Scaffold for living donor transplantation
10994054 ยท 2021-05-04
Assignee
Inventors
Cpc classification
B33Y10/00
PERFORMING OPERATIONS; TRANSPORTING
A61L2430/02
HUMAN NECESSITIES
B33Y70/00
PERFORMING OPERATIONS; TRANSPORTING
B33Y80/00
PERFORMING OPERATIONS; TRANSPORTING
A61L27/446
HUMAN NECESSITIES
International classification
B33Y10/00
PERFORMING OPERATIONS; TRANSPORTING
A61L27/54
HUMAN NECESSITIES
Abstract
Disclosed is a scaffold for living donor transplantation, which includes sintered bioglass and a biocompatible polymer and has improved properties using a 3D printer through a fused deposition modeling process.
Claims
1. A scaffold for living donor transplantation, configured such that struts, which are disposed on top of each other and cross each other in a vertical direction to thus form pores therein, are stacked layer by layer, wherein the struts include 10 to 70 wt % of sintered bioglass and 30 to 90 wt % of a biocompatible polymer, wherein the struts are configured such that sintered bioglass particles are dispersed in a biocompatible polymer matrix, wherein the scaffold is fabricated by mixing the sintered bioglass and the biocompatible polymer and then performing injection molding, and wherein the scaffold has a toughness of 50 kPa/mm to 850 kPa/mm and a stiffness of 1.5 N/mm to 20 N/mm.
2. The scaffold of claim 1, wherein the sintered bioglass particles have an average particle size of 1.5 to 2.5 m.
3. The scaffold of claim 1, wherein the struts have a diameter of 300 m to 500 m.
4. The scaffold of claim 1, wherein the struts have a stack structure of two or more layers.
5. The scaffold of claim 1, wherein the struts are provided in an interconnected or disconnected form in a same layer and are formed parallel in a regular pattern including a linear shape, a corrugated shape, a lattice shape, a zigzag shape or a spiral shape, or in an irregular pattern.
6. The scaffold of claim 1, wherein the struts have an average orientation angle of 30 to 60 between layers.
7. The scaffold of claim 1, wherein the scaffold has a bimodal pore size distribution and a porosity of 30% to 60%.
8. The scaffold of claim 1, wherein the scaffold has a roughness (Ra) of 130 nm to 260 nm and a water contact angle of 75 or less after 180 sec.
9. The scaffold of claim 1, wherein a protein proliferation absorbance after 24 hr is 0.25 O.D. (optical density) or more, a cell-seeding efficiency is 37% or more, a cell proliferation absorbance after culture for 7 days is 0.22 O.D. or more, and an F-actin area ratio is 22% or more.
10. The scaffold of claim 1, wherein the injection molding is performed using a 3D printer through a fused deposition modeling process.
Description
DESCRIPTION OF DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
DETAILED DESCRIPTION OF THE INVENTION
(9) A layer-by-layer (LBL) stacking process is performed to obtain a 3D product by stacking one layer on another. The present invention pertains to a scaffold for living donor transplantation fabricated using an LBL stacking process.
(10) Specifically, the scaffold for living donor transplantation of the present invention is configured such that struts, which are essentially disposed on top of each other and cross each other in a vertical direction to thus form pores therein, are stacked layer by layer.
(11) The external shape and dimension of the scaffold for living donor transplantation may be selected so as to be suitable for individual applications, and may meet the purpose required in the application field. The scaffold for living donor transplantation may have an external shape selected from among an extended shape, for example, a cylindrical shape, a polygonal column shape such as a triangular prism shape or an ingot shape, a planar shape, a polygonal shape, for example, a rectangular shape, a cubic shape, a square shape, a pyramid shape, a pentagonal shape, a 12-gonal shape, a 20-gonal shape, a rhombohedral shape, a prismatic shape, a spherical shape, for example, a ball shape, a hollow shape, a protruded lens shape or a cylindrical lens shape, and a disc shape or an annular shape. This shape may be achieved by stacking struts.
(12)
(13) The struts constituting the scaffold for living donor transplantation are provided in multiple layers, including at least two layers, three or more layers, four or more layers and n or more layers, and the number of layers thereof may vary depending on the end use of the scaffold. Preferably, when struts are stacked in four or more layers to form a scaffold, it is advantageous for ensuring the properties required of the scaffold.
(14) A single strut layer is configured such that a filament of substantially a single composition is extended, and also includes a structure in a disconnected form, such as a lattice pattern. The struts may be formed to extend in various shapes in the same layer and may be formed in a regular pattern such as a linear shape, a corrugated shape, a lattice shape, a zigzag shape or a spiral shape, or in an irregular pattern, in order to increase the contact area with cells and fluids. Preferably, the formation of a regular pattern, such as a lattice shape, is advantageous for controlling the properties and porosity of the scaffold.
(15) Also, in order to satisfy the properties required of the scaffold, the struts may have a diameter of 300 m to 500 m, 350 m to 470 m, or 380 m to 450 m. More preferably, the diameter thereof ranges from 390 m to 425 m. If the diameter thereof is less than the above lower limit, it is difficult to ensure properties such as strength due to the low thickness thereof. On the other hand, if the diameter thereof exceeds the above upper limit, the pores are relatively small in size and are unable to achieve sufficient porosity for cell growth or fluid flow.
(16) The struts stacked in two or more layers are configured such that adjacent layers are arranged to be rotated by a predetermined angle to thus form an orientation angle between these layers. As will be explained later, struts are formed through an injection-molding process, in which injection may be performed using a nozzle mounted on a 3D transfer mechanism, the position of which is adjusted in three directions, namely XYZ directions. Thus, the direction mentioned below means the injection direction.
