WO2019054971A2

WO2019054971A2 - Coacervate production method using coacervation and the usage of coacervates obtained by this method as tissue scaffold

Info

Publication number: WO2019054971A2
Application number: PCT/TR2018/050489
Authority: WO
Inventors: Ayşe Başak KAYITMAZER BÜYÜKOKUTAN; Özge KARABIYIK ACAR; Gamze Torun Kose
Original assignee: Yeditepe Universitesi
Priority date: 2017-09-15
Filing date: 2018-09-14
Publication date: 2019-03-21
Also published as: WO2019054971A3; TR201713643A2

Abstract

The present invention relates to production of tissue scaffolds, which are capable of cell encapsulation (macro-encapsulation) for tissue engineering using complex coacervation technique, with chitosan (CT) and hyaluronic acid (HA) polymers. The objective of the present invention is to provide a method for producing tissue scaffolds; which consist of entirely natural polymers that are biodegradable, biocompatible and have elastic feature, and are non-toxic since no crosslinking agents or organic solvents are used; and which have a porous structure providing a suitable environment for cell viability and proliferation, and also for differentiation if stem cell will be used

Description

COACERVATE PRODUCTION METHOD USING COACERVATION AND THE USAGE OF COACERVATES OBTAINED BY THIS METHOD

AS TISSUE SCAFFOLD

Field of the Invention

The present invention relates to a method for producing a tissue scaffold that enables the production of tissue scaffolds which are capable of cell encapsulation for tissue engineering using complex coacervation technique with chitosan (CT) and hyaluronic acid (hyaluronate, hyaluronan, HA) polymers.

Background of the Invention Tissue engineering is a branch of science that deals with producing organs and tissues in laboratory conditions for transplantation to patients. In tissue engineering, it is aimed to form biological tissues with cells, materials and growth factors. The problems generally encountered in tissue scaffolds used in the state of the art are the inability to increase the surface area / volume ratio sufficiently, failure to provide adequate porosity, low rate of degradation, and inability to form durable structures in terms of mechanical properties.

Tissue scaffolding is frequently used in cartilage tissue engineering. The healing capacity of the articular cartilage is limited. Therefore, cartilage tissue engineering is one of the most studied areas. Various cell scaffolds are used to protect the phenotype along with the cell regeneration. The most commonly used ones are the natural and synthetic polymers. Particularly, poly a-hydroxy acids (polylactic acid, polyglycolic acid) among the synthetic polymers are approved by the American Food and Drug Administration (FDA) and various types thereof are patented. However, the degradation products that are produced during the use of synthetic polymers are acidic and the occurrence of immune reactions against the accumulation of waste is frequently observed. In addition, there are deficiencies and limitations in the methods used, such as inability to adjust the rate of degradation, incompatibility of the scaffold due to natural elasticity problems and fixing it with suture, and failure to differentiate the cells into the correct phenotype.

Natural polymers are frequently used in the field of tissue engineering due to their structural similarity to the normal tissue and the biocompatibility of degraded materials. The most popular ones are chitosan and hyaluronic acid. Various combinations of these polymers and those prepared by different production techniques give positive results in terms of biocompatibility, biodegradability and adhesion properties. However, there are some problems related to the application of natural polymers in tissue engineering. Some of these problems are the effects of crosslinkers on the biocompatibility of natural polymers which can be used in the preparation method and the cells tend to loss their phenotypes while seeded on the tissue scaffolds.

Complex coacervation is a method based on liquid-liquid phase separation between oppositely charged macroions in a colloidal aqueous solution. The main factors which make this process applicable are the electrostatic attraction between oppositely charged macroions and the entropy gain associated with the release of small ions in the solution. The turbid phase formed upon mixing of the macroion solutions includes micrometer sized spherical droplets. The fusion of these droplets and formation of two liquid phases with different densities may be accelerated by applying centrifugation or may be carried out slowly by leaving it to self-precipitation. The lower liquid phase which is rich in macro-ions is a highly dense and viscous phase and it is referred as coacervate. The upper phase (supernatant) is a water-like liquid in terms of density and has a lower concentration even before the mixture concentrations of the macro-ions. Gel-like fluids can be obtained by changing the various factors (polymer molecular weight, charge density, elasticity, solution pH and ionic strength, temperature, ratio of oppositely charged macro-ions in the mixture) that affect the formation of the coacervate phase and can be used in tissue engineering. There are many methods utilized in the literature using oppositely charged macro- ions but they should not be confused with complex coacervation. Mainly, they are precipitate, flocculate, hydrogel and layer-by-layer assemblies. The coacervates are macroscopically different from the precipitates and flocculates. The precipitation is the solid-liquid phase separation, and the dense phase exhibits crystalline features. The flocculates are also formed by the solid-liquid phase separation but are less dense and the floes visibly remain suspended in the solution for a longer period of time.

