Technical Field of the Present Invention

The present invention relates to a microfluidic injection device for creating microbubbles using multiple fluids containing bioactive materials and biocompatible gases for use in cartilage repair.

Background of the Present Invention

Articular cartridge has limited intrinsic regeneration and healing capacity, which makes joint arthroplasty an unavoidable surgical intervention (Makris, Eleftherios A., et al. "Repair and tissue engineering techniques for articular cartilage." Nature Reviews Rheumatology 11.1 (2015): 21). There are three problems associated with regeneration of articular cartridge: First is filling the defects with tissues having the same mechanical properties as articular cartridge. Second is promoting a successful integration between the graft material and the present articular cartridge. And third is effectuating the regeneration of the original cartilage tissue. Even a small defect caused by mechanical damage can lead to osteoarthritis and become unhealable in time (Redman, S. N., S. F. Oldfield, and C. W. Archer. "Current strategies for articular cartilage repair." Eur Cell Mater 9.23-32 (2005): 23-32). The goals of cartilage repair are the restoration of function, reduction of pain and slowing, or avoidance of the development of osteoarthritis. It appears reasonable to expect these goals to be met best by a cartilage repair technique that restores a smooth, low-friction articular surface by filling the defect with tissue that resembles articular cartilage both mechanically and biologically (Gomoll, Andreas H., and Tom Minas. "The quality of healing: articular cartilage." Wound Repair and Regeneration 22 (2014): 30-38). Chondral defects are defects that do not reach the subchondral bone. Chondral defects may be caused by micro breaks in the cartilage or trauma, repeated microtrauma or injuries by foreign objects. These types of injuries cannot be repaired with sufficient regeneration via intrinsic metabolic activity without blood and will develop into osteochondral defects. Osteochondral defects are defects that go past the tidemark (the transition zone between cartilage and bone) and into the bone. The reduction in the number of chondrocytes and progenitor cells as well as in metabolic activity with advanced age causes problems in the healing of cartilage tissue.

Traditional methods such as autografts and allografts have been used clinically to treat articular cartilage lesions, however, these therapies still have many shortcomings. Autografts, which require the transplantation of a small portion of low weight-bearing cartilage from the patient into defect sites, have disadvantages such as donor site morbidity and limited cartilage tissue availability. Allografts, which are cartilage pieces obtained from tissue banks, may potentially induce immune responses. For patients with severe joint damage and osteoarthritis, total joint replacement surgery is needed.

More recently, injectable viscoelastic biomaterials are used in the treatment of defects in cartilage tissue (Athanasiou, Kyriacos A., Eric M. Darling, and Jerry C. Hu. "Articular cartilage tissue engineering." Synthesis Lectures on Tissue Engineering 1.1 (2009): 1-182). This method consists of injecting a biomaterial to the defect site via a needle. There are several disadvantages of this method:

1) Standard injectors are used during application, which leads to adhesion problems between the biomaterial to the cartilage tissue and stabilization of the biomaterial in the damaged area.

2) The original microporous structure of cartilage tissue cannot be obtained, therefore the blood flow to the area is limited, which leads to stunted cell growth and delay in the healing process.

3) There are no systems utilizing biological agents, and the composition of the injection materials do not mimic the cartilage repair pathway. Therefore, fibrous cartilage tissue is obtained in the damage site, whereas the main aim of the cartilage repair is to obtain hyaline cartilage.

4) If the injections are performed for pain relief, they need to be repeated often which leads to inconvenience and high costs for the patient.

