U.S. patent application number 14/941399 was filed with the patent office on 2016-03-10 for differentiation of adipose stromal cells into osteoblasts and uses thereof.
The applicant listed for this patent is Artecel Sciences Inc.. Invention is credited to Yuan-Di C. Halvorsen.
Application Number | 20160068813 14/941399 |
Document ID | / |
Family ID | 22075320 |
Filed Date | 2016-03-10 |
United States Patent
Application |
20160068813 |
Kind Code |
A1 |
Halvorsen; Yuan-Di C. |
March 10, 2016 |
DIFFERENTIATION OF ADIPOSE STROMAL CELLS INTO OSTEOBLASTS AND USES
THEREOF
Abstract
The invention provides methods and compositions for
differentiating stromal cells from adipose tissue into cells having
osteoblastic properties, and methods for improving a subject's bone
structure. The methods comprise culturing stromal cells from
adipose tissue in .beta.-glycerophosphate and ascorbic acid and/or
ascorbate-2-phosphate for a time sufficient to allow
differentiation of said cells into osteoblasts. Such methods and
compositions are useful in the production of osteoblasts for
autologous transplantation into bone at a surgical site or injury.
The compositions comprise adipose stromal cells, a medium capable
of supporting the growth of fibroblasts and amounts of
.beta.-glycerophosphate and ascorbic acid and/or
ascorbic-2-phosphate sufficient to induce the differentiation of
said stromal cells into osteoblasts. The invention further provides
methods of identifying compounds that affect osteoblast
differentiation. Such compounds are useful in the study of bone
development and in the treatment of bone disorders, including bone
fractures and osteoporosis.
Inventors: |
Halvorsen; Yuan-Di C.;
(Holly Springs, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Artecel Sciences Inc. |
Durham |
NC |
US |
|
|
Family ID: |
22075320 |
Appl. No.: |
14/941399 |
Filed: |
November 13, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11656571 |
Jan 23, 2007 |
9200255 |
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14941399 |
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10061214 |
Mar 14, 2002 |
7179649 |
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11656571 |
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09554868 |
May 19, 2000 |
6391297 |
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PCT/US98/25449 |
Dec 1, 1998 |
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10061214 |
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60067334 |
Dec 2, 1997 |
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Current U.S.
Class: |
424/93.7 ;
435/366; 435/377 |
Current CPC
Class: |
A61F 2310/00383
20130101; A61P 19/08 20180101; C12N 2502/1305 20130101; C12N 5/0654
20130101; A61P 43/00 20180101; C12N 2500/34 20130101; A61K 48/00
20130101; A61F 2002/2835 20130101; A61F 2002/4648 20130101; C12N
2500/42 20130101; A61F 2310/00293 20130101; C12N 2501/155 20130101;
C12N 2506/1384 20130101; C12N 2533/54 20130101; C12N 2510/00
20130101; A61F 2002/2817 20130101; A61P 19/10 20180101; A61F 2/28
20130101; A61F 2310/00365 20130101; C12N 2533/14 20130101; A61K
35/12 20130101; A61F 2002/30677 20130101; C12N 2506/13 20130101;
C12N 2500/38 20130101; A61K 38/00 20130101; A61K 35/32
20130101 |
International
Class: |
C12N 5/077 20060101
C12N005/077; A61K 35/32 20060101 A61K035/32 |
Claims
1. A method of differentiating adipose stromal cells into
osteoblasts, comprising: culturing said cells in a composition
comprising a medium capable of supporting the growth of fibroblasts
and differentiation inducing amounts of .beta.-glycerophosphate and
ascorbic acid and/or ascorbic-2-phosphate.
2. The method of claim 1, wherein said amounts are about 2-20 mM
.beta.-glycerophosphate and about 20-75 .mu.M ascorbic acid and/or
ascorbic-2-phosphate.
3. The method of claim 2 wherein said amounts are about 5-15 mM
.beta.-glycerophosphate and about 40-60 .mu.M ascorbic acid and/or
ascorbic-2-phosphate.
4. The method of claim 3, wherein said amounts are about 10 mM
.beta.-glycerophosphate and about 50 .mu.M ascorbic acid and/or
ascorbic-2-phosphate.
5. The method of claim 1, wherein said medium is selected from the
group consisting of: DMEM, aMEM and BME.
6. The method of claim 1, wherein said medium further comprises
about 5-20% fetal calf serum.
7. The method of claim 1, wherein said medium further comprises one
or more bone morphogenetic proteins.