The difference between the layer-by-layer assembly from the self-uniting polyelectrolytes (coacervate) is because of the released oppositely charged ions since coacervation includes short-distance Coulomb extraction and long-distance Coulomb repulsion forces in the same solution, therefore, the macromolecules are re-structured to acquire rheological and dynamic properties different from the multiple layers.

While gel formation in hydrogel production requires at least 5% (w/w) polymer, coacervates can be formed even at concentrations as low as 0.1% (w/w). In hydrogel production, organic solvents, or toxic chemical crosslinkers or initiators are used for the formation of gels. They adversely affect cell viability in the cell encapsulation phase. With the complex coacervation method, chemical cross- linking agent usage is not required, and thus the potential toxicity and other undesirable effects of the reagents are reduced. These properties make the coacervates more advantageous than the other polyelectrolyte systems. Summary of the Invention The objective of the present invention is to provide a method for producing coacervates which are used as a tissue scaffold that is biodegradable, biocompatible and elastic. Another objective of the invention is to provide a method for producing coacervates which are used as tissue scaffolds that are non-toxic since they consist entirely of natural polymers and no organic solvents or crosslinking agents are used. A further objective of the invention is to provide a method for producing coacervates which are used as a tissue scaffold that is permeable enough to allow cell viability (having a structure that allows diffusion of nutrients, oxygen, carbon dioxide and cell wastes), has components (e.g. hyaluronic acid) that can bind to the cell surface receptors (CD44 and RHAMM) to which the cells can easily adhere, and has a high cell entrapment capacity in a dense and viscous structure.

It is also an objective of the invention to provide a method of producing coacervates that are used as tissue scaffolds, which contain a high amount of water (> 90% by weight) and have a porous structure providing a suitable area for the proliferation of encapsulated cells, and if stem cell is used, for the differentiation of the desired cell type.

Detailed Description of the Invention "Coacervate Production Method Using Coacervation And The Usage of Coacervates Obtained By This Method As Tissue Scaffold" developed to fulfill the objectives of the present invention is illustrated in the accompanying figures, wherein

Figure 1. shows graphical and photographic representation of turbidity of the coacervate suspensions, which are formed after addition of different volumes of hyaluronic acid (HA) solution onto chitosan (CT) solution, wherein the turbidity is measured by absorption (HA and CT solutions are prepared in (A) NaCl solution; (B) CaCh solution having 300 mM ionic strength). (Visible turbidity of the coacervate suspensions can be observed in the photographs. The volumetric ratios of addition of HA solution onto CT solution are given on each cuvette). is a graphical representation of the Z average diameters (light intensity weighted average hydrodynamic diameter) obtained by titrating the cationic CT polyelectrolyte with anionic HA poly electrolyte. (300 mM ionic strength of the solution is adjusted by using (A) NaCl salt; (B) CaCh salt.) shows the optical microscopy images of the coacervate suspension formed after addition of HA solution to CT solution. ((A) Droplets in the coacervate suspension, (B) Ball-shaped formations (floes) resulting from the precipitate; The scale bars represent 20 μιη.) shows the scanning electron microscope (SEM) images of the coacervates, which are formed after phase separation, following lyophilization. ((A) Sample prepared by using NaCl salt with no cell encapsulation (empty), (B) Sample prepared by using CaC salt with no cell encapsulation (empty), (C) Sample prepared by using NaCl salt with ceil encapsulation, (D) Sample prepared by using CaCh salt with ceil encapsulation (The arrows show the cells and cell extensions). shows the viability of the encapsulated ceils inside the prepared HA/CT coacervates at the end of 1, 7, 14 and 21 days of incubation via live/dead staining. (The ratio of the number of live cells to the total number of cells gives the percentage of cell viability.) Figure 6. shows the morphological images of the encapsulated cells inside the HA/CT coacervates. (Images taken after 3 days of incubation, (A) prepared using NaCl salt solution (B) prepared using CaCh salt solution; images taken after 14 days of incubation, (C) prepared using NaCl salt solution, (D) prepared using CaCb salt solution) (The actin filaments are shown with sideward facing arrows while the ceil nuclei are shown with downward facing arrows.) (The scale bars represent 10 um in the upper images and 5 um in the lower images.)

