Highly biomimetic nested lacunocanalicular microfluidic chip and experimental method

Abstract

This invention discloses a highly biomimetic nested lacunar-canal microfluidic chip and experimental method, comprising: a microfluidic chip upper cover plate and a microfluidic chip lower cover plate bonded together; a biomimetic network structure, including a Haver main channel and multiple lacunar chambers arranged in a ring array around the Haver main channel, the multiple lacunar chambers being connected to the Haver main channel via radial flow channels of the bone canals, the lacunar chambers, the radial flow channels of the bone canals, and the Haver main channel being disposed on the microfluidic chip lower cover plate; a cell culture structure, comprising multiple cell culture chambers disposed on the microfluidic chip upper cover plate; a uniform flow loop structure for uniform introduction and uniform extraction of culture medium, comprising an introduction unit disposed on the microfluidic chip lower cover plate and an extraction unit disposed on the microfluidic chip upper cover plate; and cell culture chambers suspended inside the lacunar chambers to form a one-to-one nested biomimetic cell culture unit.

Description

Technical Field

[0001] This invention relates to the interdisciplinary fields of biomedical engineering, tissue engineering, and microfluidics, and particularly to a highly biomimetic nested bone lacunae-canal microfluidic chip and experimental methods. Background Technology

[0002] The osteoid unit (Halver system) is the basic functional unit of the skeleton, consisting of a central Haver canal and concentric bone plates. Osteocytes reside in lacunae within these bone plates and are interconnected through a network of osteotubular canaliculi. They sense the fluid shear forces generated by the flow of tissue fluid during daily activities, thereby regulating the bone remodeling process. Therefore, constructing an in vitro platform that can simulate this complex physiological microenvironment is crucial for studying the mechanical response mechanisms of osteocytes.

[0003] Currently, microfluidic chips for lacunae and canaliculi have been developed. However, most existing technologies are limited to simulating the connection relationships between Haversian canaliculi, lacunar chambers, and canaliculi channels in a two-dimensional plane. These designs typically use simple cavities to simulate lacunae, which has the following obvious drawbacks: First, the structure is too simplified and cannot reflect the fact that bone cells occupy space within the lacunae, resulting in an oversimplified flow field that cannot realistically reflect the complex surrounding flow, leading to a large deviation between the simulated fluid shear force environment and the actual in vivo situation; Second, there is a lack of detailed simulation of the convection-diffusion coupled transport process during the material exchange at the bone-blood vessel interface.

[0004] Therefore, developing a novel biomimetic chip that can more realistically simulate the in vivo environment of bone cells and enable integrated comparative studies has significant scientific research value and application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a highly biomimetic nested lacunar-canal microfluidic chip and experimental method. Through a nested structure of "lacunar chamber-cell culture chamber," a dual biomimetic "cavity within a cavity" unit is physically constructed. This nested structure guides multi-level flow splitting (flow around and seepage) of fluid, generating a complex convection-diffusion mass transport environment and fluid shear field within the chip. This provides a microenvironment for bone cell culture that more closely resembles the mechanical and material exchange conditions of real physiological conditions than traditional single-cavity structures. Simultaneously, the microporous structure on the surface of the cell culture chamber enables the exchange of nutrients and metabolic waste, providing a more biomimetic growth environment for cells.

[0006] To achieve the above objectives, the present invention provides a highly biomimetic nested bone lacunae-bone canaliculi microfluidic chip, comprising: a microfluidic chip upper cover plate and a microfluidic chip lower cover plate bonded together; The biomimetic network structure includes the Haver main channel and multiple bone lacunae chambers arranged in a ring around the Haver main channel. The multiple bone lacunae chambers are connected to the Haver main channel through radial flow channels of bone canaliculi. The bone lacunae chambers, the radial flow channels of bone canaliculi, and the Haver main channel are all located on the lower cover plate of the microfluidic chip. The cell culture structure includes multiple cell culture chambers disposed on the cover plate of the microfluidic chip; A uniform flow loop structure is used for the uniform introduction and uniform extraction of culture medium, including an introduction unit disposed on the lower cover plate of the microfluidic chip and an extraction unit disposed on the upper cover plate of the microfluidic chip. The cell culture chambers are suspended inside the bone lacunae chambers to form nested cell biomimetic culture units.