8. The method of claim 1 wherein said cells are mammalian.
9. The method of claim 8 wherein said cells are human.
10. A method of improving a subject's bone structure, comprising:
a. culturing adipose stromal cells in a composition which comprises
a medium capable of supporting the growth of fibroblasts and
differentiation inducing amounts of .beta.-glycerophosphate and
ascorbic acid and/or ascorbic-2-phosphate; and b. introducing said
osteoblasts into a surgery or fracture site of said subject.
11. The method of claim 10, wherein said amounts are about 2-20 mM
.beta.-glycerophosphate and about 20-75 .mu.M ascorbic acid and/or
ascorbic-2-phosphate.
12. The method of claim 11, wherein said amounts are about 5-15 mM
.beta.-glycerophosphate and about 40-60 .mu.M ascorbic acid and/or
ascorbic-2-phosphate.
13. The method of claim 12, wherein said amounts are about 10 mM
.beta.-glycerophosphate and about 50 .mu.M ascorbic acid and/or
ascorbic-2-phosphate.
14. The method of claim 10, wherein said adipose stromal cells are
isolated from said subject.
15. The method of claim 10, wherein said medium is selected from
the group consisting of: DMEM, aMEM and BME.
16. The method of claim 10, wherein said medium further comprises
about 5-20% fetal calf serum.
17. The method of claim 10, wherein said medium further comprises
one or more bone morphogenetic proteins.
18. The method of claim 10, wherein said subject is mammalian.
19. The method of claim 18, wherein said subject is human.
20. The method of claim 10, wherein said osteoblasts are introduced
in admixture with a composition useful in the repair of bone
wounds, bone defects and/or bone disorders.
21. The method of claim 10, wherein a nucleotide sequence of
interest is introduced into said adipose stromal cells or said
osteoblasts.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 11/656,571 filed Jan. 23, 2007, now issued as
U.S. Pat. 9,200,255; which is a continuation application of U.S.
application Ser. No. 10/061,214 filed Mar. 14, 2002, now issued as
U.S. Pat. No. 7,179,649; which is a continuation application of
U.S. application Ser. No. 09/554,868 filed May 19, 2000, now issued
as U.S. Pat. No. 6,391,297; which is a 35 USC .sctn.371 National
Stage application of International Application No. PCT/US98/25449
filed Dec. 1, 1998, now expired; which claims the benefit under 35
USC .sctn.119(e) to U.S. Application Ser. No. 60/067,334 filed Dec.
2, 1997, now expired. The disclosure of each of the prior
applications is considered part of and is incorporated by reference
in the disclosure of this application.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to methods and compositions for the
differentiation of stromal cells from adipose tissue into
osteoblasts, and uses thereof.
[0004] 2. Background Information
[0005] 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).
[0006] 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
75:213-225).
[0007] 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 775:677-680; 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.
[0008] Recent investigations have focused on the use of a variety
of matrices, either inorganic such as hydroxyapatite (Flatle 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 74:496-500; 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.
[0009] 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 373:8-18). 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.
[0010] 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 754:317-328; Grigoriadis et
al. (1988) J Cell Riot 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 757:294-307; 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 .beta.-glycerophosphate
and 20-75 .mu.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 74:31 11-3123; Dorheim et al. (1993) J Cell
Physiol 754:317-328; Asahina et al. (1996) Exp Cell Res 222:38-47;
Yamaguchi et al. (1991) Calcif Tissue Int 49:221-225).
[0011] 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 72:1335-1347). 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.
[0012] 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 some
capability of forming bone precursor cells under the proper
environmental conditions.
[0013] Ectopic bone formation in soft tissue such as fat is a rare
pathologic condition observed in patients with fibrosis ossificans
progressiva, an inherited disease. While the etiology of the
disease is not completely understood, it arises in part from the
abnormal expression of BMP by lymphocytes localized to sites of
soft tissue injury (Kaplan et al. (1997) J Bone Min Res 12:855;
Shafritz et al. (1996) N Engl J Med 535:555-561). Bone formation is
also observed on rare occasions in lipomas (Katzer (1989) Path Res
Pract 184:437-443).
[0014] The stromal-vascular fraction isolated from adipose tissue
after collagenase treatment has been demonstrated to contain a
large quantity of preadipocytes, or cells that are predisposed to
differentiate into adipocytes (Hauner et al. (1989) J Clin Invest
34:1663-1670). These cells can spontaneously differentiate into
adipocytes at relatively low frequency or respond to adipogenic
agonists such as thiazolidinediones to a much higher frequency of
differentiation (Halvorsen (1997) Strategies 11:58-60; Digby (1997)
Diabetes 4:138-141). There is evidence to suggest that stromal
cells exhibit a reciprocal pattern of differentiation between these
lineages (Gimble et al. (1996) Bone 19:421-428; Bennett et al.
