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Stem Cells. Author manuscript; available in PMC 2012 April 10.
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Stem Cells. 2011 April ; 29(4): 576–582. doi:10.1002/stem.612.

Adipose Derived Stromal Cells for Skeletal Regenerative

Medicine
Benjamin Levi, M.D.1 and Michael T. Longaker, MD, MBA1,2,3
1Hagey Pediatric Regenerative Medicine Research Laboratory, Department of Surgery, Plastic

and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, California
2Institute for Stem Cell Biology and Regenerative Medicine

Abstract
As the average age of the population grows, the incidence of osteoporosis and skeletal diseases
continues to rise. Current treatment options for skeletal repair include immobilization, rigid
fixation, alloplastic materials and bone grafts, all which have significant limitations, especially in
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the elderly. Adipose derived stromal cells (ASCs) represent a readily available abundant supply of
mesenchymal stem cells which demonstrate the ability to undergo osteogenesis in vitro and in
vivo, making ASCs a promising source of skeletal progenitor cells. Current protocols allow for the
harvest of over 1 million cells from only 15cc of lipoaspirate. Despite the clinical use of ASCs to
treat systemic inflammatory diseases, no large human clinical trials exist using ASCs for skeletal
tissue engineering. The aim of this review is to define ASCs, to describe the isolation procedure of
ASCs, to review the basic biology of their osteogenic differentiation, discuss cell types and
scaffolds available for bone tissue engineering and lastly to explore imaging of ASCs and their
potential future role in human skeletal tissue engineering efforts.

Keywords
Adipose derived stromal cells; Skeletal tissue engineering; Tissue regeneration; Multipotent
stromal cells; adipogenic differentiation; Subcutaneous fat depots

Introduction
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Over the last 50 years, the number of Americans over 65 has increased from 12 to 37 million
and continues to grow at an average rate of 2.0% per year.1 With a more aged population
comes a significant increase in people living with osteoporosis and suffering from fractures
and other skeletal disabilities. Current methods for the treatment of fractures largely rely on
immobilization, rigid fixation and bone grafts, all of which have associated drawbacks
particularly in aged patients. Concurrent with the increase in age of the population, the
weight of the average American continues to grow as does surgical procedures to address

3
Correspondence: Michael T. Longaker, MD, MBA, Hagey Pediatric Regenerative Medicine Research Laboratory, Stanford
University School of Medicine, 257 Campus Drive, Stanford University, Stanford, CA 94305-5148, Phone: (650) 736-1707, Fax:
(650) 736-1705, [email protected].
Author Contributions:
Benjamin Levi: Conception and design, manuscript writing, final approval of manuscript, collection and/or assembly of data
Michael T. Longaker: Conception and design, manuscript writing, final approval of manuscript, collection and/or assembly of data
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.
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this excess adipose tissue with over 200,000 liposuction procedures performed annually in
the United States and over a million procedures performed annually worldwide. 2
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Fat has long been felt to be an inert tissue, and for years lipoaspirate has been discarded as
surgical waste. When a surgeon harvests lipoaspirate, they are harvesting numerous cell
types, including preadipocytes, adipocytes, fibroblasts, vascular smooth muscle cells or
pericytes, endothelial cells and resident monocytes, macrophages and lymphocytes.3 Within
the stromal vascular layer, scientists have begun to investigate a vast population of cells with
the potential to differentiate into mesodermal tissues. Importantly, these multipotent adipose
derived stromal cells (ASCs) have been shown to have similar morphology, differentiation
capacity and phenotype to those mesenchymal stem cells isolated from bone marrow and
umbilical cord blood. Clinical trials have already been established for intravenous
administration of ASCs for autoimmune and inflammatory disorders such as multiple
sclerosis and arthritis.4 Despite promising preliminary results using ASCs as a therapeutic
modality for inflammatory disease applications, a large clinical trial has yet to be established
to address fractures and skeletal diseases.

