Author: Rishya Gutti
Mentor: Fabiola Munarin
Neuqua Valley High School
Over the course of many years, stem cell therapies have been developed for regenerative medicine, but one of the main challenges remains how they should be delivered. When delivering any treatment method, getting as close to the affected area and using as targeted of a therapy as possible is one of the best ways to garner results. When targeted therapies are used, the drug or therapy becomes increasingly accessible to the tissues that are targeted (Levin, et.al), making the treatment that much more effective since the cells are able to gain the most benefit.
Targeted stem cell delivery offers unique advantages related to the particular nature of stem cells, which function as blank slates, meaning they can take on the form and function as the type of cell they are needed to be (Zakrzewski, et.al). Furthermore, they are able to differentiate into a multitude of cell types, depending on the regenerative signals that are expressed by the surrounding cells or tissues. Stem cell therapies have long been looked into in terms of various tissues, and some examples of these are ocular, cardiovascular, and osseous tissues, to facilitate regrowth or regeneration (Zakrzewski, et.al).
Some of the advantages of the injection procedures compared to open-wound surgery include that these are minimally invasive which leads to a less traumatic treatment which also minimizes the post-procedure complications for the recipient due to there not being large open wounds.
In terms of delivery methods, stem cells have been injected in vehicle media, hydrogels, and in microspheres/microtissues. Injection of stem cells in a vehicle medium is the most direct method, but cells may sustain damage. However, by immobilizing them into a biomaterial, such as a hydrogel or microsphere/microtissue, the stem cells are protected in a more complex environment during the implantation process. (Ashammakhi, et.al)
Injectable Stem Cells
The basis of implantation of stem cells in a minimally invasive manner is injection. This is one of the best ways to reduce the invasiveness to deliver the cells to their target. Stem cells suspensions can be delivered directly, through a syringe and needle. This, however, can pose a threat to the cells themselves as there is nothing but a saline solution protecting them from the pressure induced by the needle during the implantation process and causing damage to the cell walls. If this happens, a portion of the cells delivered may not be viable and therefore decrease the efficacy of the treatment (Ashammakhi, et.al). Notwithstanding, the size of the needle can be altered to prevent this occurrence. An optimal size needle (16-22 gauge) is used so that the stress on the cells is minimized. Large needles, on the other hand, could cause sediment and therefore affect the viability and homogeneity of dispersion of the cells as well (Ashammakhi, et.al).
One application of direct injection is in osteoarthritic patients, who need the treatment to help their deteriorating joints. Freitag, et. al. (2019) performed a clinical trial in which a suspension of stem cells in a saline solution were injected into the knee joints of the experimental groups (n=10 individuals over 18 years old with symptomatic knee osteoarthritis received 1 injection of 100 × 10^6 ADMSCs in saline, n=10 individuals received 2 injections of 100 × 10^6 ADMSCs in saline at t=0 and 6 months). The control group (n=10 individuals over 18 years old with symptomatic knee osteoarthritis) received conservative treatment for osteoarthritis. This paper demonstrated that when ADMSCs were injected, they were largely effective in both pain relief (NPRS improvement of 69% from starting point to 12 mo. in both groups) and functional
improvement of the joint for the patients treated (KOOS scale on 1 and 2 injections with subscores for symptom improvement, activities of daily living, sport and recreation, and quality of life both significantly improved when compared to the control), as quantified with numeric pain rating scale, knee injury and osteoarthritis outcome score, and the western Ontario and McMaster universities osteoarthritis index, and MRI imaging (Freitag et. al).
Unlike direct injection, a hydrogel provides a material for the stem cells to be embedded and protected from the external environment. This method allows to retain the cells in the site of implantation for a longer period of time, compared to the direct injection of stem cells in a buffer medium. In this latter case, the injected cells are often dispersed in the surrounding aqueous milieu. However, the biomaterial component adds a level of complexity to the system, and extensive characterization of the biomaterials in vitro and in vivo is needed to ensure biocompatibility, and adequate degradation kinetics in order to prevent any immune response or rejection of therapeutics.