(1991) J Cell Sci 99:131-139; Beresford et al. (1992) J Cell Sci
102:341-351). Specifically, adipogenesis is accompanied by reduced
osteoblastic potential while osteogenesis is accompanied by reduced
adipogenic potential.
[0015] Under certain conditions, bone marrow stromal cells can be
differentiated into adipocytes. In fact, several bone stromal cell
lines have been extensively characterized with respect to this
ability (Gimble et al. (1990) Eur J Immunol 20:379-387; Gimble et
al. (1992) J Cell Biochem 50:73-82; Gimble et al. (1996) Bone
79:421-428). However, prior to the instant invention, it was not
known that stromal cells isolated from adipose tissue could be made
to differentiate into osteoblasts.
SUMMARY OF THE INVENTION
[0016] The invention provides methods and compositions for
differentiating stromal cells from adipose tissue into cells having
osteoblastic properties, and methods for improving a subject's bone
structure. The methods comprise culturing stromal cells from
adipose tissue in .beta.-glycerophosphate and ascorbic acid and/or
ascorbate-2-phosphate for a time sufficient to allow
differentiation of said cells into osteoblasts. Such methods and
compositions are useful in the production of osteoblasts for
autologous transplantation into bone at a surgical site or injury.
The compositions comprise adipose stromal cells, a medium capable
of supporting the growth of fibroblasts and differentiation
inducing amounts of .beta.-glycerophosphate and ascorbic acid
and/or ascorbic-2-phosphate.
[0017] The invention further provides methods of identifying
compounds that affect osteoblast differentiation. Such compounds
are useful in the study of bone development and in the treatment of
bone disorders, including bone fractures and osteoporosis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows the morphological changes that occur in human
adipose stromal cells induced to differentiate into osteoblasts by
treatment with osteoblast differentiation medium.
[0019] FIGS. 2A, 2B and 2C show adipose stromal cells that have
been treated with osteoblast differentiation medium or adipocyte
differentiation medium and stained with Oil Red O (FIGS. 2A and 2C)
or by the von Kossa method (FIGS. 2B and 2C).
[0020] FIG. 3 shows the time course of concentrations of
osteocalcin and leptin secreted into the media upon treatment of
adipose stromal cells with stromal cell medium (PA), adipocyte
medium (AD) or osteoblast medium (OST).
[0021] FIG. 4 shows the concentrations of alkaline phosphatase
secreted by adipose stromal cells cultured for twenty-one days in
stromal cell medium (SC), adipocyte differentiation medium (AD) or
osteoblast differentiation medium (OST).
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention provides methods for differentiating
adipose stromal cells into osteoblasts. The osteoblasts produced by
the methods of the invention are useful in providing a source
osteoblasts for research or transplantation into a subject's bone,
at the site of surgery or fracture. Thus, in one aspect, the
invention provides a method of differentiating adipose stromal
cells into osteoblasts, comprising: culturing said cells in a
composition which comprises a medium capable of supporting the
growth of fibroblasts and differentiation inducing amounts of
.beta.-glycerophosphate and ascorbic acid and/or
ascorbic-2-phosphate.
[0023] In another aspect, the invention provides compositions for
the differentiation of adipose stromal cells into osteoblasts. Such
compositions comprise: adipose stromal cells, a medium capable of
supporting the growth of fibroblasts and amounts of
.beta.-glycerophosphate and ascorbic acid and/or
ascorbic-2-phosphate sufficient to induce the differentiation of
said stromal cells into osteoblasts.
[0024] "Adipose stromal cells" refers to stromal cells that
originate from adipose tissue. By "adipose" is meant any fat
tissue. The adipose tissue may be brown or white adipose tissue.
Preferably, the adipose is subcutaneous white adipose tissue. Such
cells may comprise a primary cell culture or an immortalized cell
line. The adipose tissue may be from any organism having fat
tissue. Preferably the adipose tissue is mammalian, most preferably
the adipose tissue is human. A convenient source of human adipose
tissue is from liposuction surgery, however, the source of adipose
tissue or the method of isolation of adipose tissue is not critical
to the invention. If osteoblasts are desired for autologous
transplantation into a subject, the adipose tissue will be isolated
from that subject.
[0025] "Differentiation inducing amounts of .beta.-glycerophosphate
and ascorbic acid and/or ascorbic-2-phosphate" refers to
concentrations of .beta.-glycerophosphate and (ascorbic acid and/or
ascorbic-2-phosphate), that when supplied in a medium capable of
supporting the growth of fibroblasts (e.g., NIH-3T3 cells, human
adipose stromal cells and the like), will induce the
differentiation of said stromal cells into osteoblasts over a time
period of about five days to eight weeks. Optimal concentrations
and lengths of treatment may be determined by the practitioner
through the use of known assays for differentiated osteoblasts.
