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Welcome back to the Companion Animal Health Regenerative Medicine blog series! In today’s blog, we are going to answer three of the most frequently asked questions regarding Platelet Rich Plasma. From protocols to assessing treatment response, we will provide clarity to some of the questions surrounding this ground-breaking therapy.

Question 1: Is there any proof that Platelet Rich Plasma therapy works?

This is a very common question as there are numerous sources of information regarding the efficacy of PRP treatments. A google search will provide different answers to PRP treatments and applications, though many may not be scientifically backed or peer-reviewed (the gold standard of validating a therapy). The number one place to look for efficacy data is the pubmed.gov website. Performing a search on this website will provide the latest peer-reviewed publications which investigate efficacy (or failure) of different treatments. We have provided several of these published papers for your review, which discuss the successful treatments utilizing Platelet Rich Plasma in numerous indications and may be accessed here.

Question 2: How soon after injecting Platelet Rich Plasma do you typically see results?

It is generally accepted both in human and veterinary medicine that positive effects of this therapy will become apparent within 5-10 days post treatment.

Question 3: How do I know if the patient is responding to treatment?

Stance Analyzer_ROM Measurement 3 Cropped

There are several considerations that need to be made when trying to decipher if a patient is responding to treatment. It is important to have a method for measuring a baseline of the patient and their response to a treatment. Many practitioners using Platelet Rich Plasma therapy will have patients return to the hospital 2 weeks post-treatment and will utilize on of the following methods to measure response.

  • Canine Brief Pain Survey for Pet Owners – Created by the University of Pennsylvania, this survey asks the pet owner to grade their dog’s pain and general habits which may indicate improvement from the treatment. To access this survey (for free), click here.
  • Pain Assessment/Palpation – Palpation is an extremely valuable tool when assessing a dog’s pain and locating the area of injury. Palpation can not only serve as a useful diagnostic, but can also provide feedback post treatment as to if the pain is still present or has reduced from its original state.
    Stance Analyzer_Golden Retriever 4 Cropped
  • Stance or Gait Analysis – Having a quantifiable measurement both pre-and post-treatment will provide you the best data for if a dog is improving from a therapy. Stance and Gait analysis provide objective measurements of the dog’s percent weight bearing on each of its limbs, center of gravity and stride length (gait analysis only).
    • The Stance Analyzer is a cost-effective, compact system which can easily be integrated into a hospitals diagnostic program. Requiring a small foot print for floor space (approximately 2’ by 3’), the Stance Analyzer provides accurate readings of percent weight bearing, center of gravity and weight with a minimal time commitment. Once a reading is obtained, results are easy to interpret and can be saved for each patient to track responses to therapies.
    • A gait analysis system typically consists of a 20-foot walkway mat which provides a detailed analysis of stride, locomotion and other advanced objective readouts. This too is a great system for quantifiable readouts, but requires additional space to perform correctly. These systems are more expensive than the Stance Analyzer and are typically utilized by universities performing research and specialty hospitals. Click here to read more about gait analysis systems.
  • Gait videos – If you don’t have a stance analyzer or gait analysis but would like a visual comparison for pre- and post-treatment effects, you can use video to document your patient’s success! With the advancement of technology for smart phones, there are now applications and settings on the camera to take slow motion videos. These videos can enable you to point out changes in gait, stride length and head bobbing. They can also provide you with before and after videos to then market your regenerative treatments to your clients.

Stay tuned for our next blog where we will answer additional questions focused on Platelet Rich Plasma. If you have a question you would like answered, please contact Companion Animal Health at info@companiontherapies.com.

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As we continue to delve further into Platelet Rich Plasma and how it treats conditions, the most common questions that arise during discussions are:

  1. Do we want white blood cells in our PRP?
  2. If so, which white blood cells do we want to include?

In this post, we will explore the different types of white blood cells, their functions and whether they are beneficial or detrimental in Platelet Rich Plasma.

What are White Blood Cells?

White Blood Cells (WBCs), also known as Leukocytes, make up the majority of the body’s immune system. Their main purpose is to protect the body from foreign substances and various types of infections.

What are the different types of White Blood Cells?

  1. Neutrophils
  2. Monocytes
  3. Lymphocytes
  4. Eosinophils
  5. Basophils

 

Whole Blood Vs PRP_Small

 

What are their functions and are they beneficial when included in Platelet Rich Plasma?

1. Neutrophils

    • Basic Function: Neutrophils are the first responders during an infection or injury. When there is a site of injury, they destroy germ cells through a process called phagocytosis, which in Ancient Greek literally means “to devour”. Neutrophils also kill invading cells through the release of reactive oxygen species and antimicrobial peptides which are toxic to the invading germ cells. 1
    • Role in PRP: Neutrophils have been shown to release damaging molecules which can degrade collagen and other tissues along with extracellular matrix molecules. Neutrophils also release pro-inflammatory cytokines which leads to further damage in the tissue. Therefore, their inclusion in Platelet Rich Plasma, specifically for arthritic applications, is not recommended. 2

2. Monocytes

    • Basic Function: The three main functions of monocytes are: phagocytosis, facilitation of communication between the innate and adaptive immune systems, and cytokine production.3
    • Role in PRP: It has been found that Monocytes are associated with an increase in cellular metabolism and collagen production in fibroblasts. It has also been found that monocytes moderate the release of anti-angiogenic cytokines such as interferon-ɣ and IL-12.4 Therefore their inclusion in Platelet Rich Plasma is considered beneficial.