Figure 7. shows schematic representat on of the complex coacervation method.

The components shown in the figures are each given reference numbers as follows:

CT. Chitosan polymer

HA. Hyaluronic acid polymer

SN. Supernatant

Co. Coacervate

The method of production of coacervate with the coacervation method of the present invention is used in the formation of tissue scaffold supporting tissue regeneration and for that purpose, chitosan (KT) and hyaluronic acid (sodium hyaluronate, hyaluronan) (HA) polymers, and encapsulated cell are used for the production of this natural polymeric scaffolds and it comprises the following steps:

- dissolving chitosan (CT) and hyaluronic acid (HA) in aqueous salt solution, - adding HA solution to the cells and mixing in order to prepare the cell- containing coacervates,

- then, adding HA/cell suspension dropwise to the salt-containing CT solution, and stirring on the magnetic stirrer,

- applying centrifuge to the turbid phase resulting by means of the electrostatic attraction between the oppositely charged macroions (chitosan and hyaluronic acid) in the mixture and entropy gain associated with the small oppositely charged ions (Na⁺, Ca²⁺, CI^") released to the solution (complex coacervation), and after the coacervate droplets in this phase are coalesced, carrying out liquid-liquid phase separation,

- collecting the lower coacervate phase, wherein the macroion concentration is considerably higher than the initial macroion concentration, by centrifugation without subjecting it to any additional processes,

- obtaining the coacervates which are directly used as tissue scaffold.

The subject of the invention is to produce tissue scaffolds, which are capable of cell encapsulation for tissue engineering using complex coacervation technique, with chitosan (CT) and hyaluronic acid (HA) polymers. The invention includes natural polymer systems, which are designed to support tissue regeneration particularly in connective tissue injuries, and in which cells can be encapsulated. It is within the scope of Bioengineering and Material Engineering areas and is included in the Biotechnology sector.

The coacervate products obtained within the scope of the invention are directly used in obtaining tissue scaffolds. After the coacervate formation achieved by complex coacervation method wherein more than one (multiple) polymers are used, cell culture applications (in vitro) are carried out by taking them directly into well plates without performing any additional process. The term "coacervate" refers to the lower dense phase among the two liquid phases formed by applying centrifuge to the turbid phase mixture resulting from the complex coacervation method. When the cell number ratio per produced coacervate volume is calculated, there are 1 million cells presented in 1 mL coacervate. The number of cells entrapped within the coacervates can vary starting from thousands to as much as 10 millions.

In the scope of the invention, firstly, 0.09% (w/w) chitosan (CT) and 0.07% (w/w) hyaluronic acid (HA) biopolymers are dissolved by stirring for 24 hours with a magnetic stirrer at a constant speed at room temperature in an aqueous solution of sodium chloride (NaCl) salt or an aqueous solution of calcium chloride (CaCl₂) salt having 300 mM total ionic strength. During this process, the pH values of both polymer solutions are adjusted to 6.25 by addition of 1 M (Molar) sodium hydroxide or hydrochloric acid solution, and each polymer solution is sterilized in a laminar flow cabinet using a 0.45 micrometer pore size filter. The pH value of the HA/CT mixture used in the coacervation should support the survival and viability of the cells (since the coacervation method is applied when the cells are in the coacervate) (optimum 7.4). However, due to insolubility of chitosan polymer at high pH values, the different deacetylation degrees of CT should be optimized. It is possible to perform coacervation by using HA/CT polymers in the range of pH 5.5-7 if cell encapsulation is to be performed and in the range of pH 2-7 in cell-free samples.