(17) For example, in the case of a structure in which struts are stacked in four layers, the first layer is formed in parallel at a regular spacing in the X-axis direction, and the second layer is formed in parallel at a regular spacing at a predetermined angle relative to the X axis of the first layer. Here, the layer formed in parallel in the second layer is reset to the X axis, and the third layer is formed in parallel at a predetermined angle relative to the X axis. Further, the layer formed in parallel in the third layer is reset to the X axis, and the fourth layer is formed in parallel at a predetermined angle relative to the X axis.
(18) Such an orientation angle may be referred to as a rotation angle, and the average orientation angle between the layers of n-layered struts may fall in the range of 5 to 355, 10 to 350, 15 to 345, or 20 to 330. More preferably, it falls in the range of 5 to 180, 5 to 160, or 10 to 150. The average orientation angle is an orientation angle between layers, and when a four-layered structure is configured such that layers are arranged at a layer orientation angle of 60, the orientation angle between the first layer and the fourth layer may be 180. The orientation angle is an average numeric value, and in the n-layered structure, the orientation angle between the layers may be formed equally or unequally.
(19) In regard to the orientation angle, based on the first layer, the second layer may have an orientation angle of 5 to 180 relative to the first layer and the third layer may have an orientation angle of 10 to 340 relative to the first layer. As such, the orientation angle between the first layer and the n.sup.th layer may range from 0 to 360.
(20) According to an embodiment of the present invention, in the structure of struts stacked in four layers, the second layer may be arranged to be rotated by 30 to 60 relative to the first layer, the third layer may be arranged to be rotated by 75 to 105 relative to the first layer, and the fourth layer may be arranged to be rotated by 120 to 150 relative to the first layer.
(21) According to another embodiment of the present invention, in the structure of struts stacked in four layers, the second layer may be arranged to be rotated by 40 to 50 relative to the first layer, the third layer may be arranged to be rotated by 85 to 95 relative to the first layer, and the fourth layer may be arranged to be rotated by 130 to 140 relative to the first layer.
(22) Due to the aforementioned arrangement of the struts, the struts constituting individual layers cross each other, thereby forming pores therein. The pores are largely classified into two or more types, for example, pores formed by stacking struts in a vertical direction and pores formed by the spacing between struts arranged in the same strut layer. The pore size may vary depending on the stack structure and the spacing, and the pores have a plurality of pore distributions having the same or different pore sizes, such as bimodal and trimodal distributions. Moreover, the inter-pore regions may be independently formed, or may be in the form of vertically connected channels (i.e. interconnected pore structures) to enable free movement of the fluid.
(23) If the struts are stacked outside the above angular range, the mechanical properties of the resulting scaffold for living donor transplantation may be deteriorated, and moreover, it may be difficult to control the pore size and porosity thereof, thus being unsuitable for living donor transplantation. Hence, when the struts are arranged in multiple layers through relative rotation as described above, it is possible to fabricate a scaffold having a geometrically stacked structure and to attain mechanical properties and effects such as cell growth and the like by virtue of the above structure.
(24) According to an embodiment of the present invention, in the structure of struts stacked in four layers, the scaffold for living donor transplantation has a bimodal pore distribution having first pores having a size of 380 m to 430 m, and preferably 410 m to 420 m, and second pores having a size of 200 m to 250 m. Here, the porosity of the scaffold for living donor transplantation may vary depending on the type of stacking of struts and the diameter thereof, but may fall in the range of 30% to 60%, 35% to 55%, 40% to 50%, or 40% to 45%.
(25) The pore size and porosity of the scaffold for living donor transplantation may fall within ranges that facilitate cell proliferation, cell differentiation and bone formation, and moreover, may be freely adjusted within the above ranges so as to enable movement of fluids and attachment of cells for tissue regeneration therethrough, to prevent cell loss to thus control cell engraftment rate, and to meet the requirements for properties of the scaffold for living donor transplantation.
(26) The scaffold for living donor transplantation according to the present invention may be applied in a variety of fields. A conventional scaffold for living donor transplantation may be fabricated through various methods, among which an injection-molding process using a 3D printer based on FDM technology is performed in the present invention.
(27) In order to attain the properties required of the scaffold for living donor transplantation through the FDM process, the following two considerations should be kept in mind: application to the FDM process has to be possible, and the properties (e.g. biocompatibility, strength, etc.) of the scaffold fabricated by the FDM process have to be superior.
(28) In the present invention, the use of a mixture of sintered bioglass and a biocompatible polymer is capable of solving the problem of biocompatibility, and the stack structure of the struts makes it possible to achieve desired properties, particularly stiffness and strength.
(29) Stiffness refers to a property whereby deformation does not occur in response to external force, and toughness refers to a property of resisting strong impacts. When both are improved, the scaffold for living donor transplantation does not easily crack by virtue of the increased resistance to pressure or impact, which may compensate for the brittleness of the ceramic material of bioglass.
(30) Toughness measurement is performed through a 3-point bending test, and three parameters are obtained: toughness, maximum bending moment and maximum bending stress.
(31) The toughness may fall in the range of 50 kPa/mm.sup.3 to 850 kPa/mm.sup.3, 70 kPa/mm.sup.3 to 800 kPa/mm.sup.3, or 100 kPa/mm.sup.3 to 800 kPa/mm.sup.3, and preferably ranges from 600 kPa/mm.sup.3 to 800 kPa/mm.sup.3.
(32) Here, the maximum bending moment may fall in the range of 25 N.Math.mm to 70 N.Math.mm, 30 N.Math.mm to 65 N.Math.mm, or 35 N.Math.mm to 650 N.Math.mm, and preferably ranges from 45 N.Math.mm to 60 N.Math.mm.
