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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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With the growing area of regenerative medicine, it’s no surprise that there are dozens of systems available to process Platelet Rich Plasma (PRP). While the use of each system will result in a plasma product, the processing techniques, equipment and final sample differ quite drastically. In this installment of our Myth Buster series, we will take a closer look at the differences that one can expect to encounter when looking at PRP processing systems and how each of these components effects the PRP product.

Regen-Mythbusters-2


Processing Techniques

There are three main methods by which Platelet Rich Plasma can be processed. These methods are:

1. Centrifugation – This is the most common technique available, which involves using a centrifuge to separate the cell layers in whole blood or to pass the blood components through a gel separator (which is in the device prior to insertion of blood) to isolate the platelets from other cell types.

a. Blood Layer SeparationSpinning without gel separator – When blood is spun at a high rate of speed for a period of time, the cellular components will separate according to their density within the container/tube. These cell layers can be visualized upon removal of the device as seen in the below picture. The three distinct layers of blood include the plasma layer, the platelet buffy coat and the red blood cell layer. The plasma and buffy coat can be collected and injected or the suspension can be placed in a second container for a second spin. The second spin in the process enables further concentration of the platelets, which as we discussed in our previous posts, is important for delivering a therapeutic concentration of platelets (between 3-7-fold concentration compared to circulating blood). A sample that is obtained by a single spin process will have a lower concentration of platelets, thus possibly resulting in a sub-optimal therapy.

b. Spinning with gel separator – Gel separators provide a physical gradient for blood to pass through resulting with the final product at the top of the tubes. Gel separators are typically used for isolating either plasma or serum, but have been developed to allow smaller cell types to remain in the final product. Unfortunately, the majority of products available that use gel separation produce a low concentration of platelets due to the small volume of blood being used (typically 8- 10 mL) as well as the poor capture of platelets in the final product.

2. Flow Cytometry - This method involves a highly specialized piece of equipment that utilizes the absorption of light by a cell and further separates the cells according to the refracting light. This process relies on blood flowing in a single layer suspension through a complex network of tubes, which can be further complicated if the sample begins to clot during the process. Flow cytometry is typically automated, which provides great convenience to the user, but the equipment may require additional set up (prior to processing) and upkeep to ensure proper calibration for processing samples.

3. Gravity and Reverse Osmosis – This method involves utilizing a specially designed blood containment system with a built-in filter. The filter’s purpose is to collect the platelets while allowing the other cells to pass through. Although this method is attractive from a space and equipment perspective, performance of these systems has shown to result in an increase of neutrophils which can be detrimental when injected into a joint.

 

Volume of Blood

Blood compositionOne of the biggest differences between PRP processing systems is the volume of blood that is required to obtain the PRP. Some systems require as low as 8 mL of whole blood, while other systems, such as the CRT system, require between 25-50 mL. What does this variation mean to you? If we look at typical blood composition, as depicted in the picture below, we will see that platelets make up less than 1% of the circulating blood. In order to have a therapeutic dose of platelets in the volume which is necessary for administration, a higher volume of blood is required for processing. For example, when using the CRT system, it is recommended to collect 50 mL of whole blood to produce 4-5 mL of PRP (with concentration of platelets between 3-7 fold). For systems that require the smaller whole blood volume with the same end product volume, the concentration of platelets will be below the recommended therapeutic threshold.

 

Sterility/ Exposure to Environment

When considering any product that will be administered intra-articularly or into areas of poor blood flow, it is imperative to ensure that the processing is done with aseptic technique. It is also important that the exposure of the sample to environmental factors is minimal to reduce the likelihood of contamination. The majority of systems available are considered to be closed systems in which the sample being processed has little to no contact with the outside environment. There are some processing systems available which utilize open tubes or require passing the blood through a needle into the containment device, which may introduce additional contaminants into the sample.

Time to Process- From Collection to Administration

Depending on the technique of isolating the platelets (centrifuge vs. flow cytometry vs. gravity filter), processing times vary greatly from just under 10 minutes to as much as 45 minutes! Some processing systems require activation of the platelets, which can take up to 45 minutes. Activating platelets, however, is not a necessary step, since the platelets will activate and release their stored growth factors once they are exposed to collagen in the joint/tissue. The Companion Regenerative Therapies System takes less than 15 minutes from blood collection to administration, making it a fast, in-house therapy that can be easily scheduled even in a busy hospital.

 

To learn more about Platelet Rich Plasma and how it works as a therapy, watch this short animation:

Stay tuned for our next blog post where we “bust” another regenerative medicine myth!

 

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In the veterinary hospital, a major component revolving around the integration of a new therapy is time. How long is the treatment going to take? How many personnel will need to be scheduled to administer the therapy? How long does the patient need to be under sedation (if needed)? How long does the patient need to be in recovery?

Regen Mythbusters-01

The old saying “time is money” certainly has its place in the veterinary hospital. If a procedure requires several technicians and takes numerous hours to perform for treating one patient, that may not be an efficient use of the hospital’s or technician’s time. In the early years of regenerative medicine, many of the therapies required numerous anesthetic episodes and several hours of both the technician’s and doctor’s time. The procedures required a dog to be anesthetized to collect the sample and then return in a separate hospital visit to be administered the therapy. The collection of the tissue and administration required the doctor to be scheduled for two surgical procedures as well as the attending technician(s). In total, the time to perform this therapy could easily approach 2 hours of doctor and technician time combined, which would then necessitate a higher fee for the single therapy to cover the overhead costs. For many customers, the combination of the numerous office visits and cost of the procedures would be prohibitive, making this therapy less appealing.

Today, these therapies have become extremely efficient only requiring a single office visit with processing and administration taking less than 30 minutes. For Platelet Rich Plasma (PRP) the process takes even less time (typically 15 minutes from start to finish) with most of the procedure being performed by the attending technician(s). The steps for processing PRP are quite simple, making it a therapy that can be easily cross-trained for the supporting staff. The doctor’s time is only required for the administration of the therapy, which takes less than 2-3 minutes (depending on the number of areas being injected). With this minimal time requirement, providing regenerative therapies can easily be scheduled in-between other procedures, exams or surgeries.

To hear a first-hand account of how the Companion Regenerative Therapies System benefited a general practice and their schedule:

Stay tuned for our next blog post where we “bust” another regenerative medicine myth!

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