The dissolution of the polymers to be used in the coacervation may be carried out using only ultra-pure water or with different concentrations of salt or buffer solutions; however the properties of the coacervate (water amount inside the coacervate, its mechanical properties, etc.) vary depending on the solvent used. Furthermore, HA/CT coacervates are suitable for working under many experimental conditions (ionic strength, temperature and pH) close to the physiological conditions that provides an advantage for ensuring cell viability. In order to prepare the empty coacervates (control) that are used for comparing with the cell encapsulated samples, the solution containing only HA is added dropwise to the solution containing CT. During this addition, the HA/CT mixture is uninterruptedly continued to be stirred with the magnetic stirrer. Both HA and CT used to prepare the coacervate are required to be dissolved in the same ionic salt type. In one study, both HA and CT solutions are dissolved in NaCl solution, while in another study both HA and CT solutions are dissolved in CaCl₂. After dissolution of the polymers in the salt solution, only the HA solution for empty samples (control), and, cells suspended in HA solution for cell encapsulated samples are added dropwise onto CT solution placed on the stirrer. To prepare the cell-containing coacervates, firstly, HA solution is added onto the cells (500,000 cells / coacervate, i.e. 1,000,000 cells / mL) and stirred. Then, HA/cell suspension is added dropwise to the CT solution which contains the same salt and which is being stirred in the magnetic stirrer. Differing from the literature in general, this dropwise addition process is not performed by the method of addition with the syringe needle that is used for forming microcapsule and has a certain diameter (generally <2 mm of needle tip outer diameter), but it is performed by using electronic or manual pipettors. Thus, by adding droplets without creating any air entrapment to the chitosan solution, a turbid phase formation (coacervate suspension) is provided which is homogeneously dispersed and contains spherical droplets having a diameter of 0.5-1 micrometers. At this point, in the present invention, a turbid and colloidal dispersion with spherical droplets having a diameter of 0.5-1 micrometers is formed instead of the formation of microcapsules in the size of 1-100 mm. This turbid phase occurs by means of the electrostatic attraction between the oppositely charged macro-ions (chitosan and hyaluronic acid) and the entropy gain associated with the small oppositely charged ions (Na⁺, Ca²⁺, CI^") released to the solution (complex coacervation phenomena). After centrifuge is applied to the turbid phase formed by complex coacervation method, the coacervate droplets in this phase are coalesced and then liquid-liquid phase separation is carried out. In the centrifugation method used to accelerate the liquid-liquid phase separation, the duration and speed of centrifugation can be varied according to the resistance of the cell type used in the coacervate and the purpose of the application. Instead of using the centrifugation step, allowing the coacervation suspension to rest for several days also results in a liquid-liquid phase separation only under the influence of gravity.

Among the two phases, the upper one is called the supernatant (the concentration of the macro-ions therein is lower than the initial polymer concentrations) and the lower one is called the coacervate (the concentration of the macro-ions therein is significantly higher than the initial macro-ion concentrations). Empty or cell containing coacervates accumulated in the lower phase by using centrifugation and collecting the micro-ions at the bottom can directly be used as tissue scaffold without subjecting them to any additional processes (sonication, lyophilization, organic solvent evaporation, solvent casting, etc.). For the preparation of cell- containing coacervates, unlike the literature, instead of seeding cells onto the coacervate tissue scaffold after their formation, the cells are prepared first by being suspended in one of the polymers, and then adding the resulting cell/polymer mixture dropwise to the oppositely charged polymer solution in the present invention. In other words, the cells are confined directly in the scaffold by performing macroencapsulation instead of microencapsulation.

Characterization of the mixtures, which are obtained upon dissolving chitosan and hyaluronic acid polymers in different salt solutions (NaCl or CaCb) and mixing thereof, and which have different HA/CT addition ratios (by volume) prepared in different vessels, is performed (by using centrifuge or allowing to rest for a few days, before they are exposed to phase separation) by turbidity measurement, UV- Vis spectrometer, dynamic light scattering (DLS) and optical microscopy techniques. Phase separation is performed after centrifugation by complex coacervation method. The porosity and surface analyses of the coacervates, which are obtained after phase separation and will be used as tissue scaffolds, are performed using scanning electron microscopy (SEM). After characterization, cell encapsulation of the tissue scaffold is performed by the same method. The viability, morphology and surface properties of these cells are controlled at this stage. The difference of the prepared coacervates from the hydrogels in the current literature is that here, less than 5% by weight of polymer is used without crosslinking agent or organic solvent. The complex coacervation method can also be applied at different polymer concentrations. However, the concentrations of the polymers should be chosen to be lower than the polymer overlap concentrations which vary according to the molecular weight of the polymer. In addition, while a polymer concentration of about 1% (w/w) is required to prepare a hydrogel, this polymer concentration can be reduced to 0.01% for the preparation of coacervates. The coacervation technique is a technique that has not been used in the production of cell encapsulated tissue scaffolds for tissue engineering. This method is frequently used for encapsulation (micro-encapsulation) of various molecules (protein, growth factor, etc.) in the literature, but there is no such example in the literature as creating a tissue scaffold and entrapping cells therein (macro- encapsulation). Also, the method described in the present invention is different from macro-encapsulation of a commercial cell-containing tissue scaffold by complex coacervation method. The coacervates are coated on the top and bottom parts of the cell-containing commercial tissue scaffolds. In the present invention however, coacervates produced by our own method are used as the cell macro- encapsulated tissue scaffold instead of a commercial tissue scaffold.