[0007] Preferably, the inlet unit includes a liquid inflow channel and a liquid collection channel on the lower cover plate of the microfluidic chip. The liquid collection channel on the lower cover plate is an annular groove, and the liquid inflow channel is connected to the bottom of the Haver main pipe.

[0008] Preferably, the lead-out unit includes a liquid outflow channel and a liquid collection channel on the upper cover plate of the microfluidic chip. The liquid collection channel on the upper cover plate is an annular groove. The liquid outflow channel is connected to the liquid collection channel on the upper cover plate. The liquid collection channel on the upper cover plate and the liquid collection channel on the lower cover plate are symmetrically arranged.

[0009] Preferably, the plurality of cell culture chambers are integrally formed with the microfluidic chip cover plate.

[0010] Preferably, the surface of the cell culture chamber is provided with multiple micropores.

[0011] Preferably, the multiple lacunar chambers are arranged in several layers of a ring array around the Haver main channel. The Haver main channel, the lacunar chambers in each layer of the ring array, and the liquid collection channel of the lower cover plate are arranged in concentric circles, and the number of lacunar chambers in each layer of the ring array is the same.

[0012] Preferably, adjacent bone lacunae chambers in each layer of the annular array are connected by circumferential flow channels through bone canaliculi.

[0013] Preferably, the liquid collection channel of the lower cover plate is connected to the lacunar cavity of the outermost annular array, the lacunar cavity of the adjacent annular array, and the lacunar cavity of the innermost annular array to the Haver main channel through a radial flow channel of the bone tube. The radial flow channel of the bone tube is arranged along the radius of a concentric circle with the center of the Haver main channel as the center.

[0014] Preferably, the highly biomimetic nested bone lacunae-canal microfluidic chip is made of polydimethylsiloxane.

[0015] This invention provides an experimental method for a highly biomimetic nested lacunar-canal microfluidic chip, comprising the following steps: S1: The microfluidic chip is assembled by bonding the upper cover plate and the lower cover plate of the microfluidic chip, and a liquid flow loop is formed through the uniform flow loop structure to achieve perfusion under the drive of an external injection pump; S2: Introduce MLOY-4 cell suspension for perfusion inoculation, and allow the cells to stand to adhere to the inner wall of the cell culture chamber. S3: Introduce culture medium for perfusion culture, and adjust the flow rate and oscillation frequency of the culture medium oscillation flow through the injection pump to place MLOY-4 cells in a specific fluid shear force environment; S4: After the culture is completed, the MLOY-4 cells attached to the inner wall of the cell culture chamber are observed or detected.

[0016] The advantages and positive effects of the highly biomimetic nested lacunar-canal microfluidic chip and experimental method described in this invention are as follows: 1. Provides a more in vivo complex flow field and material exchange environment: Through a nested structure consisting of a cell culture chamber (inner chamber) and an external bone lacunar chamber, a biomimetic "cavity within a cavity" physical layout is achieved. When the culture medium flows through, this structure naturally guides the fluid to generate multi-stage flow splitting: one part forms a flow around the outer wall of the cell culture chamber, and another part forms a flow through the micropores on its wall, thereby coupling and generating a complex convection-diffusion mass transport environment within a single unit. This process simulates the material exchange characteristics of the "bone unit-bone lacunar chamber-bone tubule" path in vivo and generates complex fluid shear forces that are closer to those in vivo, providing bone cells with a mechanically biomimetic microenvironment superior to traditional hollow cavities.

[0017] 2. A more biomimetic cell growth environment and ease of observation: The cell culture chamber is integrally molded with the microfluidic chip cover plate, using a transparent polydimethylsiloxane (PDMS) material. The chamber is unobstructed from above, allowing direct observation of the cell morphology and growth status attached to its inner wall through a microscope. The microporous structure of the cell culture chamber wall provides channels for the internal cells to exchange substances with the external environment, enabling cells to grow in a relatively confined space, more closely resembling the physiological state of bone cells encapsulated by the bone matrix in vivo.

