3D bracket construction method
Regenerative medical materials, whether used as scaffolds for tissues/organs in vitro or for regenerative repair of tissues/organs in vivo, often require a certain three-dimensional (3D) spatial structure. A 3D scaffold with specific shape and microstructure, reasonable pore size and porosity can meet the reconstruction needs of specific tissues/organs, providing spatial support for cell attachment, migration, proliferation, differentiation, and the formation of new tissues. The pores are too small for cells to enter or hinder cell proliferation and expansion; If the pores are too large, the cells cannot adhere and aggregate, losing their scaffold function. Therefore, the preparation technology and process of scaffolds are crucial for the biomedical applications of materials.
There are various methods for preparing traditional 3D scaffolds, each with its own characteristics, but no method can simultaneously meet the requirements of all tissues. In response to the current problems with 3D scaffolds, people are researching and developing new preparation and processing methods to obtain more ideal scaffold materials. The emerging technologies of gas foaming and stent printing in recent years are expected to provide technical support for the preparation of a new generation of intelligent regenerative medicine 3D scaffolds (tissue engineering scaffolds).
(1) Fiber bonding method
The use of PGA fiber non-woven felt as a scaffold material is one of the earliest methods used in tissue engineering. As a classic scaffold material, it is still used in various aspects of tissue engineering research, especially in research related to bioreactors, biomechanical models, and so on. PGA non-woven felt is formed through processes such as melt extrusion, orientation, cutting and combing, and needle punching. The advantage of the skeleton composed of PLGA fibers is its large surface area, which is conducive to cell adhesion and nutrient diffusion, thus promoting cell survival and growth; The disadvantage is that the stability of the internal frame structure is not good, and its pore size is relatively large, with uncontrollable porosity and specific surface area. Therefore, it is necessary to improve this by bonding the disconnected fibers in the non-woven felt together. There are two main methods for connecting fibers.
One is the fiber fixation technology invented by Mikos et al. [35]. Immerse PGA fibers into a chloroform solution of PLLA, and after the solvent evaporates, embed the PGA fiber mesh into PLLA. Heat the mixture above the melting points of the two polymers. Due to different melting points, PLLA first melts and fills all the gaps in the PGA fiber network. Its function is to stabilize the PGA fiber and prevent the collapse of the fiber network structure during PGA melting. Continuing to heat up, the PGA fibers melt and bond together at the intersection. Select a solvent that can only dissolve PLLA (such as dichloromethane), dissolve it to remove PLLA, and obtain a highly porous PGA network scaffold. The porosity and pore size obtained by this method are generally as high as 81% and 500%, respectively μ M.
Another method is to coat the fiber surface by atomization or spray [36]. Since chloroform is not the solvent of PGA, PLLA or PLGA chloroform solution is sprayed on the surface of PGA fiber mesh by spray, and the structure of PGA fiber will not change. After the solvent evaporates, PGA fibers are bonded together by PLLA or PLGA at the intersection. This composite structure combines the mechanical properties of fibers and the surface characteristics of PLA, and its pore size is similar to the previous method.
The porous scaffold prepared by fiber bonding technology is characterized by high porosity, connectivity between pores, and certain mechanical properties, making it suitable for tissue regeneration. The disadvantage is that organic solvents are required, and the first method also requires high-temperature heating. The residual organic solvents and high-temperature processes are very detrimental to the introduction of growth factor like bioactive molecules and cells.
(2) Solution casting/particle leaching method
Solution casting/particle leaching technology is another traditional method for preparing porous scaffolds, following the PGA nonwoven scaffold. Its advantage is that by selecting water-soluble particles with a certain particle size distribution and adjusting the ratio of particles to polymers, a polymer skeleton with controllable pore size and porosity can be formed. Porogenic agents are usually soluble particles such as salts (NaCl, etc.) or sugars (sucrose, etc.).
The method is to dissolve the polymer (PLLA or PLGA) in CHCl3 or CH2Cl2, and then pour the solution onto a culture dish filled with a pore forming agent. After the solvent evaporates, the polymer/salt mixture is leached in water for two days to remove the pore forming agent, resulting in a particle free polymer skeleton.