While considering therapies for skeletal deficits, scientists must understand the composition
of bone and how to define the formation of bone de novo from ASCs. Bone is defined
broadly as a specialized cell type, the osteoblast, and surrounding extracellular matrix that
these cells secrete and remodel. Osteoblasts function to secrete calcified matrix (termed
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osteoid), proteins and growth factors necessary for osteogenesis to occur. The extracellular
matrix consists of proteins such as collagen type I, ostocalcin, osteopontin and bone
sialoprotein as well as mineralized matrix or hydroxyapatite. As mineralization occurs,
osteblasts are surrounded by its own calcifying osteoid matrix, becoming an osteocyte. The
molecular mechanisms that occur during osteoblast maturation have been thoroughly
studied. Runx2/Cfba, a helix-loop-helix nuclear factor initiates a pluripotent mesenchymal
stem cell to start differentiating down an osteoblastic lineage and Osterix (Osx) is also
required for mesenchymal cell osteogenic differentation.5

ASCs have been found to undergo osteogenesis rapidly and with minimal stimulation by
exogenous cytokines and thus represent a promising option for skeletal tissue engineering
trials.6 The current, clinical gold standard for the treatment of non-healing skeletal defects is
an autogenous bone graft. Autogenous sources from which to harvest bone grafts are limited
and thus clinicians have turned to allogenic bone substitutes such as demineralized bone
matrix which consists of extracellular matrix proteins and growth factors without any cells.
In an attempt to augment acellular options, surgeons have begun to use Bone Morphogenic
Protein-2 (BMP-2) absorbed on a collagen sponge for non-unions and spinal fusion. Despite
promising results, collagen sponges are not rigid and BMP-2 release is limited in duration
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failing to provide long term growth. The shortcomings of current methods indicate the need
to combine our understanding of what cell types can form bone and what scaffold best
facilitates the differentiation of these cells.

One osteoprogenitor cell that is easily harvested and abundant in large quantities is the ASC.
The ultimate translational goal is to harvest subcutaneous adipose tissue from the ideal
anatomic location, enrich for ASCs with an improved osteogenic potential, treat the cells
with ideal small molecules or cytokines and implant these cells on a scaffold into the
skeletal defect in the same patient without leaving to the operating room. In this review we
will discuss the definition of ASCs, review how they are harvested, examine the molecular
underpinnings of their osteogenic differentiation, analyze the effect of harvest techniques on
osteogenic differentiation in vitro and in vivo, and finally detail future clinical correlates.

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Definitions
Caplan et al. originally described mesenchymal cells when observing bone formation after
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bone marrow cells were transplanted to a heterotopic location. These cells were named
“mesenchymal” stem cells because of the belief that they could differentiate into skeletal
muscle, smooth muscle fat, cartilage, connective tissues, tendon and bone.7 Within bone
marrow, mesenchymal cells are located in the stromal compartment and are unique from the
hematopoietic compartment. Mesenchymal stem cells (MSCs) harvested from the marrow
compartment of bone, have been used for tissue engineering, including the study of bone
formation for the spine. 8 MSC harvest, however, requires aspiration from the iliac crest
which only yields 10–40 ml of marrow or from bone marrow biopsies, both of which can be
painful and yield low numbers.9 As an alternative to marrow derived MSCs, laboratories
have identified a similar multi-lineage mesenchymal progenitor cells from adipose tissue. A
wide variety of terms have been used to describe the multipotent cells derived from white
adipose tissues that adhere to plastic. Such descriptions include preadipocytes, adipose
derived mesenchymal cells, adipose derived adult stem cells, processed lipoaspirate cells,
human adipose-derived adherent stromal cells and adipose derived stromal cells (ASC).10 At
the Second Annual International Fat Applied Technology Society meeting, scientists
reached a consensus to refer to these cells as ASCs which is how they will be referred
throughout this review.11 It is crucial to differentiate ASCs from the stromal vascular
fraction which refers to the minimally processed cells that have not yet been exposed to
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plastic. These, ASCs are both mesenchymal stem cells (in that they are a postnatal progenitor
of mesoderm derived cell types) as well as stromal cells (as they are fibroblastic cell type in
vitro and likely derived from adipose connective tissue).12