[0018] 3. Improved uniformity of fluid distribution and collection: The unique uniform flow circuit structure, especially the annular collection grooves on the upper and lower cover plates, constitutes a buffer and distribution cavity for the inflow and outflow of culture medium. This structural design allows the culture medium to diffuse circumferentially through the annular grooves, thereby flowing more evenly through the bone canal channels to the bone lacunar cavity and cell culture chamber, providing a more stable and consistent perfusion environment for cells.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the internal structure of the upper and lower cover plates after bonding in a preferred embodiment of the highly biomimetic nested bone lacunae-bone canaliculi microfluidic chip of the present invention. Figure 2 This is a schematic diagram of the upper and lower cover plates after bonding in a preferred embodiment of the highly biomimetic nested bone lacunae-bone canaliculi microfluidic chip of the present invention. Figure 3 This is a schematic diagram of the lower cover plate of a preferred embodiment of the highly biomimetic nested bone lacunae-bone canaliculi microfluidic chip of the present invention. Figure 4 This is a schematic diagram of the internal structure of the lower cover plate of a preferred embodiment of the highly biomimetic nested bone lacunae-bone canaliculi microfluidic chip of the present invention. Figure 5 This is a lower view of the cover plate of a preferred embodiment of the highly biomimetic nested bone lacunae-bone canaliculi microfluidic chip of the present invention. Figure 6 This is a schematic diagram of the upper cover plate of a preferred embodiment of the highly biomimetic nested bone lacunae-bone canaliculi microfluidic chip of the present invention. Figure 7 This is a schematic diagram of the internal structure of the top cover plate of a preferred embodiment of the highly biomimetic nested bone lacunae-bone canaliculi microfluidic chip of the present invention. Figure 8 This is a top cover view of a preferred embodiment of the highly biomimetic nested bone lacunae-canal microfluidic chip of the present invention.

[0021] Figure Labels 1. Microfluidic chip top cover; 2. Top cover liquid collection channel; 3. Liquid outflow channel; 4. Outlet interface; 5. Microfluidic chip bottom cover; 6. Haver main channel; 7. Bone lacunar cavity chamber; 8. Cell culture chamber; 9. Bone canaliculus radial flow channel; 10. Bottom cover liquid collection channel; 11. Liquid inflow channel; 12. Inlet interface; 13. Bone canaliculus circumferential flow channel. Detailed Implementation

[0022] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product is in use. They are used only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," and "connect" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0023] In this application, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning set forth in this specification or derived from the content described herein shall prevail. Furthermore, the terminology used herein is for the purpose of describing embodiments of this application only and is not intended to limit the scope of this application.

[0024] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0025] like Figures 1-2 As shown, this invention discloses a highly biomimetic nested lacunar-canal microfluidic chip, comprising: a biomimetic network structure disposed on the lower cover plate 5 of the microfluidic chip, including a Haver main channel 6 and multiple lacunar chambers 7 arranged in a ring around the Haver main channel 6, the multiple lacunar chambers 7 being connected to the Haver main channel 6 via radial flow channels 9 of the bony canals; a cell culture structure comprising multiple cell culture chambers 8 disposed on the upper cover plate 1 of the microfluidic chip; a uniform flow loop structure for uniform introduction and uniform extraction of culture medium, including an introduction unit disposed on the lower cover plate 5 of the microfluidic chip and an extraction unit disposed on the upper cover plate 1 of the microfluidic chip; the cell culture structure and the biomimetic network structure are arranged vertically correspondingly, and after the lower cover plate 5 of the microfluidic chip and the upper cover plate 1 of the microfluidic chip are bonded together, the multiple cell culture chambers 8 are respectively suspended inside the multiple lacunar chambers 7, forming multiple nested biomimetic cell culture units.

[0026] This invention achieves a biomimetic "cavity within a cavity" physical layout through a nested structure consisting of a cell culture chamber 8 (inner chamber) and an outer bone lacunar chamber 7. When the culture medium flows through, this structure naturally guides the fluid to generate multi-stage flow splitting: one part forms a flow around the outer wall of the cell culture chamber 8, while another part forms a flow through the micropores on its wall, thus coupling and generating a complex convection-diffusion mass transport environment within a single unit. This process simulates the material exchange characteristics of the "bone unit-bone lacunar chamber-bone canaliculus" pathway in vivo and generates complex fluid shear forces that more closely resemble those in vivo, providing bone cells with a biomimetic microenvironment superior to traditional hollow cavities.