In order to prevent both biocompatibility and phenotype loss problems due to the usage of cross-linking agents or organic solvents, complex coacervation technique (macro-encapsulation) is carried out to produce cell encapsulated tissue scaffold for tissue engineering purposes within the scope of the invention. By means of the present invention, it is ensured that tissue scaffolds, which are formed using promising chitosan and hyaluronic acid polymers by the unique complex coacervation technique that can encapsulate cells, are introduced to the connective tissue engineering.

Preparation of polymer solutions and procedure of complex coacervation: The 0.09% by weight of chitosan (CT) and 0.07% by weight of hyaluronic acid (HA) polymers are dissolved separately in either sodium chloride (NaCl) solution or calcium chloride (CaCl₂) solution at room temperature. The complete dissolution of both polymers is achieved by constant stirring for 24 hours with a magnetic stirrer. The pH value of each polymer solution is adjusted to 6.25, and each solution is sterilized in a laminar flow cabinet using a 0.45 micrometer pore size filter. Then HA polymer solution is added dropwise onto the chitosan (CT) polymer solution provided on the stirrer. In here, HA/CT mixtures prepared in separate cuvettes for determining the amounts of polymers that give the highest amount of tissue scaffold and provide the complex coacervation properties of the tissue scaffold without transforming to different polyelectrolyte complexes or phases such as precipitate, film, spongelike structure, etc. are characterized by turbidity measurement, dynamic light scattering (DLS) and optical microscopy.

Instead of the addition of HA to the CT solution, mixing can be carried out by addition of CT on the HA solution. For this, molecular weights and concentrations of the polymers, acetylation degree of the chitosan polymer, temperature, pH, ionic strength, and the ratio of volumes of the HA and CT solutions to each other should be optimized. Addition of HA and CT solutions to each other can be carried out dropwise by the help of pipettes having an outer diameter greater than 2 mm such as electronic or manual pipettes (pipettors - macro, micro), pasteur pipettes, automatic pipettes and burettes). In the literature, microcapsule formation with drops smaller than 2 mm by using a syringe tip of 16-23 thickness (gauge) was observed. The addition of the polymer solution (e.g. HA solution) leaving the syringe tip to the oppositely charged polymer solution (e.g. CT solution) without causing any air entrainment is the difference of the present invention from the others. Otherwise, when the drops comprising the HA solution in which air is entrapped are added to the CT solution, microcapsules are formed like in the literature (in such system in the literature, coacervation takes place on the interface of the microcapsules). In our method, instead of capsule formation, a single turbid phase is formed. This phase is not subjected to any additional process such as washing or dissolving (by salt, organic solvents or acid).

Turbidity measurement:

Turbidity measurement is a method commonly used to investigate the properties of poly electrolyte complexes. The changes in the turbidity of the solution resulting from the charged or neutral droplets in the coacervate suspension formed when HA interacts with CT are measured by this method and the mixing ratios that form coacervation are determined.

The effect of the HA/CT mixing ratio ( in which volumetric ratio can be converted to the ratio of ionized carboxyls (COO ) in HA to the ionized amines in CT (N¾⁺), and thereby to the ratio of negative charges to positive charges) on the coacervation degree is investigated by turbidity measurement. Turbidity measurements are made using the UV spectrophotometer and the HA/CT mixtures are provided using a magnetic stirrer. The turbidity is measured at wavelength 630 nm (none of the polymers absorb light at this wavelength) at room temperature. The turbidity is measured in absorption units (a.u.). In all samples, used salt solutions are taken as a reference. The same measurements can be recorded as "100 - percent permeability" (100 -% T) instead of the absorption unit. For this measurement, a colorimeter can also be used instead of the UV spectrophotometer. The general purpose in these experiments is to find out at which volumetric ratios (or charge ratios) the HA/CT mixture undergoes complexation or phase separation. By combining the turbidity test results with the changes that are visible or can be observed by microcopy (details of the microscopy experiments are given below) that the mixtures undergo; information about at which volumetric ratios (or charge ratios) they undergo liquid-liquid phase separation, at which ratios they undergo liquid-solid separation (precipitate, flocculate, etc.), at which ratios they form only nanosized complexes and at which ratios they do not interact can be obtained. Such a comprehensive and systematic analysis is only available in a limited number of studies in the literature.

