There generally are two types of bone conditions: 1) non-metabolic bone conditions, such as bone fractures, bone/spinal deformation, osteosarcoma, myeloma, bone dysplasia and scoliosis, and 2) metabolic bone conditions, such as osteoporosis, osteomalacia, rickets, fibrous osteitis, renal bone dystrophy and Paget's disease of bone. Osteoporosis, a metabolic bone condition, is a systemic disease characterized by increased bone fragility and fracturability due to decreased bone mass and change in fine bone tissue structure, its major clinical symptoms including spinal kyphosis, and fractures of dorsolumbar bones, vertebral centra, femoral necks, lower end of radius, ribs, upper end of humerus, and others. In bone tissue, bone formation and destruction due to bone resorption occur constantly. Upon deterioration of the balance between bone formation and bone destruction due to bone resorption, a quantitative reduction in bone occurs. Traditionally, bone resorption suppressors such as estrogens, calcitonin and bisphosphonates have been mainly used to treat osteoporosis.
With respect to bone/spinal conditions, over 75% of the American population suffers from back pain sometime during their life. Underlying medical illnesses can contribute to back pain. These include scoliosis, spinal stenosis, degenerative disc disease, infectious processes, tumors, and trauma. The repair of large segmental defects in diaphyseal bone is a significant problem faced by orthopaedic surgeons today. Although such bone loss may occur as the result of acute injury, these massive defects commonly present secondary to congenital malformations, benign and malignant tumors, osseous infection, and fracture non-union. The use of fresh autologous bone graft material has been viewed as the historical standard of treatment but is associated with substantial morbidity including infection, malformation, pain, and loss of function (Kahn et al., 1995, Clin. Orthop. Rel. Res. 313:69-75). The complications resulting from graft harvest, combined with its limited supply, have inspired the development of alternative strategies for the repair of clinically significant bone defects. The primary approach to this problem has focused on the development of effective bone implant materials.
Three general classes of bone implants have emerged from these investigational efforts, and these classes may be categorized as osteoconductive, osteoinductive, or directly osteogenic. Allograft bone is probably the best known type of osteoconductive implant. Although widely used for many years, the risk of disease transmission, host rejection, and lack of osteoinduction compromise its desirability (Leads, 1988, JAMA 260:2487-2488). Synthetic osteoconductive implants include titanium fibermetals and ceramics composed of hydroxyapatite and/or tricalcium phosphate. The favorably porous nature of these implants facilitate bony ingrowth, but their lack of osteoinductive potential limits their utility. A variety of osteoinductive compounds have also been studied, including demineralized bone matrix, which is known to contain bone morphogenic proteins (BMP). Since the original discovery of BMPs, others have characterized, cloned, expressed, and implanted purified or recombinant BMPs in orthotopic sites for the repair of large bone defects (Gerhart et al., 1993, Clin. Orthop. Rel. Res. 293:317-326; Stevenson et al., 1994, J. Bone Joint Surg. 76:1676-1687; Wozney et al., 1988 Science 242:1528-1534). The success of this approach has hinged on the presence of mesenchymal cells capable of responding to the inductive signal provided by the BMP (Lane et al., 1994, In First International Conference on Bone Morphogenic Proteins). It is these mesenchymal progenitors which undergo osteogenic differentiation and are ultimately responsible for synthesizing new bone at the surgical site.
One alternative to the osteoinductive approach is the implantation of living cells which are directly osteogenic. Since bone marrow has been shown to contain a population of cells which possess osteogenic potential, some have devised experimental therapies based on the implantation of fresh autologous or syngeneic marrow at sites in need of skeletal repair (Grundel et al., 1991, Clin. Orthop. Rel. Res. 266:244-258; Werntz et al., 1996, J. Orthop. Res. 14:85-93; Wolff et al., 1994, J. Orthop. Res. 12:439-446). Though sound in principle, the practicality of obtaining enough bone marrow with the requisite number of osteoprogenitor cells is limiting.
The emerging field of regenerative medicine seeks to combine biomaterials, growth factors, and cells as novel therapeutics to repair damaged tissues and organs. As this specialty grows, there is a demand for a reliable, safe, and effective source of human adult stem cells to serve in tissue engineering applications. For regulatory purposes, these cells must be defined by quantifiable measures of purity. For practical purposes at the clinical level, these cells should be available as an “off the shelf” product immediately available upon demand at the point of care. From a commercial standpoint, the ability to use allogeneic, as opposed to autologous, adult stem cells for transplantation would have a significant positive impact on product development. Under these circumstances, a single lot of cells derived from one donor could be transplanted to multiple mammals, reducing the costs of both quality control and quality assurance.
Studies have demonstrated the existence of adult stem cells in multiple tissue sites. Cells derived from bone marrow, known as mesenchymal stem cells (MSC) or bone marrow stromal cells (BMSC), have been extensively characterized (Castro-Malaspina et al., 1980, Blood 56:289-30125; Piersma et al., 1985, Exp. Hematol 13:237-243; Simmons et al., 1991, Blood 78:55-62; Beresford et al., 1992, J. Cell. Sci. 102:341-3 51; Liesveld et al., 1989, Blood 73:1794-1800; Liesveld et al., Exp. Hematol 19:63-70; Bennett et al., 1991, J. Cell. Sci. 99:131-139). Recent studies have demonstrated that allogeneic bone marrow-derived MSCs can be transplanted (Bartholomew et al., 2002, Exp. Hematol. 30:42-8), and used to repair a critical sized orthopedic defect in a canine model (Arinzeh et al., 2003, J. Bone Joint Surg. Am. 85-A:1927-35). However, MSCs represent approximately 1 out of every 10,000 to 100,000 nucleated bone marrow cells or about 200 cells per ml of bone marrow aspirate (Bruder et al., 2000, Principles of Tissue Engineering, 2nd Edition, Academic Press). In order to obtain MSC numbers sufficient for tissue engineering applications, it is necessary to expand the bone marrow-derived MSCs through multiple passages in vitro.
In contrast to bone marrow, adipose tissue is easily accessible for surgical harvest and abundant in the average adult American. Recently, it has been demonstrated that adipose tissue can serve as a source of stem cells (known as adipose derived adult stem cells or ADAS cells). These cells are capable of differentiating along multiple lineage pathways. In response to specific chemicals, hormones, and/or cytokines, human and rodent ADAS cells express biochemical and histological characteristics consistent with adipose, bone, cartilage, muscle, and neuronal cells. In a recent study, murine ADAS cells accelerated the repair a critical sized calvarial defect (Cowan et al., 2004, Nat. Biotechnol. 22:560-7).
Bone grafting is often used for the treatment of bone conditions. Indeed, more than 1.4 million bone grafting procedures are performed in the world annually. The success or failure of bone grafting is dependent upon a number of factors including the vitality of the site of the graft, the graft processing, and the immunological compatibility of the engrafted tissue. In view of the prevalence of bone conditions, there is a need for novel sources of bone for therapeutic, diagnostic, and research uses. The present invention satisfies this need.