Such assays include, but are not limited to those that assess
morphological or biochemical characteristics (e.g., secreted
osteocalcin or other osteoblast-specific proteins or RNA).
[0026] The concentration of ascorbic acid and/or
ascorbic-2-phosphate refers to any combined concentration of these
compounds that total the stated concentration. For example, the
definition of "50 .mu.M ascorbic acid and/or ascorbic-2-phosphate"
includes, but is not limited to, such permutations as: 50 .mu.M
ascorbic acid; 50 .mu.M ascorbic-2-phosphate; 10 .mu.M ascorbic
acid and 40 .mu.M ascorbic-2-phosphate; or 40 .mu.M ascorbic acid
and 10 .mu.M ascorbic-2-phosphate.
[0027] Preferably the medium contains about 2-20 mM
.beta.-glycerophosphate and about 20-75 .mu.M ascorbic acid and/or
ascorbic-2-phosphate. More preferably, the medium contains about
5-15 mM .beta.-glycerophosphate and about 40-60 mM ascorbic acid
and/or ascorbic-2-phosphate. Most preferably, the medium contains
about 10 mM .beta.-glycerophosphate and about 50 .mu.M ascorbic
acid and/or ascorbic-2-phosphate.
[0028] Any medium capable a supporting fibroblasts in cell culture
may be used. Media formulations that will support the growth of
fibroblasts include, but are not limited to, Dulbecco's Modified
Eagle's Medium (DMEM), alpha modified Minimum Essential Medium
(aMEM), and Basal Medium Essential (BME) and the like. Typically,
5-20% Fetal Calf Serum (FCS) will be added to the above media in
order to support the growth of fibroblasts. However, a defined
medium could be used if the factors in FCS necessary for fibroblast
growth were identified and provided in the growth medium.
[0029] Media useful in the methods of the invention may contain one
or more compounds of interest, including, but not limited to,
antibiotics, compounds that are osteoinductive, osteoconductive, or
promote growth or differentiation, such as bone morphogenetic
proteins or other growth factors. Examples of bone morphogenetic
proteins include, but are not limited to, osteogenic protein-1,
BMP-5, osteogenin, osteoinductive factor and bone morphogenetic
protein-4 (Asahina et al. (1996) Exp Cell Res 222:38-47; Takuwa
(1991) Biochem Biophys Res Com 774:96-101; Chen (1991) J Bone Min
Res 6:1387-1390; Sampath (1992) J Biol Chem 267:20352-20362; Wozney
et al. (1988) Science 242:1528-1534, the contents of which are
incorporated herein by reference), and the like.
[0030] Preferably, the adipose tissue is treated so that the
stromal cells are dissociated from each other and from other cell
types, and precipitated blood components are removed. Typically,
dissociation into single viable cells may be achieved by treating
adipose tissue with proteolytic enzymes, such as collagenase and/or
trypsin, and with agents that chelate Ca.sup.2+. Stromal cells may
then be partially or completely purified by a variety of means
known to those skilled in the art, such as differential
centrifugation, fluorescence-activated cell sorting, affinity
chromatography, and the like. The partially or completely isolated
stromal cells may then be cultured in a media that will support the
growth of fibroblasts for a period of between eight hours to up to
five cell passages prior to treatment with media containing
13-glycerophosphate and ascorbic acid and/or
ascorbic-2-phosphate.
[0031] The stromal cells will be cultured in media containing
.beta.-glycerophosphate and ascorbic acid and/or
ascorbic-2-phosphate for a time sufficient to induce
differentiation into osteoblasts. The length of treatment with
.beta.-glycerophosphate and ascorbic acid and/or
ascorbic-2-phosphate required for differentiation of stromal cells
into osteoblasts is dependent upon a number of factors. Such
factors include, but are not limited to, the concentrations of
.beta.-glycerophosphate and ascorbic acid and/or
ascorbic-2-phosphate used, the medium used, the source of adipose
tissue or stromal cells, the initial density of plating, the
presence or absence of growth factors or bone morphogenetic
proteins and the like. The concentration of .beta.-glycerophosphate
and ascorbic acid or ascorbic-2-phosphate and other conditions and
factors may be optimized by the practitioner. Optimal
concentrations and treatment times may be determined by measuring
the percentage of cells that have differentiated into osteoblasts.
This percentage may be monitored by morphological and biochemical
assays and indices known to those skilled in the art. Such assays
and indices include, but are not limited to, those that assess
morphological or biochemical characteristics, such as the presence
of calcium deposits or osteoblast-specific proteins or RNAs, von
Kossa staining, osteocalcin secretion and alkaline phosphatase
secretion.