(33) Moreover, the maximum bending stress may fall in the range of 3 MPa to 9 MPa, 3.5 MPa to 8.5 MPa, or 4 MPa to 8 MPa, and preferably ranges from 4.5 MPa to 7.5 MPa.
(34) Stiffness may be measured using a compression tester, and three parameters are obtained: stiffness, yield displacement and yield stress.
(35) The stiffness may fall in the range of 1.5 N/mm to 20 N/mm, 1.8 N/mm to 15 N/mm, or 2.0 N/mm to 10 N/mm, and preferably ranges from 2.0 N/mm to 5 N/mm.
(36) The yield displacement may fall in the range of 0.3 mm to 0.8 mm or 0.35 mm to 0.7 mm, and preferably ranges from 0.4 mm to 0.65 mm.
(37) The yield stress may fall in the range of 3 MPa to 20 MPa, 4 MPa to 15 MPa, or 4.5 MPa to 10 MPa, and preferably ranges from 5 MPa to 10 MPa.
(38) In addition to the aforementioned properties, the scaffold for living donor transplantation of the present invention also has surface properties and various activities such as bioactivity and the like mentioned below.
(39) For example, the roughness (R.sub.a) of the surface of the scaffold measured using a surface roughness meter (Nanoview-m4151p, Korea) may fall within a range of 130 nm to 260 nm, 135 nm to 250 nm, or 140 nm to 240 nm.
(40) Also, the water contact angle, associated with hydrophilicity, has the following numerical value. The water contact angle is measured using a sessile drop process at room temperature (25 C.) by placing 10 L of a water droplet on the scaffold surface, and lower values thereof indicate greater hydrophilicity.
(41) After 1 sec: 90 or less, 40 to 90, 70 or less, 43 to 70
(42) After 30 sec: 80 or less, 0 to 80, 60 or less, 20 to 60
(43) After 180 sec: 75 or less, 0 to 75, 30 or less, 10 to 30
(44) Also, the scaffold for living donor transplantation of the present invention has a range of 0.19 to 0.6 O.D. (optical density) in view of absorbance associated with protein absorption ability using a BCA (Bicinchoninic acid) protein assay (Pierce Kit, Thermo Scientific), and has the following range over time. After 1 hr (O.D.): 0.19 or more, 0.19 to 0.27, 0.22 to 0.25 After 6 hr (O.D.): 0.23 or more, 0.23 to 0.38, 0.26 to 0.35 After 12 hr (O.D.): 0.24 or more, 0.24 to 0.43, 0.30 to 0.39 After 24 hr (O.D.): 0.25 or more, 0.25 to 0.60, 0.40 to 0.59
(45) Cell-seeding efficiency, a parameter related to cell activity, may be represented by a numeric value of 37% or more, 40% or more, or 45% or more. The higher the above numeric value, the better the cell adhesion, growth, and differentiation, which means that cell activity is increased.
(46) Moreover, the scaffold for living donor transplantation of the present invention has cell proliferation absorbance in the following range after 7-day culture related to the cell proliferation rate measured using a cell proliferation assay (MTT Assay). Here, a higher numeric value means that cell proliferation is occurring efficiently.
(47) After 1 day (%): 0.17 or more, 0.19 or more, 0.19 to 0.3
(48) After 3 days (%): 0.19 or more, 0.21 or more, 0.21 to 0.3
(49) After 7 days (%): 0.22 or more, 0.25 or more, 0.25 to 0.4
(50) Furthermore, the F-Actin area ratio of the scaffold for living donor transplantation according to the present invention may fall in the range of 22% or more, 25% or more, 28% or more, 28% to 60%, or 30% to 55%.
(51) In addition, the scaffold for living donor transplantation according to the present invention is fabricated by a) mixing sintered bioglass and a biocompatible polymer and b) performing injection molding.
(52) These steps are described in detail below.
(53) a) Mixing
(54) The properties of the scaffold described above may be achieved through mixing of sintered bioglass and biocompatible polymer and control of the content ratio therebetween.
(55) Bioglass, which is one of bioceramics, is a bioactive ceramic that is embedded in living bodies and forms a strong chemical bond by directly contacting the surrounding bone without a fibrous film therearound.
(56) Bioglass may be selected from the group consisting of CaO, SiO.sub.2, P.sub.2O.sub.5, B.sub.2O.sub.3 and combinations thereof.
(57) The bioglass of the present invention may be selected from the group consisting of CaO, SiO.sub.2, P.sub.2O.sub.5, B.sub.2O.sub.3 and combinations thereof, and is non-toxic and has the effect of inducing osteoblast differentiation in human mesenchymal stem cells better than hydroxyapatite, which is the basic mineral of bone. It also has twice the compressive strength of hydroxyapatite and may thus be used as a biocompatible material in the intervertebral space.
(58) CaO is a material that is easily fused with other ceramic components to thus contribute to the fluidity, durability and water resistance of the entire composition, and the amount of CaO is preferably 20 to 60 wt %, and more preferably 40 to 50 wt %, based on the total weight of the bioglass, but the present invention is not limited thereto. If the amount of CaO is less than 20 wt % based on the total weight of the bioglass, durability and water resistance of 3D-printed products may decrease. On the other hand, if the amount of CaO exceeds 60 wt % based on the total weight of the bioglass, the brittleness of 3D-printed products may increase, or the fluidity of the entire composition is poor, thus non-uniformly extruding the composition upon 3D printing, which is undesirable.
(59) SiO.sub.2 is a material that has transparency, viscosity, durability, and a low fusion temperature and contributes to stabilization of the entire composition, and the amount of SiO.sub.2 is preferably 15 to 40 wt %, and more preferably 30 to 40 wt %, based on the total weight of the bioglass, but the present invention is not limited thereto. When the amount of SiO.sub.2 falls within the above range, bioactivity may be improved and glass crystallization may become good.
