1Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, 2The Department of Veterans Affairs Medical Center, 3Internal Medicine, Vanderbilt University School of Medicine
Deskins, D. L., Ardestani, S., Young, P. P. The Polyvinyl Alcohol Sponge Model Implantation. J. Vis. Exp. (62), e3885, doi:10.3791/3885 (2012).
Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing studies are most comprehensive when carried out in vivo. There are many in vivo models available to study acute wound healing, including incisional, excisional, dead space, and burns. Dead space models are artificial, porous implants which are used to study tissue formation and the effects of substances on the wound. Some of the commonly used dead space models include polyvinyl alcohol (PVA) sponges, steel wire mesh cylinders, expanded polytetrafluoroethylene (ePTFE) material, and the Cellstick1,2.
Each dead space model has its own limitations based on its material's composition and implantation methods. The steel wire mesh cylinder model has a lag phase of infiltration after implantation and requires a long amount of time before granulation tissue formation begins1. Later stages of wound healing are best analyzed using the ePTFE model1,4. The Cellstick is a cellulose sponge inside a silicon tube model which is typically used for studying human surgery wounds and wound fluid2. The PVA sponge is limited to acute studies because with time it begins to provoke a foreign body response which causes a giant cell reaction in the animal5. Unlike other materials, PVA sponges are easy to insert and remove, made of inert and non-biodegradable materials and yet are soft enough to be sectioned for histological analysis2,5.
In wound healing the PVA sponge is very useful for analyzing granulation tissue formation, collagen deposition, wound fluid composition, and the effects of substances on the healing process1,2,5. In addition to its use in studying a wide array of attributes of wound healing, the PVA sponge has also been used in many other types of studies. It has been utilized to investigate tumor angiogenesis, drug delivery and stem cell survival and engraftment1,2,6,7. With its great alterability, prior extensive use, and reproducible results, the PVA sponge is an ideal model for many studies1,2.
Here, we will describe the preparation, implantation and retrieval of PVA sponge disks (Figure 1) in a mouse model of wound healing.
1. Sponge Preparation
Note: Loading sponges with a treatment as described below is optional.
2. Surgical Procedure
3. Optional Injection
Note: Once the sponges have been implanted into the animal they can be injected with a solution of cells, drugs, growth factors etc. This allows for analysis of the effects of the injected substance during varied delivery times and administration regimes. If injections are done a control should always be used.
4. Sponge Removal
Note: During the sponge removal, handle the sponge with extra caution. Avoid penetrating the sponge with the surgical instruments and prevent excessive bleeding into the sponge by avoiding the major arteries around the surrounding tissue.
5. Representative Results
Removed sponges can be stored under different condition depending on the type of analysis that will be performed. For sectioning, the retrieved sponges can be embedded in a medium for freezing such as Optimal Cutting Temperature (O.C.T.) compound or placed in 10% formalin for embedding and sectioning in paraffin blocks (Figure 3A and B). Best results for sectioning are obtained when the sponge is cut in half across the diameter and embedded cut surface down (Figure 4A and B). Sections of the sponge should be taken so that entire width of the sponge is present. Figure 3A shows two sponges, the left sponge was embedded/sectioned incorrectly and the right sponge was processed correctly.
For analysis, sponges can be placed in an Eppendorf tube and stored at -20 °C until ready for processing. For RNA and quantitative real time PCR analysis sponges should be placed in an Eppendorf tube, flash frozen, and stored at -80 °C. In addition, an RNA preservative, such as RNAlater from QIAGEN, can be used to stabilize the RNA for safe storage at higher temperatures. Wound fluid can be collected by squeezing the sponges over a tube, and pooling together the fluid from multiple sponges to obtain a representative sample. Live cells, such as fibroblast, macrophages and lymphocytes, can also be extracted from the sponges. After removal from the animal, sponges can be place in an appropriate media for the desired type of cells. Sponges can be processed to release the cells by physical (i.e. mincing) or enzymatic (i.e. collagenase) methods.
Figure 1. Dehydrated PVA sponge disks in three different sizes.
Figure 2. Image of mouse wound-healing model showing position of lateral wound and four PVA sponges.
Figure 3. (A) H & E staining at 4x and (B) Trichrome staining at 10x of sectioned paraffin embedded sponges.
Figure 4. (A) Image of sponge cut in half along the diameter. (B) PVA sponge half, embedded cut side down in OCT.
The sponge model of wound healing has multiple variations; it can be performed in many different species including humans and the sponges can also be implanted dorsally 1,2,8. For studies in humans and some other species, sponges may need to be placed in a porous silicone tubing to prevent the sponge from being encapsulated and to allow ease of removal 4,8. The location of the sponge implantation can be altered based on desired results and skill of technician. Dorsal implantation can allow the sponges to move under the skin, while ventral implants are more difficult due to probability of cutting through to the abdominal cavity. The sponges can be set up with a catheter so that drug delivery is constant and controlled, eliminating the need for multiple injections9. Smaller, tubular sponges can even be injected through a needle8. Sponges' composition is not limited to only polyvinyl alcohol. They can be prepared from many materials exclusively or in combination7.
It is crucial for sponge preparation and placement in the animal to be consistent and properly understood. If sponges are cut from sheets they must be of similar weight for comparability. If the sponge is to be loaded with cells or a treatment, the amount placed in each sponge must be exact. The healing process can vary based on the location of the placement of the sponge in the animal and healing will decrease as distance from the head increases 1,10.
We have nothing to disclose.
Funding provided by National Institutes of Health (NIH) grant R01-HL088424; Veterans Affairs merit award to PPY.
|PVA Sponge||Medtronic Inc.||CF120||The size and porosity of the sponge depends on the experiment|
|Scalpel blade size 15||BD Biosciences||371115|
|Metzembaum scissors||Thermo Fisher Scientific, Inc.||79-211|
|Hemostat Forceps||Fine Science Tools||13004-14|
|Needle holder||Fine Science Tools||12004-16|
|5-0 nylon suture||Ethicon Inc.||698|