The porosity of the skeleton is controlled by the amount of salt added, and the diameter of the pores is determined by the size of the salt grains [28]. Usually, when the weight percentage of salt reaches or exceeds 70%, the pores are highly interconnected with each other. However, the outer part of the foam exposed to the air is rougher than the part soaked in the Petri dish, that is, the two parts have different shapes, so this technology can not prepare too thick skeleton, which is generally less than 2mm. The improvement is to mold the blocky polymer/salt mixture into a cylinder shape at a melting point higher than PLLA or at the glass transition temperature of PLGA. Before the mixture is leached with water, the cylinder is cut into sheets of the required thickness. After the sheet polymer/salt complex is leached with water, the sheet skeleton is stacked together by the stacking technology, so that the thickness of the support can be more accurately controlled and the regularity of the foam surface can be improved. However, the thermal degradation of polymers during the molding process needs to be considered.
The drawback of this method is that it often has problems with closed pore structures and residual salt/sugar particles, which are detrimental to cell growth. To this end, Chen et al. [37] used ice crystals as pore forming particles and combined particle leaching with freeze-drying to develop a new method for preparing porous scaffolds. They obtained high porosity PLLA porous scaffolds and avoided the impact of salt/sugar particle residues on cell growth. Its pore structure is evenly distributed and interconnected. Then, the PLLA scaffold was further compounded with collagen, and after crosslinking with glutaraldehyde, a composite scaffold containing collagen microporous sponge in the PLLA pores was obtained.
To avoid the toxic effects of residual solvents on cells, Jung et al. [38] changed the solution casting method to sintering method to prepare porous scaffolds. The specific method is to mix 30-40mm PLA powder with NaCl particles, first squeeze at room temperature of 150Mpa for 3 minutes in the mold, then continue to squeeze at a temperature slightly higher than the PLA melting point (190 ℃) (210 ℃) for 30 minutes, cool and soak in water for 48 hours to remove salt particles, and replace the water every 6 hours. Compared with the solution casting method, the prepared porous scaffold has a more uniform pore size and significantly enhanced mechanical properties.
(3) Phase separation/freeze-drying method
The phase separation/freeze drying method refers to the method that the polymer solution, lotion or hydrogel undergoes phase separation in the process of low temperature freezing to form a solvent rich phase and a polymer rich phase, and then the solvent is removed by vacuum freeze drying to form a porous structure [39]. According to the different morphology of the system, it can be simply divided into lotion freeze drying method, solution freeze drying method and hydrogel freeze drying method. Its characteristics are: 1) avoiding high temperatures, which is conducive to the introduction and controlled release of bioactive molecules such as protein growth factors or differentiation factors; 2) The pore has a large specific surface area and is easy to operate; 3) Control the pore structure by adjusting the oil-water ratio and polymer molecular weight. The porosity of the scaffold can reach 90-95%, with a pore size range of 0.01-200um [40].
(4) Supercritical CO2 gas foaming method
Supercritical CO2 gas foaming method refers to the extrusion of polymers (blocks or sheets), soaking them in high-pressure carbon dioxide until saturation or even supercritical state, and then rapidly reducing them to atmospheric pressure. The thermodynamic instability of the gas leads to bubble nucleation and growth, forming a porous scaffold.
The advantages of this method are: 1) avoiding the use of organic solvents in the preparation of polymer scaffolds; 2) It can also avoid using high temperatures, which is beneficial for introducing growth factors under mild conditions.
The factors that affect porosity and pore structure in foaming method mainly include polymer crystallinity and molecular weight, equilibrium time, gas release rate, etc. Crystalline polymers PLLA and PGA are difficult to foam, while amorphous polymers PLGA are easy to foam, with a maximum porosity of up to 95%; The higher the molecular weight of the polymer, the more difficult it is to foam, and the lower the porosity; The longer the equilibrium time in high-pressure gas, the higher the porosity; The effect of gas release rate on porosity is relatively small. Vascular growth factors, such as endothelial cell growth factor, are successively integrated into PLGA and released in a controllable manner. Of particular importance, the activity of the released growth factors remains above 90% [41].
