Shape-complementary and porosity-matched perfusion bioreactor system for engineering bone grafts with complex geometries

Abstract

The present application discloses a perfusion bioreactor system having an inner cavity and a scaffold with matching porosity to balance the flow of fluid through the bioreactor. The scaffold can be manufactured using additive manufacturing techniques or other manufacturing techniques to match the geometry of the defect (e.g., facial bone abnormalities). The inner cavity is manufactured in a similar manner and has a cavity that matches the shape of the scaffold to form a unified structure when assembled with the scaffold. By matching the shape of the scaffold and the inner cavity, free space within the internal volume of the bioreactor is eliminated. Stem cells can flow through the bioreactor and attach to the scaffold and then be cultured to grow a tissue graft.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 079,497, filed on September 17, 2020, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present application relates generally to bioreactors. More particularly, the present application relates to a perfusion bioreactor system for producing geometrically complex bone grafts. Background Art

[0004] Autologous bone grafts are considered the current gold standard for orthopedic and cosmetic surgical interventions. While the inherent osteogenic, osteoconductive, and osteoinductive properties of autologous bone grafts provide certain advantages, they are also limited by issues such as donor site morbidity, tissue availability, and the inability to shape the donor bone to complex, defect-specific geometries. The success of bone reshaping, particularly in the craniofacial region, clearly depends on the geometric compatibility between the graft and the defect being treated. Therefore, the most realistic bone graft would possess a mixture of multiple cells and biomaterials to promote regeneration and possess the detailed geometry of the defect.

[0005] Tissue scaffolds have been used to create tissue grafts where cells attach to the scaffold and stimulate the formation of new tissue. In many cases, porous biomaterial scaffolds can be generated and implanted to stimulate cell growth in vivo. While such scaffolds can repair defects and restore function, they rely on cells that already exist around the defect to infiltrate the porous scaffold and promote bone regeneration at the defect site. However, when the scaffold is large and has critical geometry, relying solely on cell infiltration to promote bone regeneration becomes less than ideal. For defects that are larger or have complex geometries, it is beneficial to cellularize the scaffold in vitro prior to implantation, which will give the graft a better chance of regenerating bone in vivo.

[0006] Scaffolds can be made in a variety of shapes to match the shape of the defect. Additive manufacturing has emerged as a promising strategy for the fabrication of such “designer scaffolds” with complex geometries. However, the ultimate challenge is to meet the oxygen and nutrient requirements of culturing such physiologically relevant bone grafts in industrial or clinical laboratories prior to implantation. Conventional perfusion bioreactors have been considered as a countermeasure to enhance fluid flow and mass transfer in such scaffolds. Although bioreactors have conventional performance in culturing cells, when used with scaffolds of different sizes and geometries, these bioreactors are severely limited by the inability to achieve uniform perfusion across the scaffold, which is critical for the formation of robust bone tissue with uniform mechanical properties and uniform morphology across the entire scaffold. As the shape and geometry of the scaffold become more complex, the non-uniform perfusion becomes more prominent. Therefore, there is a need to create a system that facilitates the production of large and geometrically complex bone substitutes. Summary of the invention

[0007] According to an embodiment of the present application, the present application provides a perfusion bioreactor system containing a porous lumen that can be implemented as a multi-part component and customized to fill the geometry and porosity of the desired scaffold. Coordinating the geometry and porosity in this manner will produce an internal compartment within the bioreactor without preferential fluid flow, achieving uniform perfusion across the scaffold regardless of size and geometry. The resulting bioreactor has the potential to promote uniform delivery of cells, culture media, and biomaterials, thereby enabling uniform tissue growth in personalized bone grafts.

[0008] Digital images of the defect can be used to design and manufacture the desired personalized scaffold and corresponding lumen. Additive manufacturing and other manufacturing techniques can be used to create scaffolds and lumens through digital design. When used in a bioreactor, the scaffold can be coated with an extracellular matrix (ECM) biomaterial such as collagen to enhance cell attachment. In addition, the lumen can be coated with an antifouling agent to prevent unwanted cells from attaching, thereby limiting cell attachment to the scaffold. The coated components of the bioreactor can be assembled in a biosafety cabinet to maintain sterility. Stem cells can be dynamically inoculated in a bioreactor and perfused with culture medium to support cell proliferation and differentiation into the desired lineage. In one embodiment, mesenchymal stem cells (MSCs) can be differentiated into osteoblasts for bone tissue engineering using the bioreactor system disclosed in the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The components of a perfusion bioreactor are depicted.

[0010] Figure 2Shown is the tetrahedral mesh of the bioreactor used for fluid simulation.

[0011] Figure 3 A 3D-printed stent showing the inner and outer chambers.

[0012] FIG. 4A to FIG. 4D Various configurations of systems incorporating perfusion bioreactors are shown.