[0027] like Figures 3-8 As shown, the inlet unit includes a liquid inflow channel 11 and a liquid collection channel 10 on the lower cover plate 5 of the microfluidic chip. The liquid collection channel 10 is an annular groove, and the liquid inflow channel 11 is connected to the bottom of the Haver main pipe 6. The outlet unit includes a liquid outflow channel 3 and a liquid collection channel 2 on the upper cover plate 1 of the microfluidic chip. The liquid collection channel 2 is an annular groove, and the liquid outflow channel 3 is connected to the liquid collection channel 2. The upper cover liquid collection channel 2 and the lower cover liquid collection channel 10 are symmetrically arranged. The liquid inflow channel 11 is connected to the outside through the inlet interface 12, and the liquid outflow channel 3 is connected to the outside through the outlet interface 4, forming a closed path to allow the microfluidic chip to exchange substances with the outside world.

[0028] The unique uniform flow circuit structure, especially the annular collection grooves located on the upper and lower cover plates, forms a buffer and distribution cavity for the inflow and outflow of culture medium. This structural design allows the culture medium to diffuse circumferentially through the annular grooves, thereby flowing more evenly through the radial flow channel 9 of the bone canaliculus to the bone lacunar cavity 7 and the cell culture chamber 8, providing a more stable and consistent perfusion environment for the cells.

[0029] The cell culture chamber 8 is integrally formed with the microfluidic chip cover plate 1. Multiple micropores are provided on the surface of the cell culture chamber 8. These micropores are circular, with a diameter of 10-30 micrometers.

[0030] Multiple lacunar chambers 7 are arranged in two rings around Haver's main channel 6, including an inner ring array close to Haver's main channel 6 and an outer ring array away from Haver's main channel 6. There are twelve lacunar chambers 7 in both the inner and outer ring arrays, and the lacunar chambers 7 in the inner ring array are set in correspondence with the lacunar chambers 7 in the outer ring array.

[0031] In the inner annular array, the lacunar cavity 7 is connected to the Haver main tube via the radial flow channel 9 of the bony canal. Adjacent lacunar cavity 7 in the inner annular array are connected via the circumferential flow channel 13 of the bony canal. In the outer annular array, the lacunar cavity 7 is connected to the corresponding lacunar cavity 7 in the inner annular array via the radial flow channel 9 of the bony canal, and then connected to the Haver main tube 6. Adjacent lacunar cavity 7 in the outer annular array are connected via the circumferential flow channel 13 of the bony canal. The lacunar cavity 7 in the outer annular array is connected to the liquid collection channel 10 of the lower cover plate via the radial flow channel 9 of the bony canal.

[0032] The microfluidic chip is made of polydimethylsiloxane (PMDS). PDMS can be replicated using a "soft lithography" process, which involves casting the PDMS onto a photoresist mold and then curing it. This process is suitable for manufacturing complex and delicate three-dimensional microstructures in chips.

[0033] The microfluidic chip is made of polydimethylsiloxane (PDMS), which has good optical transparency, making it easy to directly observe the cell growth status in the cell culture chamber through a microscope.

[0034] In a preferred embodiment of the present invention, the parameters of each component of the microfluidic chip are as follows:

[0035] This invention discloses an experimental method for a highly biomimetic nested bone lacunar infundibulum-canal microfluidic chip, comprising the following steps: S1: The microfluidic chip is assembled by bonding the upper cover plate 1 and the lower cover plate 5 of the microfluidic chip, and a liquid flow circuit is formed through the uniform flow circuit structure to achieve perfusion under the drive of an external injection pump; S2: Introduce MLOY-4 cell suspension for perfusion inoculation, and allow the cells to stand to adhere to the inner wall of cell culture chamber 8. S3: Introduce culture medium for perfusion culture, and adjust the flow rate and oscillation frequency of the culture medium oscillation flow through the injection pump to place MLOY-4 cells in a specific fluid shear force environment; S4: After the culture is completed, the MLOY-4 cells attached to the inner wall of cell culture chamber 8 are observed or detected.

