flow cytometry and then cultured to increase the population of stem cells. The cells are grown in cell-culture medium for three weeks, until the cellular population of stem cells are in excess of 10 × 106 cells. After the cells are cultured and their numbers increased, they are suspended in the supernatant of bone marrow, which was separated initially, to prevent contamination of cells or to prevent them from getting mixed with foreign cells. Also, as the supernatant is rich in growth factors, it would aid in the growth of the stem cells. This cellular mixture is then carefully injected at the site of tendon injury. The use of bone marrow mesenchymal stem cells in the treatment of tendon repair was associated with ectopic bone formation in rabbits; the use of embryonic stem cells in tendon repair, however, has resulted in the development of teratoma. The use of tendon stem cells in the repair of tendons has been found to be the most effective and such stem cells have been identified among humans, mice, rabbits, and rats. These tendon stem cells differentiate into tenocytes under normal conditions; however, when these cells are implanted with engineered matrix of tendon, then they result in tendon-like tissues.
Isolation of human tendon stem cells
The midsubstance of patellar tendons is removed from the knee of human donors or harvested from bodies donated to research. The tissue samples are stored in phosphate buffer saline (PBS), after which they are cut into small pieces. The tiny pieces of tissue are treated with type 1 collagenase to digest the tissues, and then strained, using a cell strainer, to produce a suspension of single cells.
Growth and culture of human tendon stem cells
The isolated cells are then added to a six-well culture plate that contains 3 ml Dulbecco’s Modified Eagle Medium 20 percent fetal bovine serum, 10 U/ml penicillin, 100 αg/ml streptomycin, and 2 mM l-glutamine. An atmosphere of 5 percent oxygen is maintained throughout the process of cell culture, as tendons are present in the body in a hypoxic condition, so tendon stem cells grow better in hypoxic conditions rather than at a normal oxygen level.
On the second day of culture, the cells that do not adhere to the walls of the culture flask are removed by adding PBS. After seven to 10 days, the cells in culture are treated with trypsin to remove them from the flask. Reculture is performed by first placing the culture flask with medium under hypoxic conditions for 30 minutes. The cell population is counted on days 1, 2, 6 and 12.
Preparation of Human Tendon Stem Cells for In Vivo Implantation
Human tendon stem cells that are grown in culture have to be prepared before they are introduced into the affected area in the patient. The cells from the second day of culture are added to a 24-well culture dish using a seeding density at 6 × 104 cells per well. A hypoxic condition is maintained at 5 percent oxygen at 37 degrees C. The cells are allowed to grow for one week, after which the cells are removed from culture and mixed with 0.5 ml 5 percent engineered tendon matrix. It has been found that human tendon stem cells that are implanted with engineered tendon matrix are better at developing into tendons at the site of injury.
The stem cells that are inserted into the site of injury show greater differentiation when they are grown in hypoxic conditions, which is indicative that engineered tendon matrix should also be developed in hypoxic conditions.
Allograft or Autograft
When using stem cells to treat tissue injury another important aspect to consider is whether to use tissue from the patient’s own body or from a donor. Cell differentiation potential is slower as age advances and this leads to weakening of tissues, which is one of the main reasons why older patients are at higher risk of tendon, ligament, or cartilage injury. Using stem cells from such patients will not bring about the desired repair to tissue. However, the use of stem cells from donors would mean that there is a risk of transferring genetic diseases that may exist in the donor. When stem cells from mice that were prone to osteoporosis were transferred to mice not prone to osteoporosis, the recipient mice developed the condition, indicating that stem cells can transfer genetic diseases. The use of stem cells in the treatment of cartilage, tendon, or ligament injuries is very important, not only among humans but also among animals that suffer tissue injury.
The Use of Stem Cells to Treat Cartilage, Tendon, and Ligament Injury in Animals
Animals, especially horses, are at a high risk of injuring their ligament or tendons and are, most often, put down or sold, as the cost of treatment is high and, even after treatment, there is no restoration of full function. The use of stem cells to restore function of injured tendons and ligaments will help immensely in veterinary medicine and will aid in the treatment of injured animals. There is considerable interest in the use of stem cells in veterinary medicine and stem cells derived from animals are isolated and cultured for use in treatment.
The injection of stem cells directly into the site of injury may increase the rate of repair, but it requires the injection of specific growth factors to encourage the stem cells to follow required growth and development. This has brought into focus tissue engineering, which is the process of developing functional tissue that can be introduced directly at the injury site. There is an ever-increasing need to create better treatment procedures, and now stem cells are being genetically modified to make them function better.
Genetically Modified Stem Cells for Tissue Injury
Growth factors that are required for the growth and differentiation of stem cells or for the development of tissue-engineered matrix are expensive and need to be injected periodically. To avoid this, stem cells are being genetically modified so that they produce the growth factors on their own and would not require injection of additional growth factors. This type of tissue-engineered matrix can be mass produced and adopted for commercial use.
Amrita Duraiswamy
Sri Ramachandra Medical College
See Also: Cartilage, Tendons, and Ligaments: Cell Types Composing the Tissue; Cartilage, Tendons, and Ligaments: Development and Regeneration Potential; Cartilage, Tendons, and Ligaments: Existing or Potential Regenerative Medicine Strategies; Cartilage, Tendons, and Ligaments: Stem and Progenitor Cells in Adults.
Further Readings
Sugimoto, Y., et al. “Scx+/Sox9+ Progenitors Contribute to the Establishment of the Junction Between Cartilage and Tendon/Ligament.” Development, v.140/11 (2013).
Walsh, William R. Repair and Regeneration of Ligaments, Tendons, and Joint Capsule. New York: Springer-Verlag, 2005.
Zhang, J. and James H. C. Wang. “Human Tendon Stem Cells Better Maintain Their Stemness in Hypoxic Culture Conditions.” PLoS One, v.8/4 (2013).
Cartilage, Tendons, and Ligaments: Development and Regeneration Potential
Cartilage, Tendons, and Ligaments: Development and Regeneration Potential
187
189
Cartilage, Tendons, and Ligaments: Development and Regeneration Potential
Fibrous connective tissues such as tendons and ligaments attach muscles to bones and bones to bones, respectively. This implies a gradual variation in composition, structure, and mechanical