[0013] Figure 5 Fluid flow through a bioreactor using a lumen is described as compared to fluid flow when the lumen is omitted.

[0014] Figure 6 Confocal images of bone marrow mesenchymal stem cells seeded on the scaffold.

[0015] Figure 7 Alizarin red staining is shown to confirm that bone marrow mesenchymal stem cells differentiated on the scaffold. DETAILED DESCRIPTION

[0016] like Figure 1 As shown, according to an embodiment of the present application, a perfusion bioreactor system 100 is provided. The system 100 comprises: a bioreactor 101 having an inlet 102 and an outlet 103, a support 104 and an inner cavity 105. The inlet 102 and the outlet 103 are connected together to form an inner volume of the bioreactor 101. In operation, a fluid is pumped into the inlet 102, and then the fluid flows through the inner volume and flows out through the outlet 103. The inner cavity 105 may be composed of multiple parts and occupies the remaining part of the inner volume of the bioreactor 101 that is not occupied by the support 104. Figure 1 As shown, the lumen 105 includes two halves that, when connected, can surround the scaffold 104 and form a structure having a volume that matches the internal volume of the bioreactor 101. In other words, the scaffold 104 and lumen 105 occupy the internal volume of the bioreactor 100 and eliminate any free space in the internal volume of the bioreactor 100. When free space exists in the bioreactor 100, the fluid will preferentially flow to areas with less resistance, thereby preventing the scaffold 104 from receiving sufficient materials contained in the fluid required for tissue development.

[0017] Fabrication of bioreactor components

[0018] Computer-aided design (CAD) and / or solid modeling software, such as SOLIDWORKS design software, can be used to design the components of the bioreactor 100. In one embodiment, the inlet 102 and the outlet 103 can include commercially available components. In contrast, the stent 104 and the porous lumen 105 are custom designed, wherein the geometry of the stent 104 depends on the defect to be repaired. Imaging tools such as computed tomography (CT) and magnetic resonance imaging (MRI) can be used to obtain a three-dimensional (3D) rendering of the defect to be repaired with a graft. A custom CAD model of the stent 104 and the corresponding lumen 105 can be generated based on the rendering of the defect using Boolean tools.

[0019] Other 3D design tools may also be used during the manufacture of the stent 104. For example, a physical prototype of the stent 104 may be created and then scanned to digitize its shape and geometry. The resulting digital schematic of the prototype may then be refined through software editing tools or the prototype may be manufactured without modification.

[0020] In the design stage, the porosity of the support 104 and the inner cavity 105 depends on the intended use. In one embodiment, a porosity of 60% is achieved by using 600 μm cubic holes and 400 μm pillars in the structure of the support 104 and the inner cavity 105. In addition, algorithmic modeling methods such as those established in Grasshopper (Rhino 6) and MATLAB software can be applied to achieve larger biomimetic porosity patterns. Difference modifiers such as Boolean tools on design software can be used to create cavities in the inner cavity 105 to complement the shape of the support 104. Specifically, the inner cavity 105 is designed to have an inner cavity that matches the shape of the support 104 so that there is no free space when the two parts are combined.

[0021] Depending on the shape and complexity of the stent 104, the lumen 105 may be formed into multiple parts. Figure 1 In the illustrated embodiment, the stent 104 has a simple geometry for demonstration purposes, and the lumen 105 can easily match this shape with two parts. However, if the stent 104 has complex features such as depressions, internal bends, or non-linear features, the lumen 105 can be made of multiple parts to allow assembly without a gap between the stent 104 and the lumen 105.

[0022] After the design of the scaffold 104 and the lumen 105 is completed, a computational fluid dynamics (CFD) model can be generated to evaluate the uniformity of the fluid perfused through the scaffold 104. For example, a solid model of the lumen of the bioreactor 101 represents the area of ​​fluid flow, and a model of the scaffold 104 can be generated. These models can be used to generate velocity fields for the fluid flowing through the bioreactor 101 on computational fluid dynamics analysis software such as ANSYS Fluent, Elmer, and COMSOL Multiphysics. In an embodiment, the bioreactor system 100 is divided into four different parts: the inlet 102, the outlet 103, the lumen 105, and the scaffold 104.

[0023] Descriptions of these models are shown in Figure 2 In which the inlet 102, the outlet 103 and the inner cavity 105 are shown Figure 2 In the figure on the left, the stent 104 without the lumen 105 is shown in Figure 2 in the figure on the right. For modeling purposes, the lumen 105 and the support 104 are assumed to be porous regions. For example, the porosity of the support 104 is maintained at 94%, and the porosity of the lumen 105 is maintained at 100% or 94% (the same as the support 104). When the porosity of the lumen 105 matches the porosity of the support 104, uniform fluid flow is achieved. As described above, because the lumen 105 and the support 104 have similar resistance to the flow of fluid, matching the porosity of the support 104 and the lumen 105 reduces the fluid flow around the support 104. A further assumption of the modeling is the laminar flow of an incompressible Newtonian fluid through the porous medium. Assuming an inlet velocity of 1 mL / min, a no-slip boundary condition is applied to the wall of the bioreactor 101.