3. Lymphocytes

    • Basic Function: Lymphocytes are the patrolling cells in the circulatory system that are responsible for recognizing and responding to invading pathogens. They are characterized into three subtypes: T-Cells, B-Cells and Natural Killer Cells (NKCs). T and B cell’s main function are to recognize pathogens and moderate the inflammatory response through the release of cytokines and antibodies. They also are accountable for adaptive immunity, meaning should the same pathogen be encountered again, the body is ready to respond with antibodies. Natural Killer Cells play a major role in defending the body from both tumors and virally infected cells. They are activated when cytokines (called interferons) are released, and produce cytotoxic granules that specifically destroy the altered cells.
    • Role in PRP: Currently the significance of Lymphocytes in Platelet Rich Plasma unknown. It is known that platelets activate Lymphocytes as part of the healing cascade through elevated levels of IL-6, however their benefit or detriment to PRP is still unclear.

4. Eosinophils

    • Basic Function: Eosinophils are most commonly known as a defense mechanism against parasites and are important mediators of allergic reactions.
    • Role in PRP: Eosinophils are known to produce several beneficial growth factors including VEGF, PDGF and TGF-β. These growth factors are associated with angiogenesis, cellular proliferation and extracellular matrix formation.5 Therefore their inclusion in Platelet Rich Plasma may be beneficial in tissue repair.

5. Basophils

    • Basic Function: Basophils are also recognized to play a role in parasite defense and allergic reactions. They release several proteins that increase vascular permeation and regulation of the inflammatory process.
    • Role in PRP: The role of Basophils for PRP are relatively unknown. Basophils are known to secrete pro inflammatory cytokines but little is known if these are beneficial or detrimental to a PRP sample.

What is the bottom line?

With the current knowledge and research surrounding White Blood Cells, it is generally understood that a PRP sample should include an increase in Monocytes and a decrease in Neutrophils. Knowledge regarding the role of Lymphocytes, Eosinophils and Basophils in PRP is still limited, therefore absolute decisions on their inclusion or exclusion cannot be made. But as our understanding of White Blood Cells and their roles in PRP continue to evolve, formulations of PRP in the future may change to treat a specific condition, moving this from a personalized medicine to a precision medicine.

 

Stay tuned for our next blog where we will answer your questions about Platelet Rich Plasma. To submit a question for our next blog, email heatherw@companiontherapy.com.

 

 

References:

  1. Mayadas, T. N., Cullere, X., & Lowell, C. A. (2014). The Multifaceted Functions of Neutrophils. Annual Review of Pathology, 9, 181–218. http://doi.org/10.1146/annurev-pathol-020712-164023
  2. Helen L. Wright, Robert J. Moots, Roger C. Bucknall, Steven W. Edwards; Neutrophil function in inflammation and inflammatory diseases. Rheumatology (Oxford) 2010; 49 (9): 1618-1631. doi: 10.1093/rheumatology/keq045
  3. Nichols, B. A., Bainton, D. F., & Farquhar, M. G. (1971). DIFFERENTIATION OF MONOCYTES : Origin, Nature, and Fate of Their Azurophil Granules. The Journal of Cell Biology, 50(2), 498–515.
  4. Sundman, E.A. et al. (2013). The Anti-inflammatory and Matrix Restorative Mechanisms of Platelet-Rich Plasma in Osteoarthritis. The American Journal of Sports Medicine.Vol 42, Issue 1, pp. 35 – 41
  5. Duarte, J. et al. Platelet-Rich Plasma: Regenerative Medicine: Sports Medicine, Orthopedic, and Recovery of Musculoskeletal Injuries. Springer Science & Business Media, Oct 29, 2013 pp 220

 

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Regen Mythbusters 3

By now we have gained insight into the basics of Regenerative Medicine, such as where PRP and Stem Cells come from and their therapeutic applications for canine patients. It is generally understood that stem cells can be found in any tissue of the body. The two most common sources of stem cells for therapeutic applications are adipose tissue and bone marrow. A common misconception is that adipose derived stem cells provide a superior therapy versus bone marrow aspirate concentrate and that they are easier to collect and process. In this last segment of the Myth Buster Series, we will compare the two sources of stem cells, review their processing differences and determine if there really is a superior choice.