[0032] Osteoblasts derived from adipose tissue stromal cells may be
introduced into the bone of a human or animal subject at the site
of surgery or fracture. Introduction of osteoblasts to bone is
useful in the treatment of bone fractures and bone disorders,
including osteoporosis. Thus, in another aspect, the invention is
directed to a method of improving a subject's bone structure,
comprising: [0033] a) culturing stromal cells from adipose tissue
in a composition which comprises a medium capable of supporting the
growth of fibroblasts and differentiation inducing amounts of
.beta.-glycerophosphate and ascorbic acid and/or
ascorbic-2-phosphate; and [0034] b) introducing said osteoblasts
into a surgery or fracture site of said subject.
[0035] Preferably, the stromal cells are isolated from the adipose
tissue of the subject into which the differentiated osteoblasts are
to be introduced. However, the stromal cells may also be isolated
from an organism of the same or different species as the subject.
The subject may be any organism having bone tissue. Preferably the
subject is mammalian, most preferably the subject is human.
[0036] The stromal cells or osteoblasts may be stably or
transiently transformed with a nucleic acid of interest prior to
introduction into a surgery or fracture site of the subject.
Nucleic acid sequences of interest include, but are not limited to
those encoding gene products that enhance the growth,
differentiation and/ or mineralization of osteoblasts. For example,
an expression system for bone morphogenetic protein 4, can be
introduced into the preadipocytes in a stable or transient fashion
for the purpose of treating non-healing fractures or osteoporosis.
Methods of transformation of stromal cells and osteoblasts are
known to those skilled in the art, as are methods for introducing
osteoblasts into a bone at the site of surgery or fracture.
[0037] The osteoblasts may be introduced alone or in admixture with
a composition useful in the repair of bone wounds and defects. Such
compositions include, but are not limited to bone morphogenetic
proteins, hydroxyapatite/tricalcium phosphate particles (HA/TCP),
gelatin, poly-L-lysine, and collagen. For example, osteoblasts
differentiated from adipose stromal cells may be combined with DBM
or other matrices to make the composite osteogenic (bone forming in
its own right) as well as osteoinductive. Similar methods using
autologous bone marrow cells with allogeneic DBM have yielded good
results (Connolly (1995) Clin Orthop 373:8-18).
[0038] A further object of the invention is to provide methods for
the identification and study of compounds that enhance the
differentiation of stromal cells into osteoblasts. Compounds that
enhance the differentiation of osteoblasts may play a role in the
treatment of various bone disorders, including fractures and
osteoporosis. In addition, compounds found to induce osteoblast
differentiation are useful in for differentiating cells in vitro or
in vivo. Conversely, compounds or factors found to block osteoblast
differentiation may be useful in certain disease states where
idiosyncratic bone production, such as Paget's disease or
chondroosteoblastic metaplasia, may be treated by such compounds.
Thus, in another aspect, the invention is directed to a method of
identifying compounds that affect osteoblast differentiation,
comprising: [0039] a) culturing adipose stromal cells in the
presence and absence of a compound to be tested for effect on
osteoblast differentiation in a composition which comprises a
medium capable of supporting the growth of fibroblasts and
differentiation inducing amounts of .beta.-glycerophosphate and
ascorbic acid and/or ascorbic-2-phosphate; and [0040] b) comparing
osteoblast differentiation in said cells cultured in the presence
of said compound to that of said cells cultured in the absence of
said compound.
[0041] Any compound may be tested for its ability to affect the
differentiation of stromal cells into osteoblasts. Appropriate
vehicles compatible with the compound to be tested are known to
those skilled in the art and may be found in the current edition of
Remington's Pharmaceutical Sciences, the contents of which are
incorporated herein by reference.
[0042] The results of the tests can be compared with results using
known differentiation promoting agents, such as osteogenic protein
1 and bone morphogenetic protein-4 (Asahina et al. (1996) Exp Cell
Res 222:38-47; Takawa (1991) supra; Chen (1991) supra; Sampath
(1992) supra; Wozney et al. (1988) Science 242:1528-1534), which
are known to promote differentiation of osteoblasts by increasing
the expression of osteoblast markers such as osteocalcin, a
definitive marker of osteoblast function (Celeste (1986) Proc Natl
Acad Sci 87:9843-9872; Stein et al. (1990) FASEB J 4:3111-3123).
Also, the results of such tests may be compared to known osteoblast
differentiation inhibitors such as TNF-alpha, which results in
complete or partial blocking of the conversion of stromal cells
into osteoblasts.
[0043] The features and advantages of the present invention will be
more clearly understood by reference to the following examples,
which are not to be construed as limiting the invention.