[0024] The design files of the support 104 and the inner cavity 105 can be edited on the 3D printing software, and high-fidelity porous printing can be performed to combine into a cylindrical support. The porous component can be printed on a printer that can print out suitable materials for the support 104. In an embodiment, the printer is a M100-405nm digital light projection (DLP) printer of CADworks3D. Biocompatible materials such as photosensitive resins such as Dental SG, biocompatible and autoclavable can be used to print out the support 104 and the inner cavity 105. Since there is no need to grow tissue, as an alternative, the inner cavity 105 can also be printed by cheaper non-biocompatible materials. For post-printing processing, the printed parts can be cleaned in isopropyl alcohol (IPA) for 1 hour and dried with compressed air. At this stage, the support can be trimmed in all aspects before assembly, and the support is UV-cured and autoclaved.

[0025] While additive manufacturing provides an efficient platform for fabricating a scaffold 104 and lumen 105 having a desired porosity, other fabrication techniques may also be used. For example, fabrication may be accomplished by any of the following techniques: (1) solvent casting and particle leaching techniques, (2) gas foaming techniques, (3) vacuum drying techniques, and (4) thermally induced phase separation techniques. Although interconnectivity cannot be well controlled, these techniques provide some control over the pore size and overall porosity of the assembly 104 / 105. Despite this limitation, these alternative performance techniques are also suitable because they allow the fabrication of scaffolds 104 and lumens 105 having complementary shapes and porosities while ensuring that the pores are interconnected to facilitate perfusion.

[0026] Bioreactor Systems and Perfusion

[0027] The bioreactor system 100 and the scaffold 104 can be used to culture a variety of tissues. The following example discusses the manufacture of a bone replacement graft. In this process, human bone marrow mesenchymal stem cells (hMSCs) are passaged at 70% confluence and cultured in a mesenchymal stem cell culture medium at a density of approximately 1000 cells / mm 2 The cells were cultured at a density of 100 μg / cm2 on top of a Dental SG dish for differentiation and propagation. After 1 day of culture, the cells were maintained in a mesenchymal stem cell culture medium, or the cells were transferred to a commercial mesenchymal stem cell osteodifferentiation medium (DM). The cells were differentiated for 14 days.

[0028] Respectively, aseptic support 104 and inner chamber 105 are immersed in 50 μ g / mL collagen solution and anti-adhesion flushing solution. Under aseptic conditions, these parts are immersed in the corresponding solution in the vacuum chamber, and these parts are degassed for 3 hours. Subsequently, in a biological safety cabinet, two parts are dried to stabilize the coating. Next, both parts are immersed in Dulbecco's phosphate buffered saline (DPBS) and degassed for 3 hours.

[0029] Remove the scaffold 104 and lumen 105 from the DPBS solution and immediately assemble into the bioreactor 101 to prevent drying. Connect the inlet 102 and outlet 103 of the bioreactor 100. Once assembled, perfuse the bioreactor 101 with DPBS. Assembly can be done in a sterile manner in a biosafety cabinet.

[0030] In this embodiment, a syringe pump is used to perfuse 7.5 million bone marrow mesenchymal stem cells ( Figure 4A Then, the bioreactor 101 was turned vertically and inoculated with 7.5 million bone marrow mesenchymal stem cells ( Figure 4BUnattached cells were collected in the medium reservoir 110 and perfused in the loop using a peristaltic pump for one hour ( Figure 4C ). The medium reservoir 110 was stirred to avoid cell pooling. In addition, the cells were allowed to stand for one hour without any perfusion. After this, the unattached cells were washed out of the medium reservoir 110 with DPBS. Then, perfusion was started with fresh medium and a peristaltic pump at a rate of about 1 mL / min ( Figure 4D ). After one day of perfusion culture, scaffold 104 was obtained.

[0031] result

[0032] The effect of including an inner lumen 105 that matches the porosity and geometry of the scaffold 104 can first be evaluated by performing porous media simulations on ANSYS Fluent. Figure 5 Two separate simulations are shown, one without the lumen (top) and one with the lumen (bottom). Figure 5 The scale on the right shows different flow rates through the bioreactor 101. Without the lumen 105 (i.e., when the porosity in the lumen 105 is 100%), the fluid is most likely to flow through the void space around the scaffold 104. That is, the maximum flow rate occurs in the area around the scaffold 104. This makes the velocity field on the bioreactor 101 non-uniform, and almost no fluid flows through the scaffold 104, which in this embodiment provides a large resistance to the fluid. Conversely, the presence of the lumen 105 having the same porosity as the scaffold 104 hinders the preferential flow of the fluid, thereby obtaining a uniform velocity distribution through the scaffold 104 flowing through the bioreactor 101. Figure 5 The flow rates shown in indicate that the bioreactor 101 design emphasizes uniform flow and is likely to promote uniform bone formation.