Characterizing ASCs based on surface proteins

Previous studies have demonstrated similar phenotypes between human MSCs and ASCs as
well as similar adhesion and receptor profiles.13 Numerous investigators have described
ASC surface antigen expression profiles, but a definitive surface antigen profile that
completely defines ASCs and allows prospective isolation has been lacking. Cultured ASCs
are STRO-1 negative, however, cultured bone marrow derived MSCs (BMSCs) are reported
as STRO-1 positive despite comprising less than 5% of the total BMSC population.13
Scientists have also noted CD106 by flow cytometry,9 while others did not detect this
surface antigen. Such discrepancies underlie the need to indicate the cell passage,
proliferative stage and patient profile. Despite inconsistencies, scientists generally define
ASCs as those cells that express the surface receptor molecules CD44 (hyaluronate) and
CD90, as well as integrin β1 (CD29), endoglin (CD105) and integrin α4 (CD49) and to not
to express the hematopoietic markers CD45, CD34 and cKit (CD117).2 Despite certain cell
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surface panels being used to define ASCs, there has been observed a significant amount of
drift when looking at ASC markers immediately after harvest compared to later passage
cells making it difficult to define a “pure population.” 2

ASC Harvest
Human ASC harvest can be derived from lipoaspiration or surgically resected adipose tissue.
Once harvested, the adipose tissue settles into two layers: supernatant or processed
lipoaspirate layer, consists of the suctioned adipocytes as well as their surrounding
endothelium and stroma. The bottom layer, or liposuction aspirate fluid, consists of injected
saline, erythrocytes and denser pieces of the processed lipoaspirate layer. 2 ASCs can be
harvested from both layers, however, the yield of adherent ASCs is significantly higher in
the adipocyte layer than in the liposuction aspirate fluid cells.2

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Following harvest, adipose specimens should first be washed, undergo enzymatic digestion
followed by neutralization, straining and plating. The specimen that is plated is the stromal
vascular fraction (SVF) and those adherent cells are the ASCs. On average, 15cc of starting
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lipoaspirate can be plated onto 1x 10cm cell culture plates which three days later, can be
split to three 10cm plates. The total number of cells from adipose harvest has been described
as 308,849 per ml of lipoaspirate or 1.5 × 1010 from 1000ml of lipoaspirate after two
passages.11 At a plating density of 1.85 × 103 cells/cm2, ASCs reach confluence by day
13.14

When using human tissues in the laboratory, it is critical to use biohazard precautions and to
sterilize or discard all equipment with bleach before and after usage as this tissue is
considered Biosafety Level 2 (BSL-2). Collection containers and tubing should be disposed
or sterilized with bleach after each patient use.

Differences in hASCs based on tissue location, and preparation

Fat distribution between men and women and within each gender demonstrate significant
heterogeneity. Adipose tissue from subcutaneous locations, or depots, have different blood
supplies, cytokine signaling and gene expression profiles leading to differences in
osteogenic capacity. 15 Stimulated by the rising incidence of obesity and the need for a more
thorough understanding of adipose biology, a growing number of studies have investigated
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the differences between subcutaneous and visceral fat depots with visceral fat depots having
a greater osteogenic potential.15 The potential breakthroughs in the use of stromal cells have
prompted some individuals to store their own tissues in a fee-for-service fashion and
allowed the formation of biotechnology companies specializing in stromal cell storage. The
most prevalent example is the cryostorage of umbilical cord derived blood. The process of
cell freezing and the long-term storage under such conditions inevitably alters cellular
processes and characteristics.16 In vitro cellular parameters, including proliferation, osteo-
and adipogenic differentiation have also been assessed after the freeze-thaw process
demonstrating a decreased capacity to proliferate and differentiate after cryostorage. 17, 18
Data from our laboratory confirm that the freezing process of hASCs, has deleterious effects
on the ability of these cells to undergo osteogenic differentiation even if only in cryostorage
for two weeks.19 Thus, though cryopreserved cells can undergo osteogenic differentiation,
the fresh harvest of hASCs and immediate use may allow for a more robust osteogenic
reconstruction.

Osteogenic Differentiation In Vitro

Osteogenesis is defined by a series of events which starts with a commitment to an
osteogenic lineage by mesenchymal cells. Subsequently, these cells proliferate and
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demonstrate an up regulation of osteoblast-specific genes and mineralization. After

attachment, ASCs can be treated with osteogenic differentiation medium (ODM) containing
DMEM, 10% FBS, 100μg/mL ascorbic acid, 10mM β-glycerophosphate, and 100 U/mL
penicillin/streptomycin. Retinoic acid can be supplemented to augment mASC
differentiation but is not necessary for hASCs.

