Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
2000-05-19
2002-05-21
Fredman, Jeffrey (Department: 1655)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C435S069100, C435S173300, C536S024100, C606S077000
Reexamination Certificate
active
06391297
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods and compositions for the differentiation of stromal cells from adipose tissue into osteoblasts, and uses thereof.
BACKGROUND OF THE INVENTION
Osteoporosis is responsible for about 1.5 million fractures each year in the United States, of which about 300,000 are hip fractures. Fifty to 75% of patients with hip fractures are unable to live independently, resulting in increased costs of care. Osteoporosis is characterized by a greater than normal loss of bone density as people age. This disease occurs with a high frequency (>30% of females over age 60) in Western and Asian cultures, and is increasing in prevalence as longevity increases. While the exact cause of these bone repair disorders is unknown, it is clear the dynamic process of bone remodeling is disrupted in a process characterized by a decrease in osteoblastic (bone-producing cells) activity and an increase in osteoclastic (bone degrading cells) activity (Parfitt (1992)
Triangle
31:99-110; Parfitt (1992) In
Bone
, volume 1, B. K. Hall, ed. Teleford Press and CRC Press, Boca Raton, Fla., p. 351-429).
The use of bone grafts is conventional practice in orthopedics, neurosurgery and dentistry, as well as in plastic/reconstruction surgery and this utilization has been growing in frequency over the past two decades. With the exception of blood, bone is the most frequently transplanted tissue with an estimated 500,000 bone grafts used in the US annually. Common orthopedic uses of bone grafts include the management of non-unions and acute long bone fracture, joint reconstruction and to facilitate fusion of vertebral motion segments in treating a variety of spinal disorders (Lane (1987)
Ortho Clin N Amer
18:213-225).
Currently, the most clinically acceptable grafting material is autologous bone. So-called autografts are often obtained from a secondary operative site. There are significant issues associated with autografts. These include lack of an adequate supply for large wounds or defects. Elderly individuals with osteoporosis or osteopenia make the use of an autograft problematic. The secondary morbidity associated with the harvesting operation is high. These complications include infections, pelvic instability (the bone is often harvested from the iliac crest), hematoma, and pelvic fracture (Laurie et al. (1984)
Plas Rec Surg
73:933-938; Summers et al. (1989)
J Bone Joint Surg
71B:677-680; Younger et al. (1989)
J Orthop Trauma.
3:192-195; Kurz et al. (1989)
Spine
14:1324-1331). In addition, chronic pain at the donor site is the second most frequently reported complication (Turner et al. (1992)
JAMA
268:907-911). Finally, the ability to shape the autograft to the defect/wound site is limited due to the rigid nature of the material.
Recent investigations have focused on the use of a variety of matrices, either inorganic such as hydroxyapatite (Flatley et al. (1983)
Clin Orthop Rel Res
179:246-252; Shima et al. (1979)
J Neurosurg
51:533-538; Whitehill et al. (1985)
Spine
10:32-41; Herron, et al. (1989)
Spine
14:496-500; Cook et al. (1986)
Spine
11:305-309; the contents of which are incorporated herein by reference) or organic such as demineralized bone matrix (DBM) (reviewed in Ashay et al. (1995)
Am J Orthop
24:752-761; the contents of which are incorporated herein by reference). These matrices are thought to be osteoconductive (facilitate the invasion of bone forming cells in an inert matrix) or osteoinductive (induce the transformation of recruited precursor cells to osteoblasts). A number of successful clinical outcomes have been observed with some of these products approved for use clinically by the Food and Drug Administration. In spite of these successes, a number of issues remain for the utility of these matrices. The first is the variable subject response to DBM. Also these matrices take much longer than autologous bone transplantation to develop significant structural integrity and bear load effectively.