In addition to CO2, nitrogen (N2) and helium (He) have also been attempted for polymer foaming, but none have been successful. The disadvantage is that there are many closed pore structures, and both the porosity and pore size are relatively small [42]. If the foaming method is combined with the particle leaching method, connected porous scaffolds with open pore structures can be prepared [43].
(5) Chemical foaming method
Another method for preparing porous scaffolds is chemical foaming. Chemical foaming agents are mainly carbonate compounds. Mix the polymer solution with ammonium bicarbonate particles into a sticky state and add it to the mold. After the solvent partially evaporates, immerse it in hot water to foam, allowing ammonia and CO2 to evaporate. Finally, freeze dry to obtain a porous scaffold with high porosity and interconnectivity [44].
(6) Electrospinning technology
Electrospinning is a simple and feasible method for preparing porous scaffolds for tissue engineering. The electrospun scaffold has a unique microstructure and appropriate mechanical properties. Due to its nanoscale structure similar to natural ECM, the electrospun scaffold can mimic the structural characteristics of ECM, making it an ideal tissue engineering scaffold.
Electrospinning first appeared in 1934 when Formhals first introduced the method of obtaining polymer fibers using electrostatic repulsion in a patent in the United States [45]. The principle is to use an external electric field force to overcome surface tension in the polymer solution or melt and form a jet at the tip of the spinning nozzle capillary. When the electric field strength is high enough, under the combined action of electrostatic repulsion, Coulomb, and surface tension, the polymer jet bends along an unstable spiral track and is stretched tens of millions of times within a few tens of milliseconds. As the solvent evaporates, the jet solidifies to form submicron to nanoscale ultrafine fibers [46].
At present, more than 100 types of natural and artificially synthesized high resolution materials have been successfully electrospun into nanofibers. The resulting single fiber has a diameter ranging from 40-200nm, and can even span the range of 10-104nm, ranging from micrometers, sub micrometers to nanoscale, and is used in tissue engineering research [47].
The factors that affect electrospinning include process operating parameters (voltage, flow rate, receiving distance and temperature, humidity, air flow rate, etc.) and system parameters (polymer molecular weight, molecular weight distribution, molecular chain structure, solution concentration, solvent type and ratio, solution performance, such as viscosity, conductivity, interfacial tension, etc.). Due to the small pore size of electrospinning, usually below 100mm or even smaller, it is difficult for cells to enter the scaffold. Moreover, the thermal stability and mechanical support of electrospinning are poor.
Therefore, people are trying new spinning methods to make them suitable for the needs of tissue engineering, such as electrospinning polymers and water-soluble polymers simultaneously to prepare composite scaffolds, in order to obtain larger pore sizes.
(7) Computer assisted rapid prototyping method
In recent years, Rapid Prototyping Manufacturing (RPM) technology has received increasing attention for the preparation of tissue engineering scaffolds.
Rapid prototyping technology refers to the first step of obtaining a three-dimensional model through Computer Aided Design (CAD), and then layering it according to process requirements to divide the three-dimensional model into continuous two-dimensional cross-sectional images. Through continuous stacking and layer by layer processing, a three-dimensional porous scaffold with a pre designed hole structure and shape is obtained in one step. This includes stereolithography (SLA), laser sintering (LS), laminated object manufacturing (LOM), fused deposition modeling (FDM), and three-dimensional printing (3-DP) technologies.
Through these technologies, scaffold materials similar to human tissue microstructure and organ shape can be obtained, and personalized tissue engineering organ scaffold preparation can be achieved [48,49]. For example, for a patient with a bone defect, X-ray or CT scanning can be performed first to obtain images of the defect site. A threedimensional bone model can be designed using computer CAD software, and a specific size and shape of the bone scaffold can be prepared through printing technology for the patient's bone defect repair.
3D printing technology can also be used for selective modification of scaffolds to regulate the three dimensional spatial distribution of some special types of cells and guide the orderly regeneration of complex tissues. For this reason, Park et al. used printing technology to first modify the surface of PLLA with PEO-PPO copolymer. As a result, neither liver cells nor fibroblasts were able to adhere and grow on its surface; Then, the glycosyl ligand targeting the liver cell sialoglycoprotein receptor was covalently connected to the end of the PEO chain, resulting in selective adhesion to liver cells [50].