EXAMPLES
Example 1
Isolation of Stromal from Human Adipose Tissue
[0044] Human stromal cells were isolated from adipose tissue
according to the procedures described by Rodbell (1964) J Biol Chem
239:375 and Hauner et al. (1989) J Clin Invest 84:1663-1670.
Briefly, human adipose tissue from subcutaneous depots was removed
by liposuction surgery. The adipose tissue was then transferred
from the liposuction cup into a 500 ml sterile beaker and allowed
to settle for about 10 minutes. Precipitated blood was removed by
suction. A 125 ml volume (or less) of the tissue was transferred to
a 250 ml centrifuge tube, and the tube was then filled with
Krebs-Ringer Buffer. The tissue and buffer were allowed to settle
for about three minutes or until a clear separation was achieved,
and then the buffer was removed by aspiration. The tissue was
washed with Krebs-Ringer Buffer an additional four to five times or
until it was orange-yellow in color and the buffer was light tan in
color.
[0045] The cells of the adipose tissue were dissociated by
collagenase treatment. Briefly, the buffer was removed from the
tissue and replaced with a 2 mg collagenase/ml Krebs Buffer
(Worthington, Mass., USA, type I) solution at a ratio of 1 ml
collagenase solution/ml tissue. The tubes were incubated in a
37.degree. C. water bath with intermittent shaking for 30 to 35
minutes.
[0046] Stromal cells were isolated from other components of the
adipose tissue by centrifugation for 5 minutes at 500.times.g at
room temperature. The oil and adipocyte layer was removed by
aspiration. The remaining stromal-vascular fraction was resuspended
in approximately 100 ml of phosphate buffered saline (PBS) by
vigorous swirling, divided into 50 ml tubes and centrifuged for
five minutes at 500.times.g. The buffer was carefully removed by
aspiration, leaving the stromal cells. The stromal cells were then
resuspended in stromal cell medium (DMEM (Morton (1970) In Vitro
6:89-108; Dulbecco (1959) Virology 5:396)/Ham's F-10 medium (Ham
(1963) Exp Cell Res 29:515) (1:1, v/v); 10% (v/v) fetal calf serum;
15 mM HEPES, pH 7.4; 60 U/ml penicillin; 60 U/ml streptomycin; 15
.mu.g/ml amphotericin B), plated at an appropriate cell density and
incubated at 37.degree. C. in 5% CO.sub.2 overnight. Once attached
to the tissue culture dish or flask, the cultured stromal cells may
be used immediately or maintained in culture for up to 5 passages
before being induced to differentiate into osteoblasts as described
in Example 2 below.
Example 2
Differentiation of Extramedullary Adipose Stromal Cells into
Osteoblasts
[0047] Adipose stromal cells were isolated as described in Example
1 and then treated as follows to induce differentiation into
osteoblasts. Stromal cells were plated in 24-well and/or 6-well
tissue culture plates in stromal cell medium (see above) at a
density of about 22,000 cells/cm.sup.2. After 24 hours, the stromal
cell medium was replaced with osteoblast differentiation medium
(DMEM with 10% fetal bovine serum (v/v); 10 mM
.beta.-glycerophosphate; 50 .mu.g/ml ascorbate-2-phosphate; 60 U/ml
penicillin; 60 U/ml streptomycin; 15 .mu.g/ml amphotericin B). The
osteoblast differentiation medium was replaced with fresh medium
every 3 days for 3 weeks. When changing the media, one ml of
conditioned media was collected and stored at -80.degree. C. for
later analysis of secreted factors. Alternatively, stromal cells
isolated from adipose tissue were induced to differentiate into
adipocytes according to the method of Hauner et al. (1989) J Clin
Invest 34:1663-1670) by treatment with adipocyte differentiation
medium.
[0048] Microscopic examination of cells treated with osteoblast
medium as described above revealed morphological changes consistent
with the appearance of osteoblasts (FIG. 1). After prolonged
culture (21-28 days), several multi-cellular nodules formed in each
culture well. The grainy appearance of the osteoblast culture,
indicates the presence of calcium phosphate deposits and pre-bone
structures. Because stromal cells isolated from adipose tissue also
have the potential to differentiate into adipocytes when treated
with an adipocyte differentiation medium, the cells treated with
osteoblast medium were also examined for the presence of
adipocytes. No obvious adipocytes in the cultures treated with
osteoblast medium, as indicated by the lack of oil droplets
appearing in the cytoplasm and lack of cells having the
characteristic rounded adipocyte morphology.