While the solutions in low HA/CT mixing ratios appeared to be transparent, the solutions got turbid with the addition of increased HA (Figure 1A-B lower images). The HA/CT mixtures appeared to be turbid because they contain coacervate droplets. When a beam of light passes through the coacervate suspension, the scattering of light from the coacervate droplets increases in all directions. This increase can be due to the increase in number and size of the coacervate droplets as a result of strengthening of the interaction between the two poly electrolytes. However, the addition of excessive amount of HA forms precipitate (solid-liquid phase separation) instead of coacervate (liquid-liquid phase separation) and reduces turbidity as a result of precipitation of the particles (Figures 1A-B upper images). In view of the turbidity measurements, it is observed that the HA/CT mixture, which has a volume mixing ratio of 0.8/1 (HA/CT), remains as a coacervate suspension and higher proportion of mixing ratio produces precipitates.

Dynamic Light Scattering (DLS) measurements:

DLS measurement (also known as photon correlation spectroscopy or quasi- elastic light scattering) is a method that records the random changes in the scattering of light at a certain angle from the particles. In the literature, this method is also widely used to measure the hydrodynamic particle sizes of poly electrolyte complexes or coacervate droplets. The particle sizes in the coacervate suspension formed by the charged or neutral droplets formed when the HA interacted with CT are determined by DLS measurements and the mixing ratios providing the characteristic droplet sizes in the coacervation formation are determined. For DLS measurements, the suspensions prepared by the volumetrically increasing HA addition ratios of the HA/CT coacervate complexes are measured and the Z average diameters (light intensity weighted average hydrodynamic diameter) are shown in Figure 2. Z average diameters of the coacervate complexes (after addition of HA of 0.1 to 1 mL for 1 mL CT) are measured between 0.5-1 μπι and it is observed that they almost have a constant polydispersity index (Pdl <0.3). The polydispersity index is obtained by an equation showing the distribution of the measured particle sizes. It is provided by the measuring device in addition to the measured particle size. If Pdl value is between 0.1-0.3 it is in a narrow distribution, and if it is greater than 0.5 it is in a wide distribution. These measured droplet ("liquid aggregate") dimensions are also consistent with the optical microscopy images showing the coacervate dropleis (Figure 3). Furthermore, a sharp increase occurs in the Z average dimensions (approximately 5 μτη) of the HA/CT complexes in which precipitation is observed (Pdl> 0.5). The reason for this increase indicates that the complexes formed are not coacervate droplets but precipitates.

The fact that Pdl value is less than 0.3 indicates that the droplets have approximately the same size and meet the coacervation criteria. It has been proved by these measurements as well that coacervate droplets are different from the microcapsules which are mentioned in the literature and whose sizes are in millimeters. The sharp increase observed in the higher HA/CT volumetric mixing ratios indicates that the droplets are no longer coacervates but they transform into various types of precipitates (for example, floes), which have lost their water content. The observation of many small and big particle formations in suspensions with Pdl values of greater than 0.5 indicates that solid-liquid phase separation has taken place.

Optical Microscopy:

The coacervate droplets determined by turbidity measurement and DLS are also controlled by means of optical microscopy and the fact that the mixture to be used meets the coacervation criterion is proved with visual images. Optical microscopy is used to confirm the presence of liquid coacervate droplets in the oppositely-charged polyelectrolyte mixtures. The addition of HA to the obtained polyelectrolyte mixture is continued until precipitation is observed. The coacervate suspensions are placed on glass slides to observe the droplets. All kinds of polyelectrolyte complex formations such as precipitate or complex coacervate can be controlled by this method.

While the small spherical droplets of about 0.5-1 um in the coacervate liquids are as seen in Figure 3A; the asymmetric structures or the ball-shaped formations in the samples, where liquid-solid phase separation, i.e. the precipitate, is formed, are as viewed in Figure 3B. After the characterization of the coacervate liquids, it was decided to use the samples with a HA/CT mixture ratio of 0.4/1 (v/v) in the construction of the tissue scaffold. The phase separation is performed on the coacervate suspension to form the tissue scaffold, and the prepared samples are centrifuged at 4,000 rpm for 23 minutes to facilitate this process. After centrifugation, the supernatants (the macroion-deficient liquid phase remaining above the coacervate phase) are carefully decanted without disturbing the coacervate phase. The prepared coacervates are easy-to-form (elastic).