(60) P.sub.2O.sub.5 may inhibit the growth of bacteria such as Streptococcus mutans and may thus increase bioactivity. In particular, P.sub.2O.sub.5, which is contained in a large amount in natural teeth or bones, is able to form a glass matrix and to increase the permeability thereof. The amount of P.sub.2O.sub.5 is preferably 6 to 20 wt %, and more preferably 12 to 16 wt %, based on the total weight of the bioglass, but the present invention is not limited thereto. If the amount of P.sub.2O.sub.5 is less than 6 wt % based on the total weight of the bioglass, the effect of inhibiting bacterial propagation and the formation of the glass matrix become insignificant. On the other hand, if the amount of P.sub.2O.sub.5 exceeds 20 wt %, brittleness may increase, which is undesirable.
(61) B.sub.2O.sub.3 may improve glass crystallization to thus further increase mechanical strength and thermal expansion rate. The amount of B.sub.2O.sub.3 is preferably 1 wt % or less, and more preferably 0.5 wt % or less, based on the total weight of the bioglass, but the present invention is not limited thereto.
(62) In particular, the bioglass of the present invention uses a material crystallized through sintering, rather than using the material having the above composition as it is.
(63) Specifically, the bioglass is subjected to crystallization through sintering, thus making it possible to enhance the intrinsic strength and hardness of a 3D-printed product to be fabricated.
(64) The sintering temperature may be variously altered in consideration of the intrinsic glass transition temperature of bioglass. For example, during the sintering process, the heating step may be performed in a manner in which the temperature is gradually elevated at a heating rate of 0.01 to 0.8 C./min so as to reach a peak temperature of 800 to 1200 C., after which sintering may be carried out for 160 to 200 min at the peak temperature. A drastic change in temperature makes it difficult to maintain the shape of the sintered bioglass injected using the 3D printer, thus causing cracks and voids and significantly reducing strength.
(65) During the sintering process, the cooling step may be performed in a manner in which the temperature of the shaped product is gradually lowered from the peak temperature to 10 to 35 C. at a cooling rate of 0.01 to 0.8 C./min. If the temperature is lowered at a cooling rate exceeding 0.8 C./min, cracks or voids may form, thus significantly reducing strength.
(66) The peak temperature of the sintering process affects the strength of the final scaffold, and sintering is preferably carried out at 800 to 1200 C. in order to realize a substitute for hard biotissue. If the peak temperature is lower than 800 C., compressive strength may decrease, making the scaffold incapable of serving as a substitute for hard biotissue. On the other hand, if the peak temperature is higher than 1200 C., cracking may occur.
(67) The sintering process is performed after the injection-molding process in the fabrication of the scaffold for living donor transplantation, and thus, in the case in which non-sintered bioglass is used, rather than sintered bioglass, the resulting scaffold is configured such that the sintered bioglass alone is left behind without the biocompatible polymer. In this case, due to the high brittleness thereof, cracks are easily generated under external impact or pressure, making it unsuitable for use as a scaffold.
(68) Also, the biocompatible polymer, which is mixed with the sintered bioglass, functions as a binder. The binder is responsible for binding fine particles of sintered bioglass to each other to make them cohesive and viscous, and allows the bioglass to have excellent toughness to thus overcome the inherent brittleness problem. Moreover, when the composition composed of the sintered bioglass and the biocompatible polymer functioning as a binder, which are mixed together, is melted, the composition is imparted with fluidity and flowability to thus facilitate injection.
(69) A representative example of the biocompatible polymer may be selected from the group consisting of poly(-caprolactone) (PCL), polyethylene (PE), poly(methyl methacrylate) (PMMA), polylactic acid (PLA), poly-L-lactic acid (PLLA), polyglycolide (PGA), polylactic-co-glycolic acid (PLGA), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), polyurethane, polyacetal, polyamide, polyamide elastomer, polyester, polyester elastomer, polypropylene, polyacrylonitrile, polysulfone, polyorthoester, polyanhydride, chitosan, gelatin, collagen and combinations thereof.
(70) The biocompatible polymer according to the present invention is preferably a biodegradable polymer. The biodegradable polymer enables control of the mechanical strength thereof, is easy to process, and enables the biodegradation rate thereof to be adjusted depending on the synthesis conditions, so the material may be efficiently utilized. Preferably, the biodegradable polymer is any one selected from the group consisting of poly(-caprolactone), polylactic acid, poly-L-lactic acid, polyglycolide, polylactic-co-glycolic acid and combinations thereof, and more preferably poly(-caprolactone) is used.
(71) In particular, the amounts of the sintered bioglass and the biocompatible polymer are controlled in order to satisfy not only the toughness and stiffness of the scaffold but also the properties, such as surface roughness, hydrophilicity, protein absorption ability, and the like.
(72) Specifically, the amount of the sintered bioglass is 10 to 70 wt %, preferably 30 to 50 wt %, and more preferably 35 to 45 wt %, based on the total weight of the composition. If the amount of the sintered bioglass is less than the above lower limit, the intrinsic mechanical properties of bioglass, such as compressive strength, etc., may decrease. On the other hand, if the amount thereof exceeds the above upper limit, it is difficult to inject the resulting composition due to the high viscosity thereof.
(73) Also, the amount of the biocompatible polymer may be 30 to 90 wt %, 50 to 70 wt %, or 55 to 65 wt % based on the total weight of the composition. If the amount of the biocompatible polymer is less than the above lower limit, insufficient bonding force between sintered bioglass particles may result, or it may be difficult to solve the problem of brittleness of the sintered bioglass. On the other hand, if the amount thereof exceeds the above upper limit, the relative amount of the sintered bioglass is low, and thus there is a concern that the properties of the scaffold that is ultimately fabricated may decrease.