Multiple signaling pathways have been demonstrated to participate in the differentiation of

an osteoblast progenitor to a committed osteoblast including TGFB/BMP, Wnt/B-Catenin,
Notch, Fibroblast growth factor and Hedgehog. BMPs in particular have been demonstrated
to play a significant role in osteoblast differentiation and osteogenesis, most notably
BMP-2,4 and 7. BMP is a member of the TGF-B superfamily and initiates its signaling
cascade through BMPR receptors types I and II. These activated receptor kinases
subsequently phosphorylate transcription factors Smad 1,5 and 8.20 These phosphorylated
Smads form a heterodimeric complex with Smad4 and stimulate target genes. Such

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downstream genes include Msx2, Dlx5 and Id proteins, (inhibitor of DNA binding/
differentiation helix-loop-helix proteins) which during early osteogenesis, regulate
proliferation of osteoprogenitors.21 Investigators have demonstrated that inhibition of these
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Id proteins promote osteogenesis in vivo.22

BMPs have also been shown to activate Cbfa1/AML3 or Runt-Related Protein 2 (Runx-2)
and osteopontin as well as stimulate osteogenic differentiation. Runx-2 and Osterix (Osx)
are considered the master regulation genes for bone formation.23 There are two major
isoforms of Runx2 that vary in their tissue localization. Compared with isoform II of Runx2,
isoform I is not as tissue specific as it is also detected in sperm, brain, testis, breast, prostate,
and melanoma cancer cells.24 Runx-2 isoform II is considered one of the earliest markers of
the osteoblast lineage and is considered a key transcriptional activator of osteogenic
differentiation and is seen two to three days prior to osteogenesis.24 Binding sites for
Runx-2 are present in genes whose gene products play a role in extracellular matrix
mineralization such as collagen type I and osteopontin (OPN) as well as genes involved cell
growth, and angiogenesis. 23

Cells that express Runx-2 display characteristics of a future osteoblast or chondrocyte,

however, later in development, only those cells which differentiate into osteoblasts show
evidence of Runx-2 expression.25 Transfection of Runx-2 in fibroblasts stimulates
expression of osteoblast specific genes such as Osteocalcin whereas inhibition of Runx-2
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during development leads to the absence of osteoblasts and has a severe phenotype of
cleidocranial dysplasia that is associated with perinatal death.25

Specific to mesenchymal stem cells, studies have shown that Runx2 remains present during
mitosis and may induce lineage differentiation of multipotent cells.23 It is felt that after
mesenchymal cell differentiation down an osteoblastic pheonotype, Runx2 promotes
departure from the cell cycle and decreases proliferation but increases osteogeneic
differentiation.

Osx is a zinc-finger containing transcription factor that is expressed in osteoblasts during

mouse bone development. Up regulation of Osx has been demonstrated to stimulate the
osteogenic differentiation of ES cells in vitro and a retroviral transduction of Osx into
marrow derived MSCs has been shown to increase their osteoblastic markers such as
alkaline phosphatase, osteocalcin and osteopontin.26 Osx-null mice, similar to Runx-2 null
mice have no cortical or trabelcular bone. 5 Osx, however, appears to play a role more
downstream as Osx-null mice had normal cartilage and normal Runx-2 expression whereas
Runx-2 null mice display delayed chondrocyte maturation and decreased Osx expression.5
More recent studies using Osx knockdown demonstrated significantly impaired osteogenesis
during osteogenic differentiation of fetal bone cells.27
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To assess the osteogenic differentiation of ASCs, RNA of these osteogenic genes can be
analyzed at different time points. Specific gene markers of early ostoeogenesis include,
RUNX-2 and Alkaline Phosphatase (ALP), intermediate markers include, OPN and late
osteogenesis is best determined by Osteocalcin (OCN) expression. Alkaline phosphatase and
Alizarin Red stains also allow assessment of early osteogenic differentiation and terminal
bone mineralization respectively.