Injectable products available to be used in cartilage repair can be exemplified by BST-CarGel ve CartiFill. These products make use of injecting biomaterials into the cartilage defect area via a delivery needle (Shive, Matthew S., et al. "BST-CarGel: in situ chondroinduction for cartilage repair." Operative Techniques in Orthopaedics 16.4 (2006): 271-278). WO 2008/064487 discloses a method for repairing or regenerating tissues in a patient such as cartilage, meniscus, ligament, tendon, and bone. The method comprises the step of administering simultaneously or sequentially a pro-coagulant factor and an effective amount of a polymer composition comprising a biocompatible polymer and blood or a component thereof. When the polymer composition is in contact with the pro-coagulant factor it is converted into a non-liquid state such that the polymer composition will adhere to the site in need of repair to aid in the repair and/or regeneration of the tissue. WO 2005/060987 discloses a cartilage therapeutic composition, capable of being clinically transplanted to clinically significant, symptomatic cartilage defects in human or animal hosts. The cartilage therapeutic composition comprises a mixture of components of chondrocytes isolated and expanded or differentiated from a host such as human or animal, and thrombin and a fibrinogen matrix containing fibrinogen. The document also discloses a method of using the cartilage therapeutic composition such that a mixture of thrombin, chondrocyte components and a fibrinogen matrix is injected to a cartilage defect region followed by solidification therein.

However, these methods are not practical and because standard injectors are used, there are problems with the adhesion of biomaterials to the cartilage tissue. These problems are attempted to be overcome by adding adhesive materials such as chitosan or changing the viscosity of the material. Further, in the cases where there is a large defect, open surgery is needed in order to deliver the biomaterials to the defect site and repeat surgeries may be required.

There is a need in the art for a method of cartilage repair that has low costs and can be easily implemented by doctors that improve the chance of recovery and speed up the treatment process.

The present invention aims to improve on the problems in the prior art. Specifically, the invention makes use of microfluidics to generate microbubbles and creating a scaffold containing cells, and medicaments if needed, directly in the cartilage defect site in order to provide an improved treatment process for cartilage repair.

Using microfluidics and microbubbles to deliver therapeutic agents and generate scaffolds for cell cultures has been studied in the art. For example, US 8,679,051 discloses methods and medical devices for generating and stabilizing micro- or nano-bubbles, and systems and methods for therapeutic applications using the bubbles. The micro-bubbles may be used to enhance therapeutic benefits such as ultrasound-guided precision drug delivery and real-time verification. As another example, US 2011/091972 discloses methods and apparatuses for using microfluidics to generate bubbles and using the generated bubbles to construct scaffolds and cell-holding structures for culturing biological samples or analytes. A scaffold for growing cells is provided to include a matrix of interconnected cavities formed from mixing a gas and a liquid containing a cross linkable material to produce a matrix of gas bubbles of substantially the same size and cross linking the cross linkable material to form a structure in which cells can be grown. However, this system makes use of a micropipette technique and is most suitable for creating scaffold where cells can be cultured experimentally and not suitable to provide the precision that is required for direct application to a defect site.

The present invention discloses a microfluidic injection device that can be used in cartilage repair. The microfluidic injection device of the present invention allows the use of composite and biological agents (including but not limited to stem cells and platelet-rich plasma), which are injected using the microbubble technique. The microfluidic injection device uses CO2 gas, which allows a scaffold to be produced directly in the cartilage defect site during application in the perioperative phase. CO2 aids in the production of microbubbles and when the bubbles pop, they leave behind a porous scaffold in the defect site that cells can adhere to and propagate, and the damaged part of the cartilage can be regenerated with patient's own stem cells. The problem of slow regeneration of cartilage tissue will be boosted by stem cells and the patient will recover faster. The microfluidic injection device can be used arthroscopically, which minimizes the inconvenience caused to the patient.

The microfluidic injection device of the present invention alleviates the problems associated with the methods and devices currently available in the art and provides a system that is convenient for doctors to use, improves the chances of healing and speeds up the treatment process. The microfluidic injection device also comprises an electronic panel whereby the doctor can adjust parameters such as flowrate, gas pressure and fill rate during application. The microfluidic injection device of the invention is used to fill defects in cartilage and/or bone with a suitable scaffold architecture and provides medication and/or cells to the area as needed.

