Research on Isolation or Production of Therapeutic Cells
Bone: Current Research on Isolation or Production of Therapeutic Cells
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Bone: Current Research on Isolation or Production of Therapeutic Cells
Bone is one of the tissue types that undergo constant remodeling during the lifetime of the organism. By virtue of this property, bone is mostly able to regenerate. Healing of fractures is one example of bone regenerating, but regeneration potential is limited, and some autoimmune diseases are associated with erosion of the bone. Traumatic injuries also result in non-union fractures—fractures that do not heal, as there is no union. A fix for such situations is to create a micro-environment in which bone regeneration is more efficient. Bone morphogenic protein or BMP is one of the factors that promote efficient regeneration of the bone. This requires topical application to the site of injury, which then promotes efficient regeneration. Disadvantages of this method include that it does not prevent the invasion of soft tissue and that it promotes random regeneration of tissue surrounding the wound site. In other cases, application of BMP alone is insufficient to promote fracture or bone healing. A bone graft is necessary if the stem cells and other bone cells are present. The bone graft provides an environment of growing bone cells, and these bone grafts help regenerate the tissue to facilitate regeneration. The bone is grafted from other parts of patients’ own bodies. When bone of that nature is unavailable, doctors resort to isolating the graft from cadavers. Issues of immunogenicity or bone rejection are in play when the grafts are isolated from sources other than the patient’s body.
Bone Grafting
Bone grafting is a surgical procedure in which the bone from a healthy site is removed surgically. The bone is then carved in the shape of the wound and prepared to close the wound. This graft is then placed at the wound site and held in place with pins and plates. The bone graft provides a niche of healthy cells growing and regenerating. When the bone graft is accepted by the body, the cells regenerate to close the wound leading to union of fractures.
However, healthy individuals might not have graftable bone, and it is difficult to find a match in cadavers. This significant technical difficulty was overcome with the discovery of stem cells. Stem cells that are pluripotent can differentiate into bone stem cells when treated with appropriate hormones and these cells are differentiated on substrates, such as ceramics and polymers. These ceramics are not immunogenic and are easily integrated in the body but also provide a medium and structure for the bone cells to differentiate and grow outside the body. A synthetic bone graft is then placed in the wound. The stem cells provide a population of growing and differentiating cells that regenerate the bone. In this section, the process of stem cell isolation from the patient body and stem cells propagation in vitro for therapeutic purposes will be explained. Significant milestones in the invention of this technology and current research with clinical applications are also discussed.
Bone Marrow Aspiration
Bone marrow is the soft tissue in the joints of the bones. It contains a reserve of hematopoietic stem cells, mesenchymal stem cells, and osteoprogenitor cells with the potential to differentiate into bone cells. In order to avoid immune rejection, the stem cells are usually isolated from the patient and then later used for therapy. The procedures for isolating human bone marrow cells are standardized. The cells are usually aspirated from the hip joints. The aspirated cells are then passed through a 70 mm filter, which removes accidental bone particles that might have mixed in with the aspirate. Once the cells are isolated, they are spun down to remove the cells from the serum. The stem cells are then plated in petri dishes – which provide a surface for the cells to attach and divide. A medium with the hormones, amino acids, and other essential chemicals, such as metals (magnesium and calcium), that are required for growth is added. The medium nourishes these cells, which then rapidly divide. This proliferation results in the expansion of the population of the cells, which is necessary because a significant amount of dividing cells is required to facilitate regeneration at the wound site. The stem cells derived from the bone are a heterogeneous mixture and contain cells that are not stem cells. The stem cells that are cultured in vitro are then tested for their ability to differentiate into bone cells. Osteogenicity is induced with BMP and other hormones. At day 14, the expression of osteogenic protein markers is tested. The RNA is isolated from these cells and the gene expression profile is tested. It is essential to test the osteogenic potential of the cells before prepping them for therapy.
Current research methodologies have devised effective mechanisms to increase the yield of stem cells, enabling efficient therapies. A significant drawback in the aforementioned method is that it involves centrifugation, which is time consuming and inefficient. This group of researchers has identified a non-woven fabric that is 9 μm apart. The cells obtained from the bone marrow are filtered, and then stem cells are allowed to grow on these biomaterials in the lab. These fabrics with the stem cells growing can then be directly applied at the wound site. This model was tested in vivo in murine models and was found to be effective. This is now waiting for verification in human subjects.
Liposuction
Another significant source of pluripotent stem cells that can differentiate into bone cells are the aspirates of liposuction. The fat of the human body contains pluripotent cells that can be induced to form bone. Human liposuction aspirate is a heterogeneous population of cells that contains a subpopulation of pluripotent cells called processed lipo aspirate (PLA) cells. These cells have the capacity to differentiate to bone. This is a faster and much more efficient mechanism than bone marrow aspirate, which requires a painful and more time-consuming process of extraction. An average of 45 percent of PLA cells have osteogenic potential—the ability to form bone—and this eliminates the necessity for time-consuming processes where the stem cells have to be expanded in labs.
PLA cells are processed in lab before they can be plated to differentiate them into bone. They are washed several times with saline solutions and then treated with collagenase. Collagenase is the enzyme that breaks apart the cells, making them individual. The collagenase reaction is stopped by adding serum, and the cells are then plated. After they attach to the bottom of the petri dish, the cells are treated with BMP or bone morphogenic protein, which induces the formation of bone. Once the cells are differentiated, they are either directly applied to the wound site or are implanted with biomaterials.
Embryonic Stem Cells
Embryonic stem cells are derived directly from the embryo. An embryo is formed from the union of an egg and a sperm. The one cell zygote then undergoes mitosis to develop into the embryo. The 60 cells stage is the blastocyst stage, in which the inner cell mass contains pluripotent stem cells with the capability of developing into the three different germ layers. The cells from the ICM are aspirated to develop in vitro for various research and therapeutic purposes. The embryos that are generated as a surplus in IVF are used to aspirate cells from the blastocyst. The blastocyst phase is two weeks before implantation into the uterus. There are several concerns regarding the use of embryonic stem cells for research purposes. The rules and ethical issues vary from country to country. The European Union and the United States have very different stands on this issue. Thus the use of embryonic stem cells for research depends on the country in which the research is being performed.
The stem cells are also isolated from the genital ridge of the fetus from five to eight weeks. These cells are called embryonic fetal stem cells and are multipotent cells capable of developing into bone cells. Some of the other sources of stem cells are from the femur of the developing fetus. These bone stem cells can be isolated from the fetus at between approximately 9 and 14 weeks, and these cells can be used for treatment in vivo.