Scanning Electron Microscopy (SEM):

A scanning electron microscope (SEM) analysis is performed to the coacervate sample obtained after phase separation (the lyophilization process is carried out only at this stage for the purpose of making the samples suitable for SEM imaging) for investigating the porosity and interactions between cell and coacervate scaffolds. AH surfaces of the samples are coated with 10 nm gold just before the SEM analysis. The SEM application is applied at 10 kV and at different magnifications. AH the coacervates obtained exhibit porous structure. The SEM images reveal that the coacervates have a highly porous internal structure suitable for loading cells and a smooth surface (Figure 4 A-B). In addition, the cells which are encapsulated in coacervate samples are in contact both with each other and the tissue scaffold by means of their extracellular matrices (Figure 4 C-D). It is also observed that the coacervate tissue scaffolds formed at this stage do not form film, sponge or fiber- like structures.

Live/Dead staining:

Live/dead staining is performed for the samples containing rat bone marrow stem cells (rBMSCs) (500,000 cells/coacervate or 1,000,000 cells/mL) to determine and compare the biocompatibilities of different coacervates. Live/dead staining is a method used for determining living and dead cells in the material by detecting plasma membrane integrity and intracellular esterase activity. If the cells are alive they are stained in green color and if they are dead, they are stained in red color. ImageJ software is used to evaluate cell viability. Three different regions for each sample are chosen to calculate the number of green and red stained ceils. The ratio of the number of live cells to the total number of cells (percentage of cell viability) is calculated (Figure 5). In the live/dead staining performed one day after the encapsulation, a large number of the encapsulated cells are observed in green color in both types of coacervates prepared by using NaCl or CaC12 salt solutions. Based on this result, it is observed that the method of coacervate preparation and cell encapsulation do not cause any damage to the cells. In addition, almost all of the encapsulated cells are found to be still alive after 21 days of incubation. Based on these finding, it can be observed that the prepared coacervates do not create any toxic effect.

Morphology Analysis:

In order to analyze the morphology of the cells encapsulated in the coacervates, actin filament and nuclear stainings are applied. For actin filaments, an F-actin probe conjugated to red fluorescent dye, and for the nucleus, blue fluorescent dye (DAPI) are used. The smooth shape of the cells observed in Figure 6 show that they have undamaged plasma membranes. This also supports the results of live/dead staining. After 3 days of incubation, the stained cells are found to have a spherical cellular morphology due to encapsulation (Figures 6A and B). In the stainings after 14 days of incubation, the cells exhibit a spreading morphology and they are observed to begin interactions with each other using their extracellular matrices (Figures 6 C and D). The use of different salt solutions in the preparation stage of the coacervates does not cause distinctive differences in terms of viability and morphology of the cells. As a result, it is observed that prepared 3 dimensional coacervates support cell growth and proliferation. From these findings, it is determined that the prepared coacervates can be used as biocompatible and promising tissue scaffolds.

In the scope of the invention, chitosan and hyaluronic acid polymers are made into a unique tissue scaffold using the complex coacervation method. This method has previously been used extensively in the fields of food, agriculture, cosmetics and pharmaceuticals; but this is the first time in terms of its feature of cell encapsulation (macro-encapsulation) inside the tissue scaffold produced by using complex coacervation technique for tissue engineering. In the literature, whenever cells were used; the cells were seeded after the complexes (films, sponges, layer by layer polymer coatings, fibers, nanoparticles, micro-capsules, coacervates, etc.) from oppositely-charged polymers were formed and subjected to various processes (sonication, lyophilization, immersion into the polymer, organic solvent evaporation, solvent casting, etc.).. In addition, tests such as cell viability, cytotoxicity, morphology, etc. were also carried out by placing the prepared coacervates on the cell seeded well-plates. In fact, in these studies, molecules such as growth factors, etc. were encapsulated into coacervates in advance to ensure cell proliferation and differentiation. The major difference of our study from these studies in the literature is that the cells are dissolved in one of the polyelectrolyte solutions before the coacervate is formed and added to the oppositely-charged polymer, and then they are subjected to only centrifugation at room temperature and thereby the cell-containing coacervate phase is obtained. The prepared tissue scaffolds are biodegradable, biocompatible and have elastic feature. They are non-toxic as they consist entirely of natural polymers and no crosslinking agents or organic solvents are used. The porous structure provides a suitable environment for cell survival and proliferation, and also for differentiation if stem cell will be used, and promising results can be obtained particularly in connective tissue engineering applications by the use of macroencapsulation technique.