Brief Description of the Figures of the Present Invention

Accompanying drawings are given solely for the purpose of exemplifying a microfluidic injection device, whose advantages over prior art were outlined above and will be explained in brief hereinafter.

The drawings are not meant to delimit the scope of protection as identified in the claims nor should they be referred to alone in an effort to interpret the scope identified in said claims without recourse to the technical disclosure in the description of the present invention.

Figure 1 illustrates an exploded view of a microfluidic injection device in accordance with the present invention.

Figure 2 illustrates a side (A) and front (B) view of a microfluidic injection device in accordance with the present invention.

Figure 3 illustrates a front (A), sectional across A-A (B), bottom (C), side (D), perspective (E) and sectional across B-B(F) view of a mixing element of a microfluidic injection device with a gas inlet in accordance with the present invention. Figure 4 illustrates a sectional across B-B (A), side (B), perspective (C), front (D), bottom (E), back (F) and sectional across A-A (G) view of a microbubble production element of a two-piece mixing element of a microfluidic injection device with a gas inlet in accordance with the present invention.

Figure 5 illustrates a bottom (A), side (B), side (C) and perspective (D) view of a housing attachment element of a two-piece mixing element of a microfluidic injection device without a gas inlet as not covered by the present invention.

Figure 6 illustrates a front (A), front sectional (B), bottom (C), side (D), and side sectional (E) view of a mixing element of a microfluidic injection device without a gas inlet as not covered by the present invention.

Figure 7 illustrates a side (A), perspective (B), front (C), bottom (D) and sectional across A-A (E) view of a microbubble production element of a two- piece mixing element of a microfluidic injection device without a gas inlet as not covered by the present invention.

Referenced Parts List

1 Microfluidic injection device

2 Mixing element

3 Fluid inlet

4 Gas inlet

5 Microbubble fluid outlet

6 Syringe

7 Fixing element

8 Pressing element 9 Gear

10 Threaded shaft

11 Actuator

12 Actuator gear

13 Housing

14 Fastening hole

15 Fastening handles

16 Fluid channel

17 Gas channel

18 Microbubble channel

19 Mixing point

20 Housing attachment element

21 Microbubble production element

Detailed Description of the Present Invention

As a cell source, primary and chondrocytic cell lines have been widely used for clinical applications for cartilage regeneration. Additionally, adult and embryonic stem cells have recently received great attention as an alternative. Stem cells are well-known to exhibit unlimited proliferation and pluripotency, and are promising resources for regenerative medicine, which requires large numbers of a particular cell type including cartilage. On the other hand, signaling molecules such as growth factors (TGF, FGF, IGF, PDGF), cytokines and nonprotein chemical compounds are extensively employed to facilitate cartilage tissue growth. However, as cells are directly transplanted to the cartilage damage site, there are some issues such as the lack of cell retention, and donor site morbidity. Thus, it is very important to use three- dimensional scaffolds to facilitate cell retention and strengthen mechanical property at the transplanted site (Lee, Soo-Hong, and Heungsoo Shin. "Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering." Advanced drug delivery reviews 59.4-5 (2007): 339-359). The present invention aims to provide a microfluidic injection device (1) that can be used to deliver the cells and the building blocks of the tissue scaffold directly onto the cartilage.

Figures 1 and 2 show a microfluidic injection device (1) suitable to be used in the repair of defects in cartilage and bone tissue according to the present invention. Said microfluidic injection device (1) comprises a housing (13), an actuator (11), at least one syringe (6) fixed onto a fixing element (7) and a mixing element (2). The drive from the actuator (11) is transferred to the fixing element (8) attached to the syringes (6) via a gear assembly comprising an actuator gear (12), a gear (9) and a threaded shaft (11) and a pressing element (8). The mixing element is attached to the housing (13) via fastening handles (15) and fastening holes (14) respectively. The syringes (6) are attached to the mixing element (2) via the fluid inlets (3). In a preferred embodiment of the present invention, microfluidic injection device (1) comprises a plurality of syringes (6). In a more preferred embodiment of the present invention, microfluidic injection device (1) comprises 1 to 6 syringes (6). In a more preferred embodiment of the present invention, microfluidic injection device (1) comprises 2 or 3 syringes (6).