(74) In addition, the content ratio of the sintered bioglass and the biocompatible polymer is related to the viscosity (*, complex viscosity) in the fabrication of a scaffold using an FDM 3D printer. Specifically, the sintered bioglass has a non-viscoelastic characteristic, and the viscosity of a melt upon injection molding increases with an increase in the amount thereof. The high-viscosity melt may affect the shape and process quality of the struts after injection molding, making it difficult to ensure the diameter or thickness uniformity of the struts, and the nozzle may become clogged. The appropriate viscosity range may be obtained through the above content ratio of the sintered bioglass and the biocompatible polymer, and the viscosity preferably falls in the range of 110 to 800 Pa.Math.s, 120 to 700 Pa.Math.s, or 150 to 500 Pa.Math.s.
(75) The sintered bioglass may be pulverized before or after sintering. In particular, the average particle size of the sintered bioglass after pulverization is adjusted to the range of 1.5 to 2.5 m. Here, the average particle size is calculated from the value measured using a particle size analyzer (APA2000, MALVERN) (a cumulative 50% point in the average diameter distribution of the particles).
(76) The pulverization process may be performed using any known pulverizer, for example, a freezer mill.
(77) In order to uniformly mix the sintered bioglass and the biocompatible polymer, the biocompatible polymer may be used after first being pulverized to a particle size range similar to that of the sintered bioglass, or the pulverization process may be performed during the mixing process.
(78) b) Injection Molding
(79) Next, the pulverized composition is injection-molded to fabricate a scaffold for living donor transplantation.
(80) Injection molding may be performed through an FDM 3D-printing process.
(81) An FDM (fused deposition modeling) 3D printer is a kind of 3D printer that fabricates a three-dimensional shaped product by injecting and stacking a material having fluidity and flowability through a melting process, and includes currently commercially available FDM 3D printers and FFF (fused filament fabrication) 3D printers.
(82) The composition for an FDM 3D printer according to the present invention is in the form of a paste having fluidity, flowability and viscosity. Specifically, the composition for an FDM 3D printer according to the present invention may be applied to all 3D printers, regardless of the type thereof, so long as the 3D printer is any 3D printing device capable of injection, in addition to being applicable to commercially available FDM and FFF 3D printers.
(83) The heater provided to the 3D printer operates in a temperature range of 25 to 250 C. and functions to melt the pulverized composition. When the sintered bioglass and the biocompatible polymer, which are solid, are melted and formed into a paste, the composition may be imparted with fluidity and flowability to thus facilitate injection.
(84) In order to increase the identity or similarity of the geometric size of the struts through injection molding, optimization of processing parameters such as nozzle speed, pneumatic pressure and heating temperature is required.
(85) As the nozzle speed (mm/s) increases, the strut size (m) of each scaffold may decrease. For example, as shown in
(86) Also, as the pneumatic pressure (kPa) increases, the strut size (m) of each scaffold may increase. For example, as shown in
(87) As the temperature ( C.) increases, the strut size (m) of each scaffold may increase. For example, as shown in
(88) In a conventional fabrication of a scaffold for living donor transplantation, the sintering process is performed after injection molding, but may be excluded in the present invention because of the use of sintered bioglass and biocompatible polymer. Accordingly, the fabrication process may be simplified and the cost thereof may be reduced.
(89) The scaffold for living donor transplantation according to the present invention may be used for artificial bones, artificial joints, oral maxillofacial bones, skulls or dental artificial roots, and may be utilized as a disc-shaped artificial bone for use in spinal fusion or an artificial bone for use in facial reconstruction.
EXAMPLES
(90) A better understanding of the present invention will be given through the following examples, which are merely set forth to illustrate the present invention but are not to be construed as limiting the scope of the present invention.
Preparation Example 1
Preparation of Sintered Bioglass
(91) 139.8 g of CaO, 113.7 g of SiO.sub.2, 45.6 g of P.sub.2O.sub.5, and 0.9 g of B.sub.2O.sub.3, which were dry powder, were placed in a vessel and mixed to afford 300 g of bioglass.
(92) The bioglass thus obtained was sintered under the following conditions, thereby obtaining sintered bioglass.
(93) (Sintering Conditions)
(94) 0.fwdarw.600 C.: 720 min
(95) 600 C. (Holding): 60 min
(96) 600.fwdarw.1000 C.: 800 min
(97) 1000 C. (Holding): 180 min
(98) 1000.fwdarw.600 C. and 600 C. (Holding): 800 min
(99) 600.fwdarw.20 C.: 720 min
Examples and Comparative Examples
Fabrication of Scaffold for Living Donor Transplantation
(100) (1) Mixing
(101) The bioglass obtained in Preparation Example 1 was placed in a ball mill (High-Energy Ball Mill, FRITSCH) and pulverized so as to have an average particle size of 2.1 m. The bioglass thus pulverized was mixed with poly(-caprolactone) (PURASORB PC12, Corbion Purac, hereinafter referred to as PCL) at the content ratio shown in Table 1 below, thus preparing 100 g of a composition.
(102) The composition thus prepared was placed in a freezer mill (6875D, SPEX Sampleprep) and pulverized so as to have an average particle size of 2.1 m.
(103) (2) Injection Molding
(104) The composition thus pulverized was melted using a heater provided to an FDM 3D printer (DTR3-3315-EX, Dasa Robot System). Subsequently, the composition in a paste phase was loaded in a nozzle in the printer (diameter: 500 m), after which the composition was injected to a size of 662 mm.sup.3 via the injection port and stacked layer by layer on the working table.