Claims

A method of producing coacervate with chitosan (CT) and hyaluronic acid (HA) for production of natural polymer system, which is used in producing tissue scaffolds designed to support tissue regeneration particularly in connective tissue injuries, and in which cells are encapsulated, characterized by the steps of

- dissolving chitosan (CT) and hyaluronic acid (HA) only in water, salt solution or buffer solution,

- adding HA solution to the cells and mixing in order to prepare the coacervate-containing cells,

- then, adding HA/cell suspension dropwise to the salt-containing CT solution, which is being stirred in the magnetic stirrer,

- applying centrifuge to the turbid phase formed by the electrostatic attraction between the oppositely charged macroions (chitosan and hyaluronic acid) in the mixture and entropy gain associated with the oppositely charged small ions released to the solution (complex coacervation), and after the coacervate droplets in this phase are coalesced, carrying out liquid-liquid phase separation,

- obtaining the coacervates which are directly used as tissue scaffold.

A method of producing coacervate according to Claim 1, characterized in that the salt solution used in the step of dissolving chitosan (CT) and hyaluronic acid (HA) in salt solutions is sodium chloride (NaCl) salt solution.

A method of producing coacervate according to Claim 1, characterized in that the salt solution used in the step of dissolving chitosan (CT) and hyaluronic acid (HA) in salt solutions is calcium chloride (CaCb) salt solution.

A method of producing coacervate according to Claim 2 or 3, characterized in that, in the step of dissolving chitosan (CT) and hyaluronic acid (HA) in salt solutions, 0.09% (w/w) chitosan (CT) and 0.07% (w/w) hyaluronic acid (HA) biopolymers are dissolved in sodium chloride (NaCl) salt solution or calcium chloride (CaC12) salt solution having 300 mM total ionic strength by stirring for 24 hours with a magnetic stirrer at constant speed at room temperature.

A method of producing coacervate according to Claim 1, characterized in that adding CT solution onto HA solution instead of adding HA solution onto CT solution.

A method of producing coacervate according to Claim 1, characterized in that addition of HA and CT solutions to each other is carried out dropwise by the help of electronic or manual pipettes having an outer diameter greater than 2 mm such as pipettors - macro, micro), pasteur pipettes, automatic pipettes and burettes to the oppositely charged polymer solution (e.g. CT solution) without causing any air entrainment.

A method of producing coacervate according to Claim 1, characterized in that, in the step of dissolving chitosan (CT) and hyaluronic acid (HA) in salt solutions, the polymers are dissolved in the salt solutions by stirring at constant speed by a magnetic stirrer for 24 hours.

A method of producing coacervate according to Claim 1, characterized in that, in the step of dissolving chitosan (CT) and hyaluronic acid (HA) in salt solutions, the pH values of both polymer solutions are adjusted to 6.25 by addition of 1 molar sodium hydroxide or hydrochloric acid solution.

9. A method of producing coacervate according to Claim 1, characterized in that the pH value of the HA/CT mixture used in formation of empty coacervates (no cell) is adjusted to be in the range of 2-7.

10. A method of producing coacervate according to Claim 1 or 9, characterized in that the pH value of the HA/CT mixture used in formation of cell-containing coacervates is adjusted to be in the range of

5.5-7.

11. A method of producing coacervate according to Claim 1, characterized in that, in the process of dissolving chitosan (CT) and hyaluronic acid (HA) in sodium chloride (NaCl) solution or calcium chloride (CaCl₂) solution, both of the polymer solutions are sterilized in a laminar flow cabinet using a 0.45 micrometer pore size filter.

12. A coacervate which is produced by means of a method according to any one of the preceding claims and in which the number of entrapped cells per 1 mL of volume is 500,000 - 10 million when volumetric ratio is calculated.

13. Coacervate according to Claim 12, which is used directly as tissue scaffold without being subjected to any additional process (sonication, lyophilization, organic solvent evaporation, solvent casting, etc.).