When in operation, the actuator (11) pushes the syringes (6) and transfers the fluids therein into the mixing element (2). These fluids may be hydrogels containing bioactive materials and cells that are known in the art of cartilage repair. Some examples will be given below:

In a preferred embodiment of the invention, the fluids contain a cell solution for providing the growth of cartilage tissue in the damage site. In a preferred embodiment, the cell solution consists of materials selected from chondrocytic cell lines, pluripotent stem cell lines, platelet-rich plasma, stromal vascular fraction, bone marrow aspirate concentrate, and combinations thereof cultured in suitable buffer known in the art. In a preferred embodiment of the invention, cell solution further contains signaling molecules such as growth factors (TGF, FGF, IGF, PDGF), cytokines and nonprotein chemical compounds.

In a preferred embodiment of the invention, the fluids contain a biomaterial solution for building a tissue scaffold for cartilage tissue. Many natural biomaterials have been widely used for cartilage tissue engineering such as collagen, gelatin, polysaccharides (alginate, agarose, chitosan, hyaluronic acid), and fibrin. Collagen is any of a family of extracellular matrix (ECM) proteins occurring as a major component of connective tissue, giving it strength and flexibility. Collagen has advantages such as low antigenicity, high biocompatibility and bioabsorbability, adhesion to cells, cell growth, cell differentiation induction, blood coagulation, a styptic effect, and biocompatibility with other polymers. Also, collagen can be gelated physically or chemically so it can be used alone or with other biomaterials. In addition, synthetic biomaterials such as poly(o-hydroxy esters) such as PGA, PLA and their copolymers are the most widely investigated synthetic biodegradable polymers for cartilage tissue engineering (Lee, Soo-Hong, and Heungsoo Shin. "Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering." Advanced drug delivery reviews 59.4-5 (2007): 339-359). Preferably, the biomaterial is a biocompatible polymer selected from collagen, fibrin, chitosan, chitin, hyaluronan, alginate, glycosaminoglycan, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparin, cellulose, heparin sulfate, polyacrylamide, polycaprolactone, polylactic acid, poly lactic-co-glycolic acid, polyvinyl alcohol, polyethylene glycol and combinations thereof. In a preferred embodiment of the invention, the biomaterial is dissolved in distilled water, saline, organic or inorganic phosphate buffer, or any other suitable buffer known in the art.

The cell solution and the biomaterial solution may be mixed before loading into the syringes (6), or they may be loaded into the syringes (6) separately and mixed in the mixing element (2). In a preferred embodiment, the solutions are loaded into the syringes (6) separately and mixed in the mixing element (2). In a preferred embodiment, the fluids also contain therapeutic agents such as antibiotics and anticancer medications. In a preferred embodiment, the fluids also crosslinking agents and surfactants in order to promote the generation of or the stability of the bubbles. In a preferred embodiment, the fluids also contain citrates and EDTA.

Figure 3 shows a mixing element (2) of a microfluidic injection device (1) having a gas inlet according to the present invention. The mixing element comprises at least one fluid inlet (3), at least one gas inlet (4), at least one fluid channel (18), a mixing point (19), at least one microbubble channel (18) and a microbubble fluid outlet (5). In a preferred embodiment of the present invention, mixing element (2) comprises a plurality of fluid inlets (2) and fluid channels (18). In a preferred embodiment of the present invention, mixing element (2) comprises 1 to 6 fluid inlets (2) and fluid channel (18). In an even more preferred embodiment of the present invention, mixing element (2) comprises 2 or 3 fluid inlets (2) and fluid channel (16).