(105) Here, a scaffold composed of struts stacked in four directions, as shown in
(106) TABLE-US-00001 TABLE 1 Sintered Melting Processing Nozzle Pneumatic bioglass PCL temp. time speed pressure (wt %) (wt %) ( C.) (min) (mm/s) (kPa) Example 1 20 80 120 2 5 320 Example 2 40 60 120 2 5 450 Example 3 60 40 140 2 5 500 Comparative 100 0 25 2 5 430 Example 1 Comparative 0 100 100 2 5 480 Example 2
Test Example 1
Determination of Processing Parameter
(107) Optimal processing parameters were determined by testing the speed, pneumatic pressure, temperature and viscosity during the injection-molding process using the compositions of Examples 1 to 3.
(108)
(109) As shown in
(110) The viscosity of
(111) Based on the results of
Test Example 2
Analysis of Strut Structure
(112) (1) Pore Characteristics
(113) The scaffolds fabricated in Examples 1 to 3 and Comparative Examples 1 and 2 were measured for strut diameter, pore size and porosity. The results thereof are shown in Table 2 below. The pore size was measured using a scanning electron microscope (SEM, SNE-3000M, SEC Inc., Korea), and the porosity was calculated using the following equation and bulk density () (PCL (1.135 g/cm.sup.3)/bioglass (3.05 g/cm.sup.3)).
Porosity (%)=(1(1/.sub.s)(W.sub.s/V.sub.a))100[Equation 1]
(114) (: bulk density, W.sub.s: weight of the structure, V.sub.a: volume of the structure)
(115) TABLE-US-00002 TABLE 2 Strut diameter 1.sup.st pore size 2.sup.nd pore size Porosity (m) (m) (m) (%) Example 1 424.3 7.7 400.7 10.2 214.3 14.1 42.5 2.1 Example 2 401.4 16.3 415.7 14.4 222.9 16.7 43.1 1.0 Example 3 420.7 9.2 412.1 7.0 216.4 20.5 42.3 1.2 Comparative 392.5 20.1 391.5 9.1 207.7 20.7 46.8 3.0 Example 1 Comparative 400.7 4.7 439.3 14.3 221.4 13.6 43.1 1.5 Example 2
(116) As is apparent from Table 2, the struts had a diameter of 390 to 425 m, a bimodal pore size distribution, and a porosity of 42 to 47%.
(117) (2) Microscope Analysis
(118)
(119) The first line of
(120) The second line to the fourth line of
(121) The fifth line and the sixth line of
Test Example 3
Analysis of Composition
(122) The compositions (after pulverization) used to fabricate the scaffolds of Examples 1 to 3 and Comparative Examples 1 and 2 were subjected to thermogravimetric analysis and X-ray diffraction. The results thereof are shown in
(123) (1) Thermogravimetric Analysis (TGA)
(124) 10 mg of each composition was heated from 30 C. to 800 C. at a heating rate of 20 C./min, and was then subjected to TGA using a thermogravimetric analyzer (TGA-2050, TA-Instruments) in a nitrogen atmosphere.
(125)
(126) (2) X-Ray Diffraction (XRD)
(127) In order to measure the crystal size, an X-ray diffractometer (Siemens D500 WAXD, Siemens) using CuK.sub. radioactive rays under beam conditions of 40 kV and 20 mA was used. The X-ray diffraction test was performed under conditions of 2=15-35 and a step size of 0.1.
(128)
(129) In contrast, in the compositions of Examples 1 to 3, the above peaks were included, indicating that the sintered bioglass and PCL were appropriately mixed in the compositions.
Test Example 4
Analysis of Properties of Scaffold
(130) In order to evaluate the toughness-related properties of the scaffolds fabricated in Examples 1 to 3 and Comparative Examples 1 and 2, the following testing for toughness and stiffness was performed. The results thereof are shown in Table 3 below and in
(131) TABLE-US-00003 TABLE 3 Example Example Example Comparative Comparative 1 2 3 Example 1 Example 2 3-Point Max. Bending 55.8 47.6 38.1 74.7 37.3 bending moment (N/mm) 0.5 0.9 0.5 6.8 1.8 test Max. Bending 6.3 5.6 4.3 7.8 4.6 stress (MPa) 0.1 0.2 0.2 0.7 0.2 Toughness 760 710 100 5.9 480 (kPa/mm.sup.3) 41.7 5.48 22.7 0.9 46.2 Compression Stiffness 2.5 2.9 2.9 25.9 1.1 test (N/mm) 0.1 0.1 0.2 6.3 0.1 Yield 0.6 0.5 0.5 0.2 0.4 displacement 0.03 0.04 0.06 0.01 0.02 (mm) Yield stress 6.1 6.6 5.8 21.2 2.6 (MPa) 0.2 0.1 0.6 5.8 0.2
(132) As is apparent from Table 3 and
(133) In order to serve for a scaffold for living donor transplantation, the stiffness and toughness values have to be appropriately balanced, rather than being unbalanced, as in Comparative Examples 1 and 2. Therefore, it can be concluded that the scaffolds of Examples 1 to 3 simultaneously satisfy toughness of about 50 to 850 (kPa/mm.sup.3) and stiffness of 2 to 3 N/m.
Test Example 5
Analysis of Surface Properties of Scaffold
(134) (1) Measurement of Surface Roughness/3D Surface Topographical Images
(135) The 3D surface topographical images were obtained using a phase shift interferometer, and thus the surface roughness of the scaffolds was analyzed. As shown in
(136) For qualitative analysis of such roughness, the surface roughness (R.sub.a) was obtained based on the following equation using a surface roughness meter (Nanoview-m4151p, Korea).
