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Learn tips about Class IV laser therapy and other health related topics on the Companion Therapy Lasers blog!  Check back weekly for updated posts.

As previously discussed, an individual approach in designing treatment frequency will yield optimal results when incorporating photobiomodulation into standard of care. Thus, it is important to remember that we must be flexible in our treatment delivery and remain fluid as the patient responds to medical care.

In past years, the “3-2-1” approach showed good results, but it is clear that taking an individual approach in treatment delivery yields optimal results. With this treatment approach, the patient received three treatments the first week, two the second week, and one final treatment the third week. Although this approach will yield results, it is not an optimal approach as it allows no flexibility in treatment delivery.

Acute conditions should be approached with resolution as our final goal. Examples would include a laceration, an abscess, a muscle sprain, etc. The primary goal, regardless of the condition, is always pain relief, but the biggest value in utilizing photobiomodulation is the fact that tissues are also physically being remodeled to return to function. With resolution as our final goal, the patient is expected to undergo a short course of treatments to reduce the convalescence period.

As with any condition, the patient must be assessed comprehensively and all factors be taken into account so as to set out reasonable expectations. A superficial condition like a laceration would benefit from a single treatment, much like many clinics are performing a single post-op treatment on spays/neuters, etc. as part of their multimodal approach to pain management.

Healed Hot Spots Article ScreenshotSome acute conditions may benefit from a short series of treatment when there is a larger surface area of tissue disruption, such as with hot spots. These will respond nicely when incorporating photobiomodulation daily to every other day for a handful of treatments.

In some cases, conditions may benefit from even more frequent treatments. For example, patients in intractable pain despite standard of care (NSAID, opioids, CRI, etc.) need a more assertive delivery schedule. These patients are so debilitated that they are usually hospitalized, offering the caretaker the opportunity to treat as often as needed. Such cases could include pancreatitis, FLUTD, HGE, snake bite, severe degloving wounds, etc. In this instance, the patient may require multiple treatments daily to address for this pain. Photobiomodulation offers this flexibility to deliver two or even three treatments daily if the patient requires this amount of care. Once an effective clinical response is noted, we can then consider tapering the treatment frequency.

The frequency of treatment remains an area where photobiomodulation offers the operator the flexibility to address the patient’s needs and their conditions as individuals. As such, there is no “cookie cutter” approach to this – it is best addressed as per the patient’s presenting condition and ensuing glide path of response.

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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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