An alternative to transplantation and the use of simple matrices is the admixture of bone marrow or bone marrow stromal cells with DBM. Ideally the cells and DBM will be derived from the same subject although allogeneic DBM has already been used clinically with initial success (Mulliken et al. (1981)
Ann Surg
194:366-372; Kaban et al. (1982)
J Oral Maxillofac Surg
40:623-626). Transplantation methods using autologous bone marrow cells with allogeneic DBM have yielded good results (Connolly (1995)
Clin Orthop
313:8-18). However, issues that may impact the widespread use of these techniques include potential for contamination by non-self materials, the acceptability of the patient for donating bone marrow, and the potential complications that arise from bone marrow aspirations and depletion of bone marrow from the source.
A number of groups have shown that bone marrow stromal cells and cell lines derived thereof are capable of differentiating into cells biochemically and morphologically similar to osteoblasts (Dorheim et al. (1993)
J Cell Physiol
154:317-328; Grigoriadis et al. (1988)
J Cell Biol
106:2139-2151; Benayahu et al. (1991)
Calcif Tiss Int
. 49:202-207; the contents of which are incorporated by reference). In most cases, fibroblast-like cells were isolated from human or animal bone marrow and plated onto standard tissue cultureware. Generally, a standard media formulation, such as Dulbecco's Modified Eagle's Medium (DMEM) plus fetal calf serum 10-20% and antibiotics is used to select for the enrichment of these cells (Ashton et al. (1980)
Clin Orthop
151:294-307; Sonis et al. (1983)
J Oral Med
3:117-120). Cells were then stimulated to differentiate into osteoblasts by changing the medium to one containing 5-20% fetal calf serum, 2-20 mM &bgr;-glycerophosphate and 20-75 &mgr;M ascorbic acid or ascorbic-2-phosphate (Asahina et al. (1996)
Exp Cell Res
222:38-47; Yamaguchi et al. (1991)
Calcif Tissue Int
49:221-225; the contents of which are incorporated herein by reference). After 14-21 days in culture, many of these cell types and cell lines will mineralized matrices on the cultureware as evidenced by positive von Kossa staining. Other phenotypic indicators of osteoblast lineage include elevated secreted alkaline phosphatase activity; the presence of secreted osteocalcin in the media; and the increased expression of several genes thought to be specifically expressed in osteoblasts, including osteocalcin, osteopontin, and bone sialoprotein (Stein et al. (1990)
FASEB J
4:3111-3123; Dorheim et al. (1993)
J Cell Physiol
154:317-328; Asahina et al. (1996)
Exp Cell Res
222:38-47; Yamaguchi et al. (1991)
Calcif Tissue Int
49:221-225).
There have been a number of detailed studies carried out in several laboratories demonstrating that transplanted bone marrow stromal cells can form ectopic bone (Gundle et al. (1995)
Bone
16:597-603; Haynesworth et al. (1992)
Bone
13:81-89; Boynton et al. (1996)
Bone
18:321-329). For example, human and murine bone marrow stromal fibroblasts have been transplanted into immunodeficient SCID mice (Krebsbach et al. (1997)
Transplantation
63:1059-1069; Kuznetsov et al. (1997)
J Bone Min Res
12:1335-1347). Using antibody and histochemical markers, it was demonstrated that the donor bone marrow stromal cells account for the newly developed osteoblasts at sites of ectopic bone formation in the presence of an inductive matrix. Murine cells formed bone in the presence of hydroxyapatite/tricalcium phosphate particles (HA/TCP), gelatin, poly-L-lysine, and collagen. In contrast, human stromal cells efficiently formed bone only in the presence HA/TCP. No exogenous BMP was required in these studies.
Bone formation is not limited to the skeleton. For example, the introduction of ceramic or demineralized bone matrix into intramuscular, subrenal capsular or subcutaneous sites will result in bone formation if the area is simultaneously expressing bone morphogenetic protein (Urist (1965)
Science
150:893-899). These results suggest that cells present in these tissues have som
Artecel Sciences, Inc.
Bennett-Paris Joseph
Chakrabarti Arun Kr.
Fredman Jeffrey
King & Spalding
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