is especially true among athletes, who risk foregoing their sporting career as a result of injury.
The need to provide a more permanent solution that would restore the quality of life and is reliable is a necessity and instrumental in the quest to use stem cells in the treatment of damage to tendons, ligaments, or cartilage. Stem cell therapy has been found to increase the rate of recovery, with a reduction in recurrence and with no recorded side effects. This has resulted in considerable interest in the use of stem cells for treatment, as it can provide affordable treatment methods in both developing and developed nations, reducing the global health equity.
To understand the importance of stem cells in the treatment of damaged tendons, ligaments, or cartilage, it is important to understand the molecular changes that occur in the embryonic stem cells that lead to the formation of these tissues and the molecular pathways that are followed in adult stem cells.
Important Molecular Factors in the Formation of Tendons
During embryonic development, syndetome, which is a separate part of the somite, gives rise to progenitor cells that form tendons, aided by the expression of transcription factor scleraxis (SCX). However, the introduction of SCX alone will not result in the formation of tendons or ligaments under laboratory conditions; other factors are required, like SIX1, SIX2, and proteins called Eya, Eya 1, and Eya 2.
The effects of these molecular factors have been studied on animal tendons after trans-section or in injured human tendons, so their role in development is not fully understood. This area of research is creating a lot of interest, as an injured tendon heals with the growth of scar tissue that contains collagen, but the mechanical properties of the healed tendon are never the same as an intact tendon. There is a need to identify the exact mechanisms involved in the development of tendons so injured tendons can be treated with stem cells to gain complete recovery. During embryogenesis, stem cells develop into specialized cells on exposure to specific growth factors. Scientists attempt to create similar conditions for the growth and development of stem cells under laboratory conditions.
Sources of Stem Cells for the Treatment of Ligament, Tendon, or Cartilage Damage
Bone marrow-derived stem cells for cartilage repair. The bone marrow is an important source of stem cells and is used extensively in the treatment of various conditions. Bone marrow is present in the center of long bones, with the highest concentration of stem cells present in the pelvic bone. Other sources of bone marrow stem cells include the long bones of the arm, leg, and the thigh. The bone marrow stem cells are aspirated using a needle and then concentrated using a centrifuge. Nucleated cells are separated using flow cytometry and 15 percent of this cellular population will consist of stem cells. Platelets are also constituents of this population of cells and, as platelets contain growth factors that aid in the synthesis of collagen matrix, like fibroblast growth factor, insulin-like growth factor-1 and transforming growth factor-B, they are essential to the cellular population. Finally, thrombin is added to the cellular mixture to cleave fibrinogen, which would then give rise to a fibrin scaffold that is essential to hold the stem cells and the growth factors together. This mixture is then injected into the damaged or the injured site. Though the body signals the movement of stem cells from the bone marrow to the injured site on its own, injecting stem cells speed recovery and also increases the concentration of stem cells at the site of injury.
Bone marrow-derived stem cells for tendon repair. Bone marrow stem cells are collected and then centrifuged to obtain the cellular concentrate, while the supernatant is stored for later use. The nucleated cells are isolated using 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