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Bone: Existing or Potential Regenerative Medicine Strategies

Bone: Existing or Potential Regenerative Medicine Strategies

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Bone: Existing or Potential Regenerative Medicine Strategies

Bone is a supporting tough tissue that provides structure to the body and aids movement by cooperating with the muscles. We have over 200 bones in our bodies, forming the skeletal structure and weighing approximately 2 kilograms (kg). Bone is a unique tissue that constantly undergoes remodeling through the period of adult life. Bone defects result from tumor resection, congenital malformations, trauma, fractures, periodontitis, and diseases such as arthritis and osteoporosis. Clinically, healing of fractures is a natural phenomenon in which bone regeneration occurs as part of the healing process. Bone is the only tissue where there is no scar tissue formation in the healing process, and the constant remodeling of the bone is one of the factors that facilitate this regeneration.

Other than the liver, bone is the only organ in the human body that can regenerate. As mentioned above, the ability to regenerate is demonstrated in the human body when a fractured bone uses its potential to regrow and heal itself. Fracture healing takes place in three phases: (1) the reactive phase, in which there is inflammation and granular tissue forms; (2) the reparative phase, in which there is cartilage callus formation and lamellar bone deposition, and (3) the remodeling phase, in which the lamellar bone is remodeled to the original bone contour.

Regeneration of the bone in fractured tissue involves this three-step process. The granular tissue is a small mass of cells that contains fibroblasts and blood vessels. While learning about the different stages in the formation of bone, it is extremely important to understand the terminology. Most of the nomenclature associated with bone either starts or ends with “osteo”—meaning “bone” in Greek. The periosteum is a layer of cells that surrounds the bone. In the fractured tissue, the periosteal cells differentiate into osteoblasts, which are cellular precursors of the cartilage tissue. Once the cartilage is formed on the proximal and distal end of the bone, it grows until it unites to form a single callus tissue. The next phase in this cascade is the mineralization of the extracellular matrix in the callous tissue. Osteoblasts in the callus tissue form the lamellar bone when they come in contact with the mineralized matrix, and this process is called ossification. This stage in bone regeneration is the trabecular bone or the spongy bone. This bone is then reabsorbed by osteoclasts, a class of bone macrophages that reabsorb bone, to create the reabsorption pit. The osteoblast then deposits compact bone into this pit, which closely resembles the original structure.

In some fractures, the healing process is impaired because of delayed union or nonunion of the bones, and 13 percent of fractures do not heal due to this impaired process. Bone regeneration is necessary in situations of skeletal reconstruction in events of trauma and injury. The gold standard until now is autologous or autogenous bone grafting, meaning bone graft from the same individual’s body. The difficulty in this method is the availability of bone tissue to be grafted. To solve this issue, bone from cadavers was resorted to, but this resulted in immunogenic graft rejection. Stem cells and their potential to differentiate into different cell types was an alternate solution to these challenges. The initial solution to this problem was to directly administer BMP—bone morphogenic proteins—to the site of the wound where the stem cell progenitors would use this hormone to differentiate into the bone. Another solution was to directly administer stem cells obtained from the patient to the site of the wound, where the stem cells would use the local systemic cues to differentiate into bone cells that would lead to wound closure. All of these options did not result in successful union of the fracture and resulted in formation of scar tissue. Though this attempt was not as successful, it led to expansion of the knowledge base whereby scientists discovered that the site of the wound needed to be closed off from the surrounding environment to facilitate efficient closure. The current methods include extracting stem cells from the patient’s bone marrow, differentiating them into osteogenic progenitors outside the body, and packaging them appropriately to insert into the body later. The technology used, the potential therapeutic solutions, and the drawbacks of those technologies are discussed below. Several of these therapies have reached the stage of clinical trials in which the therapeutic option is tested on human patients. Some of the options are outlined in the following paragraphs.

Percutaneous Injection

Percutaneous injections are those in which the stem cells derived from the bone marrow of a patient are injected directly at the wound site and the stem cells enhance the healing of the bone by differentiating. A drawback to this method is obtaining a sufficient number of cells that can differentiate to actually heal the bone. To circumvent this technical difficulty, the cells are spun down and the mononuclear cells are isolated. This helps to concentrate the cells before they can be injected. Other methods are to differentiate and populate the cell in vitro in culture.

In addition to bone marrow, pluripotent adipose tissue is also capable of differentiating into cells that are progenitors for bone. The adipose tissue can be derived from the skin layers of the patient. Another popular source is by the liposuction of fat. The stem cells in that aspirate are pluripotent, with differentiating capacities that are useful. Frozen adipose tissue is also a great source of adipose pluripotent stem cells.