R.sub.a=[|Z(x)|dx]/L(Z and L of FIGS.6(a) to 6(e) are the height and length of the rough structure)[Equation 2]
(137) TABLE-US-00004 TABLE 4 Surface roughness (R.sub.a) (unit: nm) Example 1 144.7 31.9 Example 2 168.1 26.3 Example 3 234.3 41.7 Comparative 284.6 55.4 Example 1 Comparative 118.5 14.9 Example 2
(138) As is apparent from Table 4, compared to Comparative Example 2 using the biocompatible polymer alone, when the amount of the bioglass that was contained was higher, the surface roughness was increased, indicating that the surface roughness can be adjusted depending on the content ratio of sintered bioglass/PCL.
(139) (2) Measurement of Water Contact Angle
(140) The hydrophilicity of the scaffolds fabricated in Examples 1 to 3 and Comparative Examples 1 and 2 was evaluated using a water contact angle meter. The results thereof are shown in Table 5 below.
(141) Hydrophilicity evaluation was carried out by placing 10 L of a water droplet on each scaffold surface and measuring a water contact angle using a sessile drop process at room temperature (25 C.).
(142) TABLE-US-00005 TABLE 5 Water contact angle 1 sec 30 sec 180 sec Example 1 67 2 58 5 26 3 Example 2 46 3 34 4 20 3 Example 3 45 2 26 3 14 3 Comparative 39 3 0 0 Example 1 Comparative 93 4 82 4 78 3 Example 2
(143) As is apparent from Table 5, compared to Comparative Example 2 (783) using PCL alone, the contact angle was decreased with an increase in the amount of the sintered bioglass. The scaffold of Example 1, containing 20 wt % of the sintered bioglass, exhibited a water contact angle of 263, indicating that the water contact angle, that is, the hydrophilicity of the scaffold, can be adjusted depending on the content ratio of sintered bioglass/PCL.
Test Example 6
Tissue Regeneration Applicability of Scaffold
(144) In order to evaluate the bio-application of the scaffolds fabricated in Examples 1 to 3 and Comparative Examples 1 and 2, the analysis was carried out as follows, and the results thereof are shown below.
(145) (1) Measurement of Protein Absorption Ability
(146) In order to measure protein absorption ability, a BCA (Bicinchoninic acid) protein assay (Pierce Kit, Thermo Scientific) was used. Each scaffold was placed in a 24-well plate including -minimum essential medium (Life Sciences, USA) containing 10% fetal bovine serum (Gemini Bio-Products, USA) and 1% antibiotic (Antimycotic, Cellgro, USA). Next, the scaffold was cultured at 37 C. for 1, 6, 12, and 24 hr. Before measurement of absorbance, the scaffold was cleaned with PBS (phosphate buffer saline) and lysed in 0.1% triton X-100. Next, 25 L of the lysate was added to 200 L of a BCA reagent. Finally, the mixture was cultured at 37 C. for 30 min. The absorbance was measured at 562 nm using a microplate reader (EL800, Bio-Tek Instruments, Winooski, Vt.).
(147) The results of measurement of the protein absorption ability of Examples 1 to 3 and Comparative Examples 1 and 2 after 1, 6, 12 and 24 hr are shown in Table 6 below.
(148) TABLE-US-00006 TABLE 6 Absorbance (O.D.) 1 hr 6 hr 12 hr 24 hr Example 1 0.2215 0.274 0.31167 0.41133 0.03339 0.00726 0.05856 0.04027 Example 2 0.22733 0.2945 0.36433 0.48367 0.0344 0.000866 0.01872 0.05622 Example 3 0.24717 0.3425 0.389 0.57733 0.02434 0.03339 0.03205 0.03921 Comparative 0.28033 0.38967 0.44533 0.62867 Example 1 0.01892 0.04119 0.8969 0.04027 Comparative 0.179 0.22327 0.23383 0.24183 Example 2 0.02498 0.02839 0.04221 0.02811
(149) As is apparent from Table 6, compared to Comparative Example 2 using PCL alone, the scaffolds of Comparative Example 1 and Examples 1 to 3 including sintered bioglass exhibited increasing protein absorption ability over time. In particular, the protein absorption ability increased with an increase in the amount of the sintered bioglass. This is deemed to be due to the irreversible electrostatic interaction between the amine group of the protein and the negatively charged sintered bioglass component in the scaffold.
(150) (2) Cell Activity in Scaffold in Vitro
(151) Cell-Seeding Efficiency
(152) The scaffold (662 mm.sup.3) of each of Examples 1 to 3 and Comparative Examples 1 and 2 was sterilized with 70% ethanol under UV light. The mouse preosteoblast tissue (MC3T3-E1) was seeded at a density of 110.sup.5 cells/mL into the scaffold, after which the scaffold was placed in a 24-well plate including -minimum essential medium (Life Sciences, USA) containing 10% fetal bovine serum (Gemini Bio-Products, USA) and 1% antibiotic (Antimycotic, Cellgro, USA). The specimen was cultured under conditions of 5% CO.sub.2 and 37 C., and the medium was replaced every day.
(153) TABLE-US-00007 TABLE 7 Cell-seeding efficiency (%) Comparative 36.97689 3.32317 Example 2 Example 1 45.75389 2.59645 Example 2 57.15178 6.38773 Example 3 62.14866 5.10196
(154) As is apparent from Table 7, the cell-seeding efficiency of the scaffold was increased with an increase in the amount of the sintered bioglass. This is deemed to be due to the protein absorption ability and biochemical properties by increasing the surface roughness and hydrophilicity of the scaffold by virtue of the sintered bioglass, indicating that, depending on the content ratio of the sintered bioglass/PCL, protein absorption ability can be improved and various cellular activities, such as initial cell adhesion, growth, and differentiation, are increased, thereby enabling control of cell adhesion ability.
