Regenerative Treatment of SUI by Autologous Macrophage Therapy

My name is Snigdha Cingireddi and I am a senior at Hathaway Brown School, and my project is The Regenerative Treatment of SUI by Autologous Macrophage Therapy. I chose this topic because, as someone who attends an all-girls school, I have a lot of female role models who I admire and care about, as I'm sure many of you do as well. Many women in my life have informed me about their negative experiences with various types of tissue reinforcement operations that resulted in the use of various meshes to prevent SUI. That's why I wanted to be a part of a project that looked into possible solutions to this problem. When I first presented a background poster on my topic, I recall many women approaching me and telling me how their doctor had recommended vaginal meshes for their conditions (the majority of which were SUIs caused by pregnancy), and how terrified they were to get one because of what they had heard and seen from the media and other women. The more I researched, the more information I discovered that piqued my curiosity. I noticed that vaginal meshes had a high rate of erosion (10%), which means that 1/10 of women who have this mesh implanted have experienced erosion. Erosion occurs when the mesh breaks, infects the surrounding tissue, and weakens the tissue over time. Regulatory agencies (such as the FDA) were forced to issue warnings as a result of this. In addition, sling and mesh manufacturers have faced and are still facing ongoing legal action. For example, vaginal meshes were prohibited in the United Kingdom two years ago. Women have the right to medical treatment that makes them feel safe and at ease in their own skin. That's why I wanted to contribute to the creation of an alternate mesh that addressed these issues. I didn't want to hear about the negative stigma surrounding meshes from the people I admired, and I wanted to ensure that they felt secure and safe seeking treatment for SUI and other mesh-related disorders.

Rationale: The project is Polarization of M0 macrophages to an M2 phenotype seeded upon genipin-crosslinked collagen mesh for treatment of Stress Urinary Incontinence (or SUI). Stress Urinary Incontinence is the unintentional loss of urine caused by physical movement or activity such as coughing and sneezing. SUI most commonly occurs during pregnancy because the fetus puts pressure on the urethra. In order to prevent this hospitals use Mid-Urethral and Polypropylene sling in an estimated 750,000 patients/year in order to prevent this however these slings aren’t ideal and have a lot of negative publicity.

Research Question: Focusing on developing an alternative solution, which is a mesh using a biodegradable biomaterial called collagen. Collagen is known for strengthening body tissues, and cells. The mesh was made out of collagen solution, cross-linked with Genipin (which is a plant extract that has anti-inflammatory properties), and macrophages were seeded onto the mesh. A macrophage is a white blood cell which is important to our immune system.

Hypothesis: Delivery of autologous-blood derived M2 macrophages via genipin crosslinked collagen meshes will result in faster regeneration, e.g. earlier deposition of collagen, earlier inception of neovascularization and earlier attainment of mechanical robustness than the collagen meshes which are implanted without M2 macrophages.

Expected outcomes: 1. To demonstrate the feasibility of seeding macrophages on the cross linked mesh for implantation and 2. To illustrate M0 macrophages on scaffolds undergo a transition to M2 macrophages. M0 macrophages are bad, M1 are neutral, and M2 are good and important because they are pro regenerative which makes them a regulator for collagen. 3. Make sure the mesh is structurally robust and the body promotes collagen deposition around the tissue.

Material & Methods/Results

Scaffold fabrication

Electrochemical compaction was used to transform collagen solutions on spools into aligned threads of continuous length. Collagen solution was dialyzed against DI (deionized) water 24 hours prior to electro compaction at pH 7.0. The collagen solution was then loaded into a syringe and dispensed in the space between an electrode wheel in a regulated manner. The wheel spun the collagen and used electro compaction, a process that compresses the collagen using electrical potential, to create the solution into threads. The threads were collected in a solution of 80% isopropanol and 20% water.

After the collection of the collagen threads, they were twisted together to form a yarn to increase mechanical robustness of the structure. To combine the collagen threads, L-ascorbic acid solution (2.7 mg/ml in DI water) was used to stick the ends of the thread together to create the yarn. The yarn was then carefully spun onto a spool and dried in a fume hood before it was stored at 4 °C.

In order to turn the yarn of collagen threads into scaffolds a CNC, computer numeric controlled machine (Sherline Inc. CA), was used to make and position the structure of the scaffolds. The CNC machine used a software (LinuxCNC software, v. 2.6.11) programmed to create two axes to wind the yarn around a mandrel. The two axes created a pore pattern to create the collagen scaffold which were 5 mm in length and 1 mm in width. During the fabrication of the scaffold making process the yarns were fused to each other by applying L-ascorbic solution, followed by a drying period after formation of the complete structure. Overall the dimensions of the rectangular scaffold were approximately 20 x 5 x 0.5 mm.

Collagen scaffolds were crosslinked in genipin solution (2% w/v in 90% ethanol) for 72 h at 37°C. Scaffolds were sterilized prior to further use by treatment for 3 h in peracetic/ethanol solution (1%/22.5% v/v in DI water).

Immunofluorescence

Macrophages were seeded on glass bottom fluorodishes at 5 ×104 cells. They were incubated with three different antibodies: mouse anti-rat CD68, mouse anti-rat NOS2, and mouse anti-rat arginase overnight. The next day the samples were incubated with secondary antibodies: Alexa Fluor 488, Alexa Fluor 555 and Alexa Fluor 595. After being rinsed twice with PBS the samples were placed under fluorescent microscope and an immunofluorescence staining occurred. The images produced were used to see the embedding of the macrophages onto the collagen scaffolds.

Subcutaneous sling implantation

The rats were given standard treatment prior to surgery, such as being kept in a clean environment with food and water. Prior to seeding on the mesh, the rat macrophages will be manipulated/polarized/treated into classically activated (M1) and alternatively activated (M2) macrophages in culture. These conditions have little to do with silence or the expression of genes. The rats were given ketamine/xylazine anesthesia to the point that they were unable to react to toe pinching. Toe pinching was used to record anesthesia every 15 minutes. Observation of respiration and body temperature is used to keep track of the animals.Under K/X anesthesia, a 3 cm long longitudinal midline incision will be made, followed by bilateral subcutaneous dissection to establish a space above the rectus abdominis muscle or the dorsal area. For all rats, 1x1 cm of each mesh material was implanted in the four different positions (upper right, upper left, lower right, lower left). Fixation of the implanted meshes from the sides with non-absorbable monofilament sutures. Skin staples or 4-0 non-absorbable monofilament sutures are used to close the wound.

Bone marrow extraction from donor rats

The rats will be euthanized. Femur and tibia will be harvested immediately after euthanasia for cell extraction.