The actuator (11) pushes multiple syringes (6) simultaneously in a controlled manner and the material within the syringes are transferred to the mixing element (2) via fluid inlets (3), wherein they flow through fluid channels (16) mix with each other in the mixing point (19). The mixing element has a gas inlet (4) whereby CO2 gas is introduced into the mixing point (19) via a gas channel (17) from a gas supply with a regulator, which can be controlled by the operator of the microfluidic injection device (1). The gas inlet (4) may be positioned at different angles vertically or horizontally and may be facing the front or the back. The gas supply may be connected to the gas inlet (4) via a locking or a luer fitting. In a preferred embodiment of the invention, CO2 gas is medical grade CO2. Use of CO2 gas is beneficial to the growth of cartilage tissue.

Microbubbles are formed at the mixing point (19) where the fluids and gas are injected vertically, horizontally or at an angle via the fluid channels (16) and gas channels (17) respectively. An important advantage of not mixing the different solutions with each other before adding the gas and instead forming the microbubbles as the different fluids are mixing for the first time is that it is possible to obtain bubbles that are covered by the different solutions in a layerwise manner without mixing with each other. This is advantageous with fluids which may have an undesirable interaction with each other, such as precipitating out of the solution and blocking the channels of the mixing element (2). In a preferred embodiment of the invention, fluid channels (16) and gas channel (17) enter the mixing point (19) at an angle of 20-70°. The size of the microbubbles can be controlled by controlling the gas pressure, fluid flowrate, the diameter of the fluid channels (16) and the angle by which fluid channels (16) and gas channel (17) enter the mixing point (19). Microbubble size is also dependent on the surface pressure and the viscosity of the fluid. For example, microbubble size decreases with increasing gas pressure. As another example, microbubble size decreases by increasing the angle by which fluid channels (16) and gas channel (17) enter the mixing point (19). Microbubbles used in biomedical applications are preferably at least 10 pm in diameter, which is equivalent to the diameter of a red blood cell, in order to obtain similar rheology in the micro vessels and capillary vessels of the subject. In a preferred embodiment of the invention, the microbubbles formed are 10-500 pm in diameter. In an even more preferred embodiment of the invention, the microbubbles formed are 10-300 pm in diameter. Preferably, the fluid flowrate in fluid channels (16) is 600 pl/min and the gas pressure is 30-600 kPa to form microbubbles of desired size.

The fluid containing microbubbles is transferred to the microbubble fluid outlet (5) via the microbubble channel (18) and injected to the desired damage site. Microbubble fluid outlet (5) can be attached to a needle whereby the fluid containing microbubbles, can be applied directly to the damage site, creating a tissue scaffold containing bioactive materials that is suitable to be used in cartilage repair, and adheres there more efficiently due the gas pressure. In alternative embodiments of the invention, microbubble fluid outlet (5) can be attached to other injection means, such as a catheter, tube, or other surgical instrument that can be inserted within a body to a point of interest.

In an alternative embodiment of the invention, the microfluidic injection device (1) comprises a two-piece mixing element (2") comprises a housing attachment element (20) and a microbubble production element (21) (Figures 4 and 5).

In embodiment not covered by the invention, mixing element (2') of a microfluidic injection device (1) does not comprise a gas inlet (Figures 6 and 7). Here, the device can be used to mix the fluids containing biomaterials and/or cells without generating microbubbles.

A doctor can use the handheld microfluidic injection device (1) to position the needle, or other injection means, at the desired cartilage damage site and deposit the microbubble fluid directly thereonto in a practical and efficient manner.

In a preferred embodiment, when in operation, the actuator (11) pushes the syringes (6) at a speed of 20-40 ml/s on average.

In a preferred embodiment, the actuator (11) is a 5 N/m 12 V reductor motor, servo motor, brushed de motor, a step motor or any suitable motor known in the art.

In a preferred embodiment, the syringes (6) can be removed to be refilled with the desired fluids.