Adipocyte Differentiation of ASCs

There has been increasing interest in examining the interdependency between adipogenesis
and osteogenesis as it is thought there was an inverse relationship between adipocytes and
osteoblasts in bone marrow.28 Peroxisome proliferator activated receptor gamma (Ppar γ)
has been shown to play a key role in adipogenic differentiation as scientists have

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demonstrated that Ppar γ-deficient ES cells failed to differentiate into adipocytes, but
spontaneously differentiated into osteoblasts. Furthermore, these ES had restoration of their
adipogenic potential with reintroduction of the Ppar γ gene.29 Subsequent findings indicate
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that a shared co-activator protein, transcriptional co-activator with PDZ binding motif
(TAZ) functions as a link between Runx2 and PPAR γ and that TAZ activated Runx2 and
osteogenesis while suppressing Ppar γ and adipogenesis.28 Specific mutations in Ppar γ
allowed scientists to demonstrate that young heterozygous Ppar γ +/− mice had increased
bone mass and elevated mRNA of Runx2 and Osx.30 These studies into adipogenesis and
osteogenesis indicate that Ppar γ down-regulation may be a future target in order to enhance
the osteogenic capability of ASCs.

In vivo models
To adequately translate in vitro findings to the clinical realm, robust in vivo data must be
obtained to demonstrate the osteogenic capacity of ASCs. Long bone skeletal defect models
offer the benefit of using bones that are under load bearing stress. Many groups use femoral
defects as well as tibial defects as a load bearing bone.31 The ideal model should represent
that clinical condition of the bone injury or defect. Thus, if interested in treating long bone
non-unions, similar appendicular skeletal non-union models should be used in animals.
Alternatively, if treating a calvarial defect where other tissues such as dura mater play a role,
a similar calvarial model should be utilized.
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When studying long bone non-unions, injuries should be created in a load bearing location,
and stable fixation must be used. Benefits of the femoral bone are its larger shaft than the
tibia thus tolerating a larger defect and allowing for the placement of an external fixator
device. Several groups have used this defect model to seed ASCs on an osteoconductive
scaffold. Peterson et al demonstrated the use of ASCs genetically modified to over-express
BMP-2 to heal a femoral critical sized defect, though this same study showed that BMP-2
alone on a scaffold also allowed for significant healing.32 Other laboratories attempting to
enhance osteogenesis using ASCs seeded on a BMP-2 releasing scaffold failed to
demonstrate improved healing over using the BMP-2 laden scaffold alone, however, the
authors failed to demonstrate viability of the ASCs in vivo.33 Thus, it is known that BMP-2
stimulates surrounding tissues, however, more robust data is needed to demonstrate that
BMP-2 also augments the osteogenic potential of implanted ASCs. Human ASCs promoted
fracture healing and improved biomechanical function in a rat femur non-union, however,
ASCs failed to improve healing in the spinal fusion model.8

Several models exist to assess calvarial defects, and we believe a 4mm mouse parietal bone
model offers a reliable, easily replicated and easily followed defect model (Figure 1). This
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model functions as a critical sized defect (4mm) in that it will undergo less than 5% healing,
even if followed for 8 weeks. Thus, if any healing is seen, it is likely due to the treatment
used. When a hydroxyappatite coated PLGA scaffold is seeded with 150,000 human or
mouse ASCs, we have observed over 80% healing at 8 weeks.6 In comparison, those treated
with a PLGA scaffold alone healed less than 20% proving the osseous healing is likely due
to the implanted ASCs.

When using human cells, nude or athymic animals should be used to decrease the
inflammatory response which may confound results. The pro-inflammatory environment is a
significant feature in bone repair and is initiated by the innate immune system. Similar to
injuries to other tissues, an increase in intereukin-1 and 6 as well as tumor necrosis factor
alpha is seen during bone remodeling.34 Injured bone repair requires the participation of
both the immune and hematopoeitic niche of immune marrow cells as well as osteogenic
precursor cells from the surrounding periosteum and surrounding osteoblasts and soft tissue.

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Nude or athymic animals may demonstrate a blunted inflammatory response, however, they
only lack a T-cell response and can still mount an inflammatory response with regards to B-
cells and NK-cells and still possess the surrounding osteogenic precursor cells from the
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periosteum.

