Application of Mesenchymal Stem Cells in the Regeneration of Musculoskeletal Tissues

Edward J. Caterson, BS, Leon J. Nesti, PhD, Todd Albert, MD, Keith Danielson, PhD, Rocky Tuan, PhD, Department of Orthopaedic Surgery, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania

In This Article

Abstract and Introduction

Mesenchymal stem cells are a rare population of undifferentiated cells, isolated from adult tissue sources, that have the capacity to differentiate into mesodermal lineages, including bone, fat, muscle, cartilage, tendon, and marrow stroma. These cell populations may be expanded in culture and subsequently permitted to differentiate into the desired lineage. This directed differentiation may be reached by the application of bioactive molecules, specific growth factors, and signaling molecules. Understanding the functional potential of these cells and the signaling mechanisms underlying their differentiation should lead to innovative protocols for clinical orthopaedic interventions. Clinically applicable techniques to isolate, expand, and reimplant these autogenous cells will become part of the repertoire of orthopaedic therapy. In the presence of extrinsic signaling molecules, provided by both the clinician and the local cellular environment, the intrinsic multipotential nature of the stem cells may be realized for applications such as the replacement of bone graft for segmental defects, nonunions, and spinal fusions. Additional applications may include treatment of full-thickness articular defects and articular resurfacing by site-specific delivery of stem cells. The ultimate goal is directed cellular regeneration of damaged or diseased musculoskeletal tissue. Currently, the limitation is our knowledge and ability to direct this differentiation, but with further study molecular orthopaedic interventions should become a reality. [MedGenMed, MedGenMed, February 5, 2001. © Medscape, Inc.]

As the scientific, practical and ethical debate on embryonic stem cells continues, a growing body of information about a different set of adult progenitor stem cells, often referred to as bone marrow stromal cells or mesenchymal stem cells, is being assembled.[1,2,3] Mesenchymal stem cells have the potential to differentiate into a variety of cell types, including osteoblasts, chondrocytes, adipocytes, myoblasts, fibroblasts, and marrow stroma. A stem cell by definition is a cell type which, in the adult organism, can continue to proliferate in spite of the physiologic or artificial removal of cells from the population.[4] Stem cells, by their normally asymmetric division, are not governed by or limited to a fixed number of cell divisions. Stem cells seem to be under negative control by contact inhibition with other cells or suppressed by negative control regulators from their differentiated progeny.[5,6] Their progeny in turn are affected by a number of factors both intrinsic and extrinsic. As the stem cell moves toward a more differentiated phenotype, it is the interaction between intrinsic genomic potential and extrinsic local signaling which combine at each lineage step in order to complete the developmental pathway of the emerging tissue.[3]

Mesenchymal stem cells have been harvested from marrow, periosteum, and muscle connective tissue.[2,7,8,9,10,11] At present, bone marrow is the most accessible source of mesenchymal stem cells. One of the relevant orthopaedic applications for mesenchymal stem cells is bone induction. Currently, bone grafting is used in fracture treatment, to induce joint fusion, and to fill segmental defects in bone. Autologous cancellous bone is the most effective graft material at present. Bone grafting achieves its purpose by placing mesenchymal stem cells and their differentiated progeny at a repair site. Clinical studies in support of this mode of action have shown that percutaneous injections of 100 to 150 milliliters of nonheparinized bone marrow were successful as adjuvant treatment in tibial nonunions.[12,13] In practice, mesenchymal stem cells are often obtained by aspiration of the anterior superior iliac crest. The age of the patient, the aspiration site, and the systemic disease state all can affect the number of mesenchymal stem cells recovered.[14] The aspirate is drawn into a syringe containing heparinized normal saline. The syringe is detached and then inverted to ensure complete mixing of aspirate and heparin. The cells are isolated from the marrow aspirate by density-gradient centrifugation, followed by further separation by virtue of their differential adhesion characteristics. Mesenchymal stem cells adhere to polystyrene while marrow cells of the hemopoieitic lineage are nonadherent on tissue culture polystyrene. There is approximately 1 mesenchymal stem cell for every 100,000 nucleated marrow cells in a young healthy donor.[15] The adherent colony forming units of mesenchymal lineage exhibit alkaline phosphatase activity using this enzyme marker, and on the basis of the number of nucleated cells in each aspirate, the aspiration volume may be specified according to the goals of mesenchymal stem cell collection.

The periosteum is another repository for mesenchymal stem cells. This cell layer clearly responds to injury by cellular expansion and can form woven bone quickly. The periosteum contains cells capable of differentiating into chondrocytes when the periosteum is transplanted into an articular cartilage defect.[16,17,18] This property indicates that the mesenchymal stem cells in the periosteum, like those in the marrow, are developmentally multipotential and able to form complex and different tissues of the mesodermal lineage.

As conceptual and technical advances continue, the use of autologous stem cell therapy as the starting material for tissue engineering should become a reality. These rare cells, once isolated, may be expanded in number mitotically, and could then be reintroduced back into the same donor, thus eliminating immunorejection. By the addition of extrinsic bioactive factors, acting as cues, mesenchymal stem cell differentiation into specific tissues of mesodermal lineage may be achieved. Additional study of the properties of these cells and understanding the mechanisms governing each step of differentiation will help to define the action and requirements of the extrinsic differentiation cues. Ideally, these extrinsic cues will bolster signaling, leading to selective expression of specific genes to direct the cell through the desired differentiation pathway. In this manner, mesenchymal stem cells can be considered to have obvious orthopaedic value for bone, cartilage, tendon, and ligament regeneration. Mesenchymal stem cells may also be utilized as gene therapy vehicles in genetically based diseases such as osteogenesis imperfecta.[19] In addition, these cells also may have a role in the skeletal maintenance of patients at risk for osteoporosis. Clearly, the applications for mesenchymal stem cells will be expanded as the science and understanding move forward. One problem that must be overcome with transplantation of marrow-derived stem cells is that they may not home to their functionally required site. This is in contrast to hemopoietic progenitor cells, now being used clinically for transplantation. At present, differentiation into the osteoblastic lineage has been the most studied area of mesenchymal stem cell biology.

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