In a preferred embodiment, parameters including but not limited to fluid flowrate and gas pressure can be monitored using the OLED screen of the microfluidic injection device and the actuator (11) can be controlled by an electronic card having an H-bridge, a DC-DC boost converter and a microprocessor. Control systems such as PID are controlled by PWM. The direction of the motor is determined by the H-bridge and depending on the clockwise and/or counterclockwise direction of the motor, the syringes are moved forward or backward. In a preferred embodiment, a limit switch is utilized to determine if the syringes (6) have reached the start and end points. In a preferred embodiment, the microfluidic injection device (1) operates using a rechargeable lithium battery.

In a nutshell, the present invention proposes a microfluidic injection device (1) for generating microbubbles, comprising a housing (13) housing an actuator (11), at least one syringe (6) fixed onto a fixing element (7) and a mixing element (2), wherein said syringe (6) is preloaded with a biocompatible fluid component, said mixing element (2) comprises at least one fluid inlet (3) whereby at least one syringe is attached (6) to said mixing element (2), at least one fluid channel (16), a mixing point (19) and a microbubble channel (18) in fluid communication with said fluid inlet (3) and syringe (6), said mixing element (2) comprises a gas inlet (4) and a gas channel (17) in fluid communication with said mixing point (19) whereby said gas component is loaded into said mixing element (2), said actuator (11) is configured to facilitate the pushing of said fixing element (7) against said syringe (6) whereby the contents of said syringe (6) are transferred into and travel along said fluid channel (16) to said mixing point (19) where said biocompatible fluid component and gas component mix and microbubbles are generated and the microbubble fluid is pushed along said microbubble channel (18) to a microbubble fluid outlet (5), and said microbubble fluid outlet (5) is configured to be attached to an injection means.

In a further embodiment of the invention, said mixing element (2) comprises between 1 and 6 fluid inlets (3) whereby between 1-6 syringes (6) can be attached to said mixing element (2).

In a further embodiment of the invention, said fluid channels (16) enter the mixing point (19) at an angle between 20-70°.

In a further embodiment of the invention, the microbubbles formed by said microfluidic injection device (1) are 10-500 pm in diameter.

In a further embodiment of the invention, the microbubbles formed by said microfluidic injection device (1) are 10-300 pm in diameter.

In a further embodiment of the invention, said biocompatible fluid component is a cell solution, a biomaterial solution, or a combination thereof.

In a further embodiment of the invention, said cell solution comprises materials selected from chondrocytic cell lines, pluripotent stem cell lines, platelet-rich plasma, stromal vascular fraction, bone marrow aspirate concentrate, and combinations thereof cultured in a suitable buffer solution.

In a further embodiment of the invention, said cell solution comprises materials selected from growth factors, cytokines and combinations thereof.

In a further embodiment of the invention, said biomaterial solution comprises a biocompatible polymer selected from collagen, fibrin, chitosan, chitin, hyaluronan, alginate, glycosaminoglycan, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparin, cellulose, heparin sulfate, polyacrylamide, polycaprolactone, polylactic acid, poly lactic-co-glycolic acid, polyvinyl alcohol, polyethylene glycol and combinations thereof dissolved in suitable buffer solution.

In a further embodiment of the invention, said biocompatible fluid component comprises therapeutic agents, crosslinking agents, surfactants, or combinations thereof.

In a further embodiment of the invention, said biocompatible fluid component comprises citrates, EDTA, or combinations thereof.

In a further embodiment of the invention, said gas component comprises medical grade carbon dioxide gas.

In a further embodiment of the invention, said mixing element (2") has a two-piece structure comprising a housing attachment element (20) and a microbubble production element (21).

In a further embodiment of the invention, said injection means is a needle.

The present invention also proposes a method for generating tissue scaffolds for use in cartilage repair, comprising the steps of:

- loading a microfluidic injection device (1) according to any one of Claims 1 to 14 with at least one biocompatible fluid component and at least one gas component,

- using said microfluidic injection device (1) to generate bubbles within said biocompatible fluid component to be introduced to a point of interest in a cartilage defect.