Guided Bone Regeneration (GBR)

Guided bone regeneration is defined as the process whereby the wounded region is closed off from the surrounding soft tissue with the use of barrier membranes. This mechanically impedes the growing soft tissue and provides an enclosed socket for the new regenerating tissue. Expanded polytetra-fluorothylene (e-PTFE) is one of the materials that has been used until now to facilitate GBR. Acceptance of an external foreign material and association of the human body with these biomaterials is one of the significant issues. e-PTFE is regularly used in dentistry when implants are drilled into bone, which then heals from the trauma and grows around the membrane. Other options that have been experimented with for this purpose are biodegradable materials such as collagen gels; polyurethane, polyglycolic acid, polyorthoester, and several polymers of polyactic and polygalactic acid were tested. After insertion of the polymer within the tissue, the degradation rate and curve are incumbent on the wound site and cannot be controlled. The rate at which the phagocytes absorb the polymer is a variable that is different every time the therapy is administered. In light of this, e-PTFE was chosen as the most efficient vector for delivery of these stem cells into the site of the wound. The stem cells are a mixture of osteogenic progenitor cells and mitogenic mesenchymal stem cells that are derived from the marrow of the patient. Dentistry is a field that avidly uses GBR for bone regeneration.

GBR was tested first in animal models of bone regeneration of cranofacial skeletal tissue. With e-PTFE and osteogenic progenitor cells, mice were able to complete the union of the bone tissue without the formation of scar tissue. With the success of in vivo models, several clinical trials were approved for testing the GBR technology. In 2009, a clinical trial was approved to test the effect of GBR in the presence of BMP and in the absence of it. Eleven patients were recruited for the trial. They received 34 implants in total at sites exhibiting lateral bone growth. A collagen membrane with xenogenic bone material was used. The use and efficiency of BMP was tested in the process. This is a five-year trial in which the patients were followed for acceptance of the implant and tested to determine if the efficiency of healing was higher in the presence of BMP. It was found that there was no significant difference between the two groups, suggesting that BMP is not essential to GBR.

Scaffolds and Bone Substitutes

Scaffolds and bone substitutes are biomaterials that are similar to bone, which, when inserted at the site of the wound, stimulate regeneration in addition to providing structure for the growing tissue. A significant advantage of this method is that it does not trigger immunogenicity, though it is an invasive process in which the scaffold is inserted surgically. Bone substitutes such as β-TCP, calcium phosphate cement, and glass ceramics are used as bone substitutes because it has been proven that these agents promote cellular proliferation, migration, and differentiation to bone cells. Other non-biological materials used include porous tantalum, which provides structure and a robust substrate for the growth of bone.

The REBORNE project (Regenerating Bone Defects Using New Biomedical Engineering Approaches), funded by the European Commission and coordinated by Inserm, recently gained approval for a clinical trial in which stem cells from patients will be isolated from the bone marrow. These cells will consist of a heterogeneous mixture of mesenchymal stem cells and osteogenic progenitor cells. These cells will be treated with growth hormones in the lab to facilitate the differentiation to bone cells. The cells will then be applied to a scaffold that provides a structure for the progenitor cells to differentiate and proliferate. These scaffolds will be surgically inserted at the wound site and the cells will continue to grow and heal the fracture, resulting in the union of the bone. The trial kicked off in January 2010; it is a five-year trial for which patients with fractures from trauma will be recruited. The effective healing of the wound and union of the fracture will be assessed to ascertain the efficiency of the scaffold.

Clinical Trials

Clinical trials are devised to test the effectiveness of the treatment option that has been developed. The efficiency and robustness of the method is judged based on the healing time, the quality of the regenerated bone, and the efficacy of acceptance of the bone graft/biomaterial in the body. Some of the important considerations are to test patients who have given their informed consent and to determine that the variables that will interfere with the treatment options are constant. Different lengths and periods of time are tested to analyze the best form of therapy administration. When designing clinical trials, it is also important to consider the ethical policies of each country. For example, the policies on the use of human embryos and stem cells derived from them are very different in the United States compared to the European Union (EU). For studies dealing with bone regeneration clinical trials, the period for assessing the immune acceptance of the graft/biomaterial is between six and 12 weeks. The efficacy of the bone regeneration is tested by analyzing the bone density and constitution of the fresh bone. Of course, healing of the fractures is the main goal. Another factor is assessing the formation of scar tissue that might get in the way of function restoration. This is important in dealing with therapies for regeneration of the hip and joints. And, last but not least, infections during the procedure need to be prevented, in addition to targeting a therapy with a reduced pain factor.

Some of the future technologies and patents include use of porous metals that form the basic structure on which the stem cells form the regeneration. The metal provides support and structure to the developing cells.

Sharanya Kumar

Independent Scholar

See Also: Bone: Cell Types Composing the Tissue; Bone: Current Research on Isolation or Production of Therapeutic Cells; Bone: Development and Regeneration Potential; Bone: Major Pathologies; Bone: Stem and Progenitor Cells in Adults; Bone Marrow Transplants.

Further Readings

Dimitriou, R., E. Jones, D. McGonagle, and P. V. Giannoudi. “Bone Regeneration: Current Concepts and Future Directions.” BMC Medicine, v.9/66 (2011).

La, W. G., et al. “Delivery of Bone Morphogenetic Protein-2 and Substance P Using Graphene Oxide for Bone Regeneration.” International Journal of Nanomedicine, v.9/Supp. 1 (2014).