[0049] Cells treated with the osteoblast differentiation medium
were stained by the von Kossa method to determine whether the
stromal cells had differentiated into osteoblasts. Briefly, fetal
calf serum was serially diluted out of the medium by exchanging 80%
of the medium several times with serum-free medium. The cells ware
fixed in 5% formaldehyde and then washed several times with PBS to
remove any remaining serum. The fixed cells were incubated in 100%
ethanol at 4.degree. C. for about 10 minutes. The ethanol was then
removed and the fixed cells were incubated in 0.5 ml 5% silver
nitrate for 10 minutes under UV light at 254 nm. The cells were
then rinsed 2-3 times in distilled water, incubated in 5% sodium
thiosulfate for 5 minutes and then rinsed with water. The stained
cells can be stored in 50% glycerol indefinitely. The results of
von Kossa staining are shown in FIGS. 2B and 2C. Only cells
receiving osteoblast medium stained positive and turned dark.
[0050] Oil Red O staining was performed as follows. Plates were
rinsed with phosphate buffered saline several times to remove serum
or bovine serum albumin in the culture medium. The cells were then
fixed in methanol or 10% formaldehyde in phosphate buffered saline
for 15 minutes to 24 hours. An Oil Red O working solution was
prepared by adding 6 ml of a stock solution (0.5 g Oil Red O in 100
ml isopropanol) to 4 ml of dH.sub.2O. The working solution was kept
for an hour at room temperature before filtering through a Whatman
#1 filter. Cells were stained with approximately 3 ml/100 mm plate
or 1 ml/well in 6-well plate for 1 hour at room temperature and
then rinsed several times with H.sub.2O. All of the remaining wash
water was removed. 150 .mu.l/well isopropanol was added and the
plate was incubated at room temperature for 10 minutes. The
isopropanol was pipetted up and down several times, to ensure that
all of the oil red O was in solution. The optical density was
measured at 500 nM.
[0051] Cells receiving adipocyte differentiation medium, stained
with oil red O as described below displayed the characteristic red
color indicating accumulation of lipid (FIG. 2A). Preadipocytes
showed very small oil droplets accumulating after 4 weeks in
culture. Cells receiving osteoblast medium showed some non-specific
background oil red O, however, the staining was not associated with
cells (FIG. 2C). These results suggest that stromal cells
differentiating into osteoblasts do not have detectable adipocyte
morphology. Lack of such neutral lipid accumulation is an indicator
of loss of adipocyte function and lineage.
[0052] Biochemical changes indicative of osteoblast differentiation
were also assessed. Osteocalcin is an osteoblast-specific secreted
protein that can be measured by ELISA (Intact Human Osteocalcin K
EIA kit, catalog number BT-460, Biomedical Technologies, Inc.,
Stoughton, Mass.). Media obtained from osteoblasts, preadipocytes,
and adipocytes were examined for the presence of secreted
osteocalcin. Briefly, conditioned medium (2 ml of medium from
40,000 cells after 72 hours) was collected and 20 .mu.l of each
medium was used for the assay. As shown in FIG. 3, there is
increasing osteocalcin found in the media of the osteoblasts, while
little or no osteocalcin secretion is seen in the adipose stromal
cells and adipocytes.
[0053] Secreted leptin peptide was measured by a commercially
available ELISA kit. Conditioned medium (2 ml of medium from 40,000
cells was conditioned for 72 hours) was collected and 100 .mu.l of
each medium was used for the assay following protocol suggested by
the manufacturer. As expected, leptin is secreted into the media is
increased during adipocyte differentiation but not in during
osteoblast differentiation. The presence of leptin, an
adipocyte-specific secreted protein, in the conditioned media is a
clear marker for adipocyte activity. Osteoblasts and stromal cells
fail to secrete any detectable leptin into the media, indicating a
lack of adipocyte lineage, while only cells undergoing adipocyte
differentiation secrete leptin after 2 weeks.
[0054] Another indicator of osteoblast lineage is the ability to
secrete alkaline phosphatase. Cells were prepared and incubated as
described above and the conditioned media assayed for alkaline
phosphatase activity using a commercially available alkaline
phosphatase assay kit (Sigma Diagnostic Inc., catalog number
104-LS, St. Louis, Mo.). As shown in FIG. 4, the preadipocytes had
a basal level of secreted alkaline phosphatase activity. Upon
differentiation, the adipocytes lost this basal level of secreted
activity while the osteoblasts showed an increase in the levels of
alkaline phosphatase.
Example 3
Identification of Compounds Affecting Osteoblast
Differentiation
[0055] Cultures of stromal cells isolated from adipose tissue
according to the method described in Example 1 were used to study
the effect compounds of interest on osteoblast differentiation.
Osteoblasts were differentiated from adipose stromal cells as in
Example 2, but in the presence and absence of a compound of
interest. Differentiation was measured by assays for secreted
osteocalcin. Compounds found to positively affect osteoblast
differentiation using the method of this example include bone
morphogenetic protein 4 and osteopontin-I (data not shown). These
results are in agreement with the previous findings of Wozney et
al. (1988) Science 242:1528-1534; Asahina et al. (1996) Exp Cell
Res 222:38-47; and Cook et al. (1996) Clin Orthop. 324:29-38.