[0036] Therefore, by employing the highly biomimetic nested lacunar-canal microfluidic chip and experimental method described in this invention, a unique nested structure of "lacunar chamber 7 - cell culture chamber 8" is formed, physically constructing a dual biomimetic unit of "cavity within a cavity". This nested structure guides the multi-level flow splitting (flow around and seepage) of fluid, generating a complex convection-diffusion mass transport environment and fluid shear field inside the chip, providing a mechanical and material exchange microenvironment for bone cell culture that is closer to real physiological conditions than traditional single-cavity structures; at the same time, the microporous structure on the surface of the cell culture chamber can realize the exchange of nutrients and metabolic waste, providing a more biomimetic growth environment for cells.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A highly biomimetic nested lacunar-canal microfluidic chip, characterized in that, include: The upper cover plate and the lower cover plate of the microfluidic chip are bonded together; The biomimetic network structure includes the Haver main channel and multiple bone lacunae chambers arranged in a ring around the Haver main channel. The multiple bone lacunae chambers are connected to the Haver main channel through radial flow channels of bone canaliculi. The bone lacunae chambers, the radial flow channels of bone canaliculi, and the Haver main channel are all located on the lower cover plate of the microfluidic chip. The cell culture structure includes multiple cell culture chambers disposed on the cover plate of the microfluidic chip; A uniform flow loop structure is used for the uniform introduction and uniform extraction of culture medium, including an introduction unit disposed on the lower cover plate of the microfluidic chip and an extraction unit disposed on the upper cover plate of the microfluidic chip. The cell culture chambers are suspended inside the bone lacunae chambers to form nested cell biomimetic culture units.

2. The highly biomimetic nested lacunar-canal microfluidic chip according to claim 1, characterized in that: The inlet unit includes a liquid inflow channel and a liquid collection channel on the lower cover plate of the microfluidic chip. The liquid collection channel on the lower cover plate is an annular groove, and the liquid inflow channel is connected to the bottom of the Haver main pipe.

3. The highly biomimetic nested lacunar-canal microfluidic chip according to claim 2, characterized in that: The extraction unit includes a liquid outflow channel and a liquid collection channel on the upper cover plate of the microfluidic chip. The liquid collection channel on the upper cover plate is an annular groove. The liquid outflow channel is connected to the liquid collection channel on the upper cover plate. The liquid collection channel on the upper cover plate and the liquid collection channel on the lower cover plate are symmetrically arranged.

4. The highly biomimetic nested lacunar-canal microfluidic chip according to claim 1, characterized in that: The multiple cell culture chambers are integrally formed with the microfluidic chip cover plate.

5. The highly biomimetic nested lacunar-canal microfluidic chip according to claim 1, characterized in that: The surface of the cell culture chamber is provided with multiple micropores.

6. The highly biomimetic nested lacunar-canal microfluidic chip according to claim 1, characterized in that: The multiple lacunar chambers are arranged in several layers of a ring array around the Haver main channel. The Haver main channel, the lacunar chambers in each layer of the ring array, and the liquid collection channel of the lower cover plate are arranged in concentric circles. The number of lacunar chambers in each layer of the ring array is the same.

7. A highly biomimetic nested lacunar-canal microfluidic chip according to claim 6, characterized in that: Adjacent bone lacunae chambers in each layer of the ring array are connected by circumferential flow channels via bone canaliculi.

8. A highly biomimetic nested lacunar-canal microfluidic chip according to claim 7, characterized in that: The liquid collection channel of the lower cover plate is connected to the bone lacunae chambers of the outermost annular array, the bone lacunae chambers of adjacent annular arrays, and the bone lacunae chambers of the innermost annular array to the Haver main pipe through radial flow channels of bone tubes. The radial flow channels of bone tubes are set along the radial direction of concentric circles with the center of the Haver main pipe as the center.

9. The highly biomimetic nested lacunar-canal microfluidic chip according to claim 1, characterized in that: The highly biomimetic nested bone lacunae-canal microfluidic chip is made of polydimethylsiloxane.

10. An experimental method for a highly biomimetic nested lacunar-canal microfluidic chip, characterized in that, A highly biomimetic nested lacunar-canal microfluidic chip based on any one of claims 1-9 comprises the following steps: S1: The microfluidic chip is assembled by bonding the upper cover plate and the lower cover plate of the microfluidic chip, and a liquid flow loop is formed through the uniform flow loop structure to achieve perfusion under the drive of an external injection pump; S2: Introduce MLOY-4 cell suspension for perfusion inoculation, and allow the cells to stand to adhere to the inner wall of the cell culture chamber. S3: Introduce culture medium for perfusion culture, and adjust the flow rate and oscillation frequency of the culture medium oscillation flow through the injection pump to place MLOY-4 cells in a specific fluid shear force environment; S4: After the culture is completed, the MLOY-4 cells attached to the inner wall of the cell culture chamber are observed or detected.

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