Imaging
Although histology and histomorphometry remain the basis of in vivo analysis of pathways
and defining tissues at a specific time point, such methods are less able to demonstrate
physiologic whole body effects.35 Furthermore, such hostologic methods require chemical
fixation and examination of tissues under nondynamic conditions.35 Imaging techniques,
such as Live micro-computed tomography (microCT), however, allow scientists to serially
follow osseous healing without sacrificing the animal and 3-D reconstructions allows for
high resolution images. Unlike histology, imaging techniques allow the repetitive study of
the same animal model allowing a dynamic analysis.35 Simultaneously, cells can be tagged
with reporters allowing studies of the implanted cells to continue while osteogenesis occurs.
Along with osseous healing, scientists and surgeons must demonstrate the viability of the
ASCs in vivo to prove that the donor ASCs participate in healing of the recipient defect
rather than just secreting cytokines to stimulate the surrounding host tissues. One way to
demonstrate viability is to stably transduce ASCs with a lentivirus carrying triple fusion
reporter genes, including firefly luciferase (Fluc), red fluorescence protein (RFP) and herpes
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simplex virus truncated thymidine kinase (HSV-ttk) genes.35 Stably expressed ASCs can
then be purified by fluorescence activated cell sorting based on the RFP expression (Figure
1). Bioluminesence allows monitoring in vivo whereas the RFP or GFP allows for marking
of these cells during histologic examination to determine if the host or implanted cells are
located in the region of osteoid formation.

Bone Tissue Engineering: Scaffolds

Stem cells exist in tightly controlled environmental niches and alterations in this
microenvironment can dramatically modify their behavior and differentiation capacities.
Furthermore, stem cells are often utilized in the setting of disease or injury where
inflammatory signals may be prevalent altering their function. Biomaterial scaffolds can
potentially provide a controlled environment protecting implanted cells from harmful
stimuli. Biomaterial matrices are also used to deliver genetic material and/or inductive
biochemical cues which allow for some degree of developmental control over the delivered
stem cells.

Numerous studies have demonstrated the benefits of utilizing stem cell-scaffold constructs
in regenerative medicine. Scaffolds provide a highly modifiable vehicle for inductive factors
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as well. One cytokine of great interest and promise in skeletal tissue engineering is BMP-2.
In a laboratory setting, several investigators have used BMP-2 delivery methods to enhance
the osteogenic capability of ASCs. 32 BMP-2, however, is osteoinductive and likely
enhances the osteogenesis of surrounding osteoblasts, periosteal cells and underlying dura in
calvarial defects. Thus, further studies must determine if the enhanced osteogenesis seen
with BMP-2 is due to its paracrine effect on implanted cells or host tissues.

In a clinical setting, InfuseR, a rBMP-2 with an absorbable collagen sponge carrier has been
approved and is frequently used in spine surgeries. Original clinical studies in 2002 analyzed
a prospective randomized analysis of 279 patients with degenerative lumbar disc disease
who were treated with either InFUSE, or autogenous iliac bone grafts. The authors
demonstrate that at 24 months, 94.5% fusion rate in the InFUSE treated group compared to
88.7% in the bone graft treated group. Furthermore, mean operative time and blood loss
were shown to be lower in the InFUSE treated patients.36 A more recent randomized,

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controlled trial of lumbar spine fusion in patients over sixty, also demonstrated that the
InFUSE group had lower complication rates likely related to the lack of a secondary defect
created by harvesting iliac bone. Interestingly, this study also demonstrated a similar total
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cost in care between the two groups despite the expense of BMP-2.37

Other studies analyzing the interaction of scaffolds and cells include a study by Fang et al.
where the authors utilized degradable matrices containing a BMP-4 plasmid DNA and
demonstrated successful in vivo genetic manipulation of fibroblasts to induce bone
formation in rats.38 Schek et al. demonstrated that bone formation was greater with hydrogel
delivery of BMP-7-expressing adenovirus in mice compared to non-hydrogel controls.39
These are just a handful of examples of the benefits of using scaffold-based applications to
enhance the regenerative potential of stem cells.

Bone Tissue Engineering: Cells

Investigations into cell-based skeletal tissue engineering must identify a cell type with the
ability to generate bone. Shortcomings of cell types often involve their availability, viability,
vascularization and elicitation of immune response upon implantation, and directed
differentiation. Options for skeletal tissue engineering include osteoblasts, embryonic stem
cells and other postnatal mesenchymal cells. Though osteoblasts have already undergone
further differentiation than MSCs down an osteogenic pathway, osteoblasts are limited in
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availability and their harvest requires loss of bone from the donor site. Attempts to enhance
the osteogenic capability of osteoblasts using mitogens and cytokines also failed to improve
osteogenesis in aged osteoblasts.

