porosity was guided by isolating the gel in all the sides except the bottom part in contact with a frozen metal plate.
  • the applied temperatures were varied to vary the pore size. By decreasing the temperature, the pore size decreased.
  • a system was specifically conceived to control the pore orientation using the same principle (figure 8B). Radial and linear porous orientation (vertical and horizontal) were formed based on the features of such created device.
  • the vertical and horizontal porous architecture were continuous and integrated in a single structure.
  • gels and gradients of particles inside the gels were fabricated in combination with the pore guidance technique. Gradients of particles were formed with isotropic and anisotropic porosity distribution (figure 10).
  • X-ray computed microtomography (micro-CT) reconstruction and analysis were performed showing the porosity orientation, either forming isotropic or anisotropic structure organizations (figure 11).
  • the ceramic phase was composed of HAp which followed the porosity orientation, demonstrating the generation of specific bioactive microparticle distribution patterns.
  • Quantitative data were graphically traced revealing the gradient of HAp.
  • anisotropic degree of the porous architecture significantly increased.
  • anisotropic porosity was formed in the structures, meaning that its pores were vertically aligned, while a degree closer to 0 (zero) represented isotropic porous structures, where a randomly organized porous architecture was formed. Strikingly, the structural gradient was maintained both in the fabricated anisotropic and isotropic porous structures.
  • the mechanical properties of isotropic and anisotropic porous structures were compared.
  • a decrease in storage modulus (E’) was registered while a constant range of values was observed for the anisotropic and isotropic porous structures, respectively.
  • the absolute value of E’ was lower for the isotropic porous structures than the anisotropic ones (about twofold decreased), meaning that these structures presented higher deformability.
  • Assessing the tan d the isotropic porous structures presented higher absolute value (about twofold increased), showing lower difference between the storage modulus and the loss factor.
  • the isotropic porous structures presented higher ability to dissipate energy, under dynamic load in a range from 0 Hz to 10 Hz frequency, thus being more viscous and less prone to mechanical deformation.
  • the cells cultured in the anisotropic structure were organized in a specific orientation at a higher frequency, as evidenced by the distribution peaks formed, which were absent in the isotropic condition, correlated to a random organization. Further, the cell shape clearly varied per region, depending on the differentiation process happening in different regions of a single construct.
  • SOX9 chondrogenic marker significantly increased in the region with reduced or absent HAp concentration, named the chondrogenic region (top part, marked in blue).
  • Osteogenic markers OCN, OPN, and RUXN2 were highly expressed in the HAp concentrated region, named the osteogenic region (bottom part, marked in red.
  • Cells in the interface region presented significantly higher expression of SOX9 (2.4-fold increase, p ⁇ 0.05) compared to the osteogenic, but lower (4.1-fold decrease) in comparison with the chondrogenic one. Further, significantly lower expression of OCN (2.1-fold), RUNX2 (5.5-fold), and OPN (4.3-fold) were observed in the interface as compared to the osteogenic region (p ⁇ 0.05).
  • isotropic porous organization significantly increased the expression of the chondrogenic marker SOX9 (1.5-fold) and the osteogenic marker OCN (2.6-fold) on the chondrogenic region of the construct when compared to the anisotropic ones (p ⁇ 0.05).
  • hAMECs human adipose-derived microvascular endothelial cells
  • Figure 14 fat pad-ASCs
  • This biochemical change at day 5 demonstrated a spatial-temporal control over prevasculature, inducing its formation.
  • Cell adhesion, shape, and distribution of pre- stained hAMECs demonstrated a cell morphology change from day 5 to 7.
  • the endothelial cells were dispersed and rounded in the basal condition and were arranged in an elongated shape creating networks in the endo-osteogenic proangiogenic medium.
  • the tubular-like segments were identified in both conditions and quantified, showing a significant increase of »35% in the region cultured with the endo-osteogenic cocktail condition.

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Abstract

La présente invention concerne un procédé de régulation de la distribution d'agents bioactifs, tels que des biomolécules, dans un gel à base de gomme gellane par convection thermique induite; en particulier, à l'aide d'un réacteur à chambres multiples.
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CN113145186A (zh) * 2021-03-01 2021-07-23 清华大学 多隔室自编码的微球、制备方法、微流控装置及应用
CN113274412A (zh) * 2021-04-28 2021-08-20 山东大学 通用型钙制剂在神经干细胞分化调节上的应用
CN116650724A (zh) * 2023-05-18 2023-08-29 东南大学 一种基于冰模板技术的具有拓扑结构的导电神经导管
CN116763995A (zh) * 2023-06-20 2023-09-19 中国矿业大学 一种具有定向孔隙结构的骨软骨一体化支架及其制备方法

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CN113145186A (zh) * 2021-03-01 2021-07-23 清华大学 多隔室自编码的微球、制备方法、微流控装置及应用
CN113070108B (zh) * 2021-03-01 2022-05-06 清华大学 图案化水凝胶微粒制备方法及微流控装置
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