(155) Cell Proliferation Efficiency
(156) A viable cell count was determined through a cell proliferation assay (MTT Assay) (Cell Proliferation Kit I, Boehringer Mannheim). The sample was added with 0.5 mg/mL of MTT and cultured at 37 C. for 4 hr. Thereafter, the absorbance was measured at 570 nm using a microplate reader (EL800, Bio-Tek Instruments, Winooski, Vt.).
(157) TABLE-US-00008 TABLE 8 Absorbance (O.D.) After 1 day After 3 days After 7 days Comparative 0.16675 0.00768 0.1845 0.0058 0.21375 0.01053 Example 2 Example 1 0.19175 0.0075 0.21725 0.00806 0.25025 0.01415 Example 2 0.243 0.02574 0.26775 0.01276 0.334 0.02099 Example 3 0.265 0.03238 0.2885 0.02927 0.368 0.03735
(158) As is apparent from the results of MTT analysis shown in Table 8, the cell proliferation also increased with an increase in the amount of the sintered bioglass, similar to the cell-seeding efficiency of Table 6. Thereby, it can be concluded that the cell proliferation behavior can be adjusted depending on the content ratio of sintered bioglass/PCL.
(159) F-Actin
(160) In order to detect cell nuclei, the scaffold was exposed to fluorescent staining of Diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, Calif.). Moreover, in order to visualize an actin cytoskeleton, staining of Phalloidin (Alexa Fluor 594; Invitrogen, Carlsbad, Calif.) was carried out, and the stained cells were observed using a confocal microscope (LSM 700; Carl Zeiss, Germany).
(161)
(162) TABLE-US-00009 TABLE 9 F-Actin area ratio (%) Comparative 21.52767 2.47724 Example 2 Example 1 30.97167 6.79412 Example 2 43.029 3.09069 Example 3 53.644 5.98447
(163) As is apparent from
(164) (3) Measurement of Gene Expression
(165) In order to measure the relative expression of Type-I collagen (Col-I), Runt-related transcription factor (Runx2), Alkaline phosphatase (ALP), Osteopontin (OPN), Osteocalcin (OCN), and Bone Morphogenic Protein 2 (BMP2), real-time polymerase chain reaction of the MC3T3-E1 cells cultured for 7 days in each scaffold was carried out.
(166) Total RNA was isolated from the cultured scaffold using a TRIzol reagent (Sigma-Aldrich), and cDNA was then synthesized therefrom. For reverse transcription, ReverTra Ace qPCR RT Master Mix (Toyobo, Japan) was used. Moreover, cDNA was amplified using THUNDERBIRD SYBR qPCR Mix (Toyobo, Japan) and ABI StepOnePlus. For amplification of cDNA, denaturation at 95 C. for 1 min was performed, after which the cycle of 15 sec at 95 C., 60 sec at 60 C., and 15 sec at 72 C. was repeated 40 times, and finally, cDNA was extended at 72 C. for 5 min.
(167) Gene-specific primers were as follows:
(168) TABLE-US-00010 runx2(forward:5-ACATCCCCATCCATCCAT-3, reverse:5-GGTGCTGGGTTCTGAATCTG-3), OPN(forward:5-GGAGGAAACCAGCCAAGG-3, reverse:5-TGCCAGAATCAGTCACTTTCAC-3), OCN(forward:5-CCCTCCTGAAGGTCTCACAA-3, reverse:5-GCTGTCTCCCTCATGTGTTG-3), Col-I(forward:5-ACTCAGCCGTCTGTGCCTCA-3, reverse:5-GGAGGCCTCGGTGGACATTA-3), ALP(forward:5-GCCCAGTGCCTTCTGATTT-3, reverse:5-GGGCAGCGTCAGATGTTAAT-3), BMP2(forward:5-AGATCTGTACCGCAGGCACT-3, reverse:5-GTTCCTCCACGGCTTCTTC-3), andthehousekeepinggenemouse GAPDH(forward:5-CCTTGAGATCAACACGTACCAG-3, reverse:5-CGCCTGTACACTCCACCAC-3).
(169) TABLE-US-00011 TABLE 10 No. Relative expression ALP/GAPDH Comparative Example 2 1 0.22087 Example 1 1.33828 0.38901 Example 2 2.77655 0.09479 Example 3 3.58484 0.40055 BMP-2/GAPDH Comparative Example 2 1 0.35537 Example 1 2.9804 0.58307 Example 2 4.81805 0.66508 Example 3 6.10637 0.86176 Col/GAPDH Comparative Example 2 1 0.06532 Example 1 13.14175 0.60236 Example 2 18.40076 0.88642 Example 3 21.20035 1.86857 OPN/GAPDH Comparative Example 2 1 0.04191 Example 1 3.93551 0.31252 Example 2 10.18773 0.5399 Example 3 13.43262 3.01044 RUNX-2/GAPDH Comparative Example 2 1 0.13509 Example 1 18.59516 0.55741 Example 2 40.86318 5.59465 Example 3 48.70381 10.47568 OCN/GAPDH Comparative Example 2 1 0.02964 Example 1 1.27795 0.06102 Example 2 2.40561 0.24953 Example 3 3.0789 0.22572
(170) As is apparent from Table 10, in the scaffolds of Examples 1 to 3 and Comparative Examples 1 and 2, the extent of gene expression was also increased with an increase in the amount of the sintered bioglass. Thereby, it can be concluded that the bioglass affects bone formation, differentiation and biological activity and that it is necessary to control the content ratio of sintered bioglass/PCL in order to ensure high gene expression levels.
Test Example 7
Optimal Content Ratio of Sintered Bioglass/PCL
(171) The toughness, cell-seeding efficiency, cell proliferation rate, and Runt-related transcription factor of the scaffolds for living donor transplantation fabricated in Examples and Comparative Examples were evaluated and plotted, and respective optical images thereof are represented.
(172)