[0033] In addition, the feasibility of dynamic seeding of bone marrow mesenchymal stem cells in the bioreactor system 100 can be evaluated. In one embodiment of the evaluation, the scaffold 104 can be stained with 4',6-diamidino-2-phenylindole (DAPI) and phalloidin, and the cell distribution can be evaluated using a confocal microscope (λ 405nm, 455nm, respectively). Figure 6 This confocal image confirms that the bone marrow mesenchymal stem cells successfully adhere to the scaffold 104 in a relatively uniform distribution under dynamic conditions. Further, the cells are attached at a relatively high confluence, which is conducive to the initiation of differentiation into the skeletal lineage.

[0034] like Figure 7As shown, static seeding and differentiation of bone marrow mesenchymal stem cells can also be performed on the scaffold 104. After 14 days, alizarin red staining confirmed that the scaffold 104 can help bone marrow mesenchymal stem cells differentiate into the osteogenic lineage ( Figure 7 ).

[0035] This application discloses the technical features in the specification, claims and drawings of the specification in a specific form, in the form of a device for performing the disclosed function, or in the form of a method or step for obtaining the disclosed effect. In an appropriate manner, these technical features can be implemented independently or in any combination, thereby implementing this application in different forms. In particular, one or more technical features in any embodiment described in the specification of this application can be combined with one or more features in any other embodiment described in the specification of this application.

[0036] The present application also claims protection for any technical features disclosed in combination with the present application specification and / or with reference to any one or more publications disclosed in the present application specification.

Claims

1. A bioreactor system, characterized in that: include: a bioreactor including an inlet and an outlet; a support disposed within the interior volume of the bioreactor, wherein the scaffold has a plurality of interconnected pores, the plurality of pores defining porosity and enabling fluid flow through the scaffold; and near the inner cavity of the stent, wherein the lumen has a shape complementary to that of the stent, wherein the porosity of the lumen matches the porosity of the scaffold; The support and the lumen occupy an interior volume of the bioreactor.

2. The bioreactor system according to claim 1, wherein the scaffold is made using additive manufacturing technology. 3 . The bioreactor system of claim 1 , wherein the support comprises a plurality of struts arranged to form the plurality of holes.

4. The bioreactor system of claim 1, wherein the scaffold has a complex geometry with concavities, internal curvatures, or non-linear features. 5 . The bioreactor system according to claim 1 , wherein the inner chamber has a cavity matching an outer shape of the scaffold.

6. The bioreactor system of claim 1, wherein the lumen comprises a plurality of components, wherein the plurality of components are adapted to assemble a scaffold having a complex geometry.

7. The bioreactor system of claim 1, wherein the porosity of the scaffold is in the range of 60% to 94%.

8. The bioreactor system of claim 1, wherein fluid flows uniformly through the bioreactor.

9. The bioreactor system of claim 1, wherein the fluid resistance of the scaffold is similar to the fluid flow resistance of the lumen.

10. The bioreactor system of claim 1, wherein the scaffold is fabricated using one of solvent casting and particle leaching techniques, gas foaming techniques, vacuum drying techniques, and thermally induced phase separation techniques.

11. The bioreactor system of claim 1, wherein the scaffold is coated with an extracellular matrix biomaterial and the lumen is coated with an antifouling agent.

12. A method of making a bone graft, comprising: Designing stents based on digital representation of the defect; manufacturing the scaffold using additive manufacturing technology, wherein the scaffold has a porosity of 60% or more; fabricating an inner cavity having a porosity that matches the porosity of the scaffold, wherein the inner cavity has a cavity that matches the outer shape of the scaffold; placing the scaffold and the lumen in a bioreactor comprising an inlet and an outlet; flowing a fluid through the bioreactor, wherein the fluid comprises stem cells and a stem cell culture medium; and The stem cells attached to the scaffold are cultured.

13. The method according to claim 12, further comprising: rotating the bioreactor to reverse the directions of the inlet and the outlet; and Other liquid is allowed to flow through the outlet.

14. The method according to claim 13, further comprising: collecting the stem cells not attached to the scaffold into a culture medium reservoir; and The collected stem cells in the culture medium reservoir are perfused through the bioreactor.

15. The method according to claim 14, further comprising: Fresh culture medium was perfused through the bioreactor.

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