The capability of ESCs to differentiate into almost any cell type found in the human body
has led to many promising areas of investigation which may yield deeper understanding of
cellular biology and potential cures for diseases. Previous studies have demonstrated the
ability of ESCs to undergo osteogenic differentiation in vivo and in vitro, however, their
pluripotency predisposes them to differentiating into any three of the embryonic germ layers
(ectoderm, endoderm or mesoderm) thus forming a teratoma. 40 Use of ESCs in a clinical
capacity, also potentially raises the challenging issue of graft-versus-host disease associated
with allogeneic stem cell transplantation. While investigations continue to reduce donor-host
rejection, concerns regarding xenogeneic contamination also exist. ESCs have been
traditionally cultured in vitro in the presence of mouse embryonic fibroblast (MEF) feeder
layers because in their absence, ESCs have been found to undergo rapid differentiation.40
Eliminating this MEF layer necessitated the creation of a stem-cell line under animal-free
conditions. To accomplish this, scientists developed extracellular matrix coated plates from
MEFs and sterilized prior to use.41 Other strategies proposed to avoid animal products and
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graft versus host reactions include the use of autologous donor adult stem cells or the more
recently described induced pluripotent stem (iPS) cell.42

Periostium derived progenitor cells may also serve as a cell source for tissue engineering.
Periosteum is a specialized cell type that covers bone surfaces and has the potential to
differentiate into multiple mesenchymal tissues including bone. These cells have been of
particular interest to dentists and oral surgeons given the increased ease of harvesting
periosteal cells compared to bone marrow cells during periodontal surgery. Furthermore,
studies have demonstrated an improved proliferation rate of periosteal cells compared to
MSCs and demonstrate a robust capability to undergo osteogenesis.43 Despite this
proliferative and osteogenic capability and promising use in alveolar bone treatment,
periostium is limited in availability and this cell type may not be ideal if treating a large non-
union or calvarial defect.

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Bone Marrow Derived MSCs have also been used successfully for osteogenic differentiation
in vitro and in vivo. MSCs implanted on Matrigel and placed into the lumbar fusion bed of
rats have been shown to result in enhanced fusion compared to animals receiving mixed
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marrow stromal cells alone.44 A study following 51 long bones (tibia and femur) undergoing
distraction osteogenesis of long bones, and injection of MSCs along with platelet-rich
plasma into the callus during both lengthening and consolidation phases, resulted in
accelerated mature bone formation and a reduction in the incidence of complications in the
femur.45 Horwitz and colleagues have also investigated the use of MSCs in the treatment of
osteogenesis imperfecta, a genetic disorder of type I collagen resulting in fragile bones and
skeletal deformities.46 While there is no current cure for osteogenesis imperfecta,
preliminary reports have shown MSCs to be potentially successful in enhancing bone
formation in these patients.46 Thus, the success of marrow derived MSCs can hopefully be
translated to the much more readily available and more easily accessible ASCs.

Scientists have demonstrated that ASCs can be easily isolated from human adipose tissue
and can undergo differentiation into bone, cartilage, tendon, muscle and fat cells when
grown in the appropriate conditions. 10 Though the number of ASCs harvested per gram is
the same as marrow derived MSCs, adipose tissue is more readily available in larger
quantities.14 With regards to skeletal tissue engineering, some groups have shown ASCs to
have similar growth kinetics, cell senescence, and osteogenic differentiation capacity in vitro
to bone marrow derived MSCs, though others have shown marrow derived MSCs to have
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superior growth kinetics and differentiation capacities.14 Similarly, some have demonstrated
the increased osteogenic potential of marrow derived MSCs whereas others have shown
ASCs have been shown to be more osteogenic in vivo.6, 14 In vitro studies of ASCs have
demonstrated their robust capability of undergoing osteogenic differentiation as early as 3
days after induction and those ASCs from humans have proven more osteogenic than those
harvested from mice.47 In vivo studies have demonstrated that ASCs loaded on a PLGA
scaffold led to the development of osteoid material. Subsequent studies have demonstrated
the ability of ASCs from mouse and human sources to heal critical sized defects with those
treated with human derived ASCs showing significant bone formation as early as 2 weeks
post implantation. 6