First, let’s consider how Adipose and Bone Marrow Derived Stem Cells are similar:

  • They are both derived from the patient’s own body and are known as autologous adult-derived Mesenchymal stem cells
  • Both can differentiate into cartilage, bone, tendon and ligament cell types.1
  • They can treat certain similar indications with little to no clinical difference.2
  • Both bone marrow and adipose derived stem cells can produce growth factors and anti-inflammatory proteins. These proteins have been shown to contribute to improved healing and reduced inflammation in injured tissues.3
  • Both can be cultured to provide higher cell concentrations.
  • There are commercially available systems to process either sample type.
  • Collection of both Adipose tissue and Bone Marrow require anesthesia.
  • Administration of either Adipose or Bone Marrow derived stem cells is typically done in combination with Platelet Rich Plasma.
  • Fresh samples typically include a heterogenous mixture of several cell types.

Now let’s investigate how Adipose and Bone Marrow Derived Stem Cells are different:

Time frame for processing

  • Fresh Bone Marrow Aspirate Concentrate takes less than 30 minutes from collection to processing and can be done under one anesthetic episode.
  • Fresh Adipose Derived Stem Cells take approximately 3-4 hours to process and require multiple anesthetic or sedative episodes.

Processing requirements

  • Fresh Bone Marrow Aspirate Concentrate involves collecting at least 25 mL of bone marrow from either the femur or humerus. It is then filtered and spun in a processing system for approximately 10 minutes. Once the spin is complete, the plasma is removed and 10% of total volume is collected.
  • Fresh Adipose Derived Stem Cells involve taking approximately 20 grams of adipose under anesthesia, typically harvested from the falciform. The adipose is then mechanically and enzymatically disrupted to separate fat cells, blood cells and the Stromal Vascular Fraction.
    • Stromal Vascular Fraction includes other cell types including white blood cells, fibroblasts, endothelial cells, hematopoietic stem cells and smooth muscle cells.

♦ Concentration of cells

  • In a typical 25 mL collection of bone marrow aspirate, one can expect to have approximately 30,000 stem cells in 3 mL.
  • A typical 20 gram collection of adipose (fresh) will yield 600,000 cells that are considered the Stromal Vascular Fraction.
    • Please Note: There is little evidence in current research that suggests the ideal number of stem cells for treating certain conditions. In one such case, investigated in this peer reviewed paper by B. Carr, et. al., the small number of stem cells found in Bone Marrow Aspirate resulted in similar clinical effects compared to the higher concentration used from Adipose tissue. As our knowledge continues to evolve surrounding indications and specific protocols, these numbers may vary depending on what is being treated.

♦ Open vs. Closed System

  • With Bone Marrow Aspiration, commercially available in-house systems are fully enclosed since the cells are collected via syringe and placed directly into concentrating devices.
  • For Adipose processing, conical tubes are used for adipose digestion and processing, which is classified as an “open system”. This generally means that the samples are exposed to the outside environment which may affect the contents that are being processed. In academic and industry research facilities, it is typically recommended that samples processed utilizing open containers be placed in a fume hood to limit exposure to the surrounding environment.

♦ Equipment Necessary to Process Samples

  • Processing Bone Marrow Concentrate requires a specialized centrifuge designed for concentration of the aspirate. Commercially available kits typically provide syringes, bone marrow collection needles, anticoagulant and concentrating devices.
  • Processing Adipose Tissue requires a specialized centrifuge along with additional equipment including incubator water bath and agitator. Commercially available kits also provide syringes, anticoagulant, enzymes and concentrating devices/ processing tubes.

 

AND THE WINNER IS………………………Well, there really isn’t a winner or a loser in this case. Both Adipose and Bone Marrow Derived Stem Cells can be collected, processed and administered in the same day. Granted, there are differences in the processing times, cell concentrations and collection techniques, however, both therapies provide clinically effective results for similar indications. Deciding which tissue to collect stem cells from is dependent on personal preferences and training. There are numerous educational courses that provide hands-on instruction for either collection technique. If you are interested in learning bone marrow collection and processing, check out one of our upcoming Companion Regenerative University courses. The most important question to ask is “What is my goal for providing this therapy?”. If you answer that question, you may come out with your own Adipose vs. Bone Marrow Derived Stem Cell winner.

Stay tuned for our next blog which will investigate the role of White Blood Cells and their inclusion or exclusion in Platelet Rich Plasma!

 

References:

  1. The comparison of multilineage differentiation of bone marrow and adipose-derived mesenchymal stem cells. Xishan Zhu, Jing Du, Gang Liu. Clin Lab. 2012; 58(9-10): 897–903.
  2. Partial Cranial Cruciate Ligament Tears Treated with Stem Cell and Platelet-Rich Plasma Combination Therapy in 36 Dogs: A Retrospective Study. Canapp S.O. Jr, Leasure C.S., Cox K., Ibrahim V. and Carr B.J. (2016) Front. Vet. Sci. 3:112. doi: 10.3389/fvets.2016.00112
  3. Y.-M. Pers, M. Ruiz, D. Noël, C. Jorgensen, Mesenchymal stem cells for the management of inflammation in osteoarthritis: state of the art and perspectives, Osteoarthritis and Cartilage, Volume 23, Issue 11, November 2015, Pages 2027-2035, ISSN 1063-4584, http://dx.doi.org/10.1016/j.joca.2015.07.004.

 

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