One of the hallmarks of a hydrogel is that it can mimic a cell’s natural environment, therefore leading to a healthier, more viable sample or stem cells (Ma Y, et.al). By providing this type of environment for the cells, they are able to proliferate and act in the intended method, due to the biophysical cues provided by the hydrogel. Different types of hydrogels can promote different behaviors, such as adhesion, migration, proliferation, or differentiation, depending on the gel’s characteristics, including degradability, shape memory, hydrophilicity/hydrophobicity (Ma J, et.al).
Additionally, hydrogels can be either natural (taken from the extracellular matrix) or synthetic (artificially manufactured with chemical materials). In general, if a very specific chemical composition is needed or a durable and stable gel is required for the tissue engineering application, a synthetic gel might be best, but if a quickly degrading, non-immune-inducing method is needed, a natural gel may work better (Choe, et.al).
Below is a table organizing various applications of minimally invasive stem cell delivery by way of hydrogels.
Injectable 3D Scaffold-Free Microtissues
Continuing on, in this methodology of delivery, the cells are cultured in the lab into a 3D spheroid or piece of microtissue, without the use of a supporting biomaterial. There are numerous benefits to this practice in the context of cell protection and proliferation as well. In terms of cell protection, these spheroids/microtissues help greatly in combating damage caused by the pressure induced in the syringe when delivery occurs (Li, et.al). Additionally, these spheroids and microtissues can simulate an in-vivo environment and can be made into different shapes depending on their final location’s needs. This can lead to them being more responsive and productive. In the trials of Li, et. al, 3D microtissues were implanted into mice in a minimally invasive approach, and were trying to deduce whether when the hMSC (human mesenchymal stem cells) would make an impact on necrosis through a mouse limb ischemia model. The results showed that no limb salvage was observed among the control group, but out of those that received the microtissue treatment (density of 10^5 hMSCs), 75% showed salvage, and only 25% resulted in spontaneous limb necrosis (Li, et.al). Moreover, cell sheet technology, which is a type of scaffold-free microtissue, shows promising results. Many times before, when stem cells were just directly injected, the level of cell loss outweighed the benefit of using this method, but with the cell sheet, this problem of cell retention is no longer a problem (Narita et.al). Additionally, because the cell sheets do not involve a scaffold, they do not elicit an immune response, making it safer for the patient. Finally, the work of Yamada, et.al, shows that adipose derived stem cells (ADSCs) show promising results for bone and cartilage regeneration. Due to their rapid regeneration/differentiation rate, they function well for bone and cartilage repair. In this study, the ADSCs were cultured and made into 3D scaffold-free spheroids. They were then implanted (via injection) into rats that had defects in their calvarial bones (Yamada, et. al). Through this study, it was found that the 3D scaffold-free spheroids had lower rates of cell apoptosis compared to ADSC- single cells in both the in-vitro conditions of the laboratory they were manufactured in and in the in-vivo model (for 12 weeks) of the rat. Thus, it was concluded that injectable ADSC-spheroids are a viable option to minimally invasive stem cell delivery for bone and cartilage regeneration (Yamada, et. al).
All together, there are various aspects to delivery of stem cells. Over the course of this review, three important methods of minimally invasive stem cell delivery have been discussed. There are advantages and disadvantages to each. For example, direct injection uses nothing but a saline solution, meaning that it is the most cost-effective way, but it also does not do much in terms of protecting the cells or helping them proliferate; hydrogels can mimic a stem cell’s ideal environment, but deciding between a natural or synthetic hydrogel may prove to be challenging; spheroids and microtissues are the newest method, and are extremely effective since they seamlessly mimic an in-vivo environment, but they have to be grown in a lab first and may not be the most cost-effective. In summary, depending on the need of the research or patient, different methods can be used, but all aspects should be considered to choose the best one.
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