To achieve clinical translation of hASCs, ideally surgeons would harvest the cells, seed
them on an osteoinductive scaffold and reconstruct the skeletal injury/defect in the same
patient without leaving the operating room. Similar cell saving and re-use devices such as
the Cell SaverR, are used in trauma and complicated surgeries in which blood loss gets
cycled through this FDA approved machine and the erythrocytes can be transfused back into
the patient. One example of a potential clinical strategy would be to harvest lipoaspirate,
isolate ASCs intraoperatively, place them on a BMP-2 laden scaffold and place the BMP-2
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and ASC loaded scaffold back into the patient’s skeletal defect in one step, without leaving
the operating room. Interestingly, ASCs have been shown to up-regulate osteogenic genes in
response to shear stress making them capable of responding to a mechanical load.48 Such
ability to undergo osteogenesis without in vitro differentiation and responsiveness to
mechanical stress makes ASCs an appealing cell source for skeletal tissue engineering.

Clinical correlates
Case report studies have examined the potential use of hASCs to heal skeletal defects in the
human patient. Defects of facial bones and the cranium49 have been demonstrated to heal or
stimulate healing with the use of ASCs. Though promising, current studies are limited as
many countries have not yet approved the use of ASCs. Furthermore, the methods of ASC
usage have varied dramatically and have included combination with bone grafts, the use of
various osteoconductive scaffolds as well as recombinant proteins. Mesimaki et al. used a

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 Levi and Longaker Page 10

microvascular flap with hASCs, beta-tricalcium phosphate and BMP-2 to heal a large defect
in the maxilla, reporting promising outcomes up to 8 months post-operatively. 50 Though
encouraging, these early studies offer only level 4 and 5 data and lack significant power to
NIH-PA Author Manuscript

help tailor clinical practice. Larger scale studies including prospective, randomized control
trials, must verify these findings, and to determine the optimum cell delivery method and
cytokine stimuli for hASC driven osteogenesis.

Conclusions
ASCs are a readily available, multipotent, abundant cell type with the capability to undergo
robust osteogenesis. The fact that they can undergo osteogenic differentiation so rapidly in
vitro makes them an exciting candidate for in vivo studies. Even more importantly, their
ability to undergo osteogenic differentiation without any stimulation when placed on an
osteoconductive scaffold in vivo make ASCs a promising candidate for skeletal tissue
engineering. Further studies of the mechanism of osteogenic differentiation and ways to
improve ASC osteodifferentiation by eliminating the heterogeneity and stimulating the BMP
pathway offer potential for future studies.

Acknowledgments
We would like to thank the following people for their outstanding work in our laboratory: Emily R. Nelson, B.S.,
NIH-PA Author Manuscript

Victor Wong, M.D. and Aaron W. James, M.D.

Sources of Support:

This study was supported by National Institutes of Health, National Institute of Dental and Craniofacial Research
grant 1 R21 DE019274-01, 1 RC2 DE020771-01, the Oak Foundation and Hagey Laboratory for Pediatric
Regenerative Medicine to M.T.L. B.L was supported by the National Institutes of Health, National Institute of
Arthritis and Musculoskeletal and Skin Diseases grant 1F32AR057302-02. National Endowment of Plastic Surgery

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Figure 1.
In vivo model for skeletal tissue engineering.(Left, Top) Superior view of mouse left
inguinal fat pad. Red arrow points to the fat pad which is dissected and subsequently
digested for ASC harvest. (Left, Bottom) Human lipoaspirate in sterile collection canister
settled into two layers. The upper yellow layer (Green Arrow) is the adipose tissue from
which ASCs are harvested. Subsequently ASCs are isolated in vitro. 150,000 cells can then
be seeded on an osteoconductive scaffold (Middle) and then the ASC laden scaffold can be
placed inside a critical sized calvarial defect in syngeneic for mASCs or nude athymic mice
for hASCs (Right). Mice can then be imaged using IVIS systems to detect cell viability and
MicroCT to quantify osseous healing (Right).
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