Compounds that enhance osteoblast differentiation may be used to
enhance the bone structure of a subject, as discussed below.
Example 4
Use of Osteoblasts in Bone Repair
[0056] Stromal cells are isolated from adipose tissue using
liposuction from patients suffering from non-healing fractures or
osteoporotic fractures using the method of Example 1. The
preadipocytes are induced to differentiate to osteoblasts in vitro
using the method described in Example 2. After 7-21 days, the
differentiated osteoblasts are harvested, for example, by trypsin
treatment or mechanical scraping of the differentiated cells from a
tissue culture plate and then are concentrated by centrifugation at
3000.times.g for 10 min at 4-20.degree. C. under sterile
conditions. The harvested cells are resuspended in a collagen or
Matrigel.TM. solution and are then injected directly into the
fracture or surgery site using a 20 gauge or larger bore needle.
Alternatively, before injection, the cells are mixed with DBM or a
ceramic matrix such as ProOsteon 2000.TM. (Interpore Cross, Irvine,
Calif.) or Collagraft.TM. (Zimmer Inc., Warsaw, Ind.). The amount
of cells used will depend upon the surface area of the fracture and
the nature of the fracture. Multiple treatments may be necessary
depending upon the speed of recovery of the fracture desired. The
result is decreased time to healing and increased bone density
around the fracture site.
Example 5
Use of Genetically Altered Osteoblasts for Fractures or
Osteoporosis
[0057] Stromal cells are isolated as described in Example 1 and
genetic material (e.g., DNA encoding useful gene products, such as
bone morphogenetic proteins, operably linked to a promoter) is
introduced into the stromal cells using standard transfection
methods such as calcium chloride (Maniatis et al. (1982) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring
Harbor, N.Y.). Such a protocol has been developed using Effectene
reagent and is described herein. In a microcentrifuge tube, 0/1-2.0
ug pCMV-.beta.gal (Stratagene, Inc. La Jolla, Calif.) is added to
150 .mu.l of Buffer EC (Qiagen Effectene.TM. kit; catalog number
301425, Qiagen Corp.). This allows the DNA to condense. 8 .mu.l
enhancer (Qiagen Effectene kit) is added to the condensed DNA. The
tube containing the condensed DNA is then vortexed for one second
and allowed to incubate at room temperature for 2-5 minutes. The
tube is centrifuged briefly to remove drops at the top of the tube.
10 .mu.l Effectene.TM. is added, the tube is vortexed for 10
seconds, and incubated at room temperature for 5-10 minutes.
Following a 5-10 minute incubation time, 1 ml of medium is added to
the DNA mixture.
[0058] 120 .mu.l of the old medium is removed from the cells and 70
.mu.l of fresh medium is added. 25 .mu.l of the DNA mixture is then
to each well. The cells are incubated at 37.degree. C. for about 5
hours. However, Effectene is not toxic and may be left on the cells
for any period of time.
[0059] The cells are then rinsed once with 80 .mu.l fresh medium
and assayed at 72 hours post-infection for .beta.-galactosidase
activity using the method described by Maniatis et al. (1982). Such
cells may be differentiated into osteoblasts under the methods
described in Example 1. Alternatively or the DNA maybe introduced
directly into cells differentiated into osteoblasts.
[0060] Other methods of introducing nucleic acid sequences into
cells may also be used. For example, adenovirus, may be used to
introduce DNA into the stromal cells similarly to the protocols
described by Becker et al. (1994) Meth Cel Biol 43:161-189 and
Meunier-Duimont et al. (1996) Eur Biochem 237:660-667. The cells
are then treated so they differentiate into osteoblasts as
described above in Example 1. Alternatively, the differentiated
osteoblasts will be amenable to infection by viral particles. An
addition of an antibiotic selection marker allows enrichment for
cells bearing the introduced genetic material. The derived
osteoblasts bearing the introduced genetic material are then
introduced to fracture and osteoporotic bone marrow as described
above in Example 3.
[0061] All publications mentioned in the specification are
indicative of the level of those skilled in the art to which this
invention pertains. All publications are herein incorporated by
reference to the same extent as if each individual publication was
specifically and individually indicated to be incorporated by
reference.
[0062] While the invention has been described with reference to
specific embodiments, it will be appreciated that numerous
variations, modifications, and embodiments are possible, and
accordingly, all such variations, modifications, and embodiments
are to be regarded as being within the spirit and scope of the
invention.
* * * * *