nih public access medicine stem cells adipose derived stromal cells for skeletal … b, stem cells....

Adipose Derived Stromal Cells for Skeletal RegenerativeMedicine

Benjamin Levi, M.D.1 and Michael T. Longaker, MD, MBA1,2,3

1Hagey Pediatric Regenerative Medicine Research Laboratory, Department of Surgery, Plasticand Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, California2Institute for Stem Cell Biology and Regenerative Medicine

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

KeywordsAdipose derived stromal cells; Skeletal tissue engineering; Tissue regeneration; Multipotentstromal cells; adipogenic differentiation; Subcutaneous fat depots

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

3Correspondence: Michael T. Longaker, MD, MBA, Hagey Pediatric Regenerative Medicine Research Laboratory, StanfordUniversity School of Medicine, 257 Campus Drive, Stanford University, Stanford, CA 94305-5148, Phone: (650) 736-1707, Fax:(650) 736-1705, [email protected] Contributions:Benjamin Levi: Conception and design, manuscript writing, final approval of manuscript, collection and/or assembly of dataMichael T. Longaker: Conception and design, manuscript writing, final approval of manuscript, collection and/or assembly of dataDisclosure of Potential Conflicts of InterestThe authors indicate no potential conflicts of interest.

NIH Public AccessAuthor ManuscriptStem Cells. Author manuscript; available in PMC 2012 April 10.

Published in final edited form as:Stem Cells. 2011 April ; 29(4): 576–582. doi:10.1002/stem.612.

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this excess adipose tissue with over 200,000 liposuction procedures performed annually inthe United States and over a million procedures performed annually worldwide. 2

Fat has long been felt to be an inert tissue, and for years lipoaspirate has been discarded assurgical waste. When a surgeon harvests lipoaspirate, they are harvesting numerous celltypes, including preadipocytes, adipocytes, fibroblasts, vascular smooth muscle cells orpericytes, endothelial cells and resident monocytes, macrophages and lymphocytes.3 Withinthe stromal vascular layer, scientists have begun to investigate a vast population of cells withthe potential to differentiate into mesodermal tissues. Importantly, these multipotent adiposederived stromal cells (ASCs) have been shown to have similar morphology, differentiationcapacity and phenotype to those mesenchymal stem cells isolated from bone marrow andumbilical cord blood. Clinical trials have already been established for intravenousadministration of ASCs for autoimmune and inflammatory disorders such as multiplesclerosis and arthritis.4 Despite promising preliminary results using ASCs as a therapeuticmodality for inflammatory disease applications, a large clinical trial has yet to be establishedto address fractures and skeletal diseases.

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

ASCs have been found to undergo osteogenesis rapidly and with minimal stimulation byexogenous cytokines and thus represent a promising option for skeletal tissue engineeringtrials.6 The current, clinical gold standard for the treatment of non-healing skeletal defects isan autogenous bone graft. Autogenous sources from which to harvest bone grafts are limitedand thus clinicians have turned to allogenic bone substitutes such as demineralized bonematrix 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 MorphogenicProtein-2 (BMP-2) absorbed on a collagen sponge for non-unions and spinal fusion. Despitepromising results, collagen sponges are not rigid and BMP-2 release is limited in durationfailing to provide long term growth. The shortcomings of current methods indicate the needto combine our understanding of what cell types can form bone and what scaffold bestfacilitates 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 idealanatomic location, enrich for ASCs with an improved osteogenic potential, treat the cellswith ideal small molecules or cytokines and implant these cells on a scaffold into theskeletal defect in the same patient without leaving to the operating room. In this review wewill discuss the definition of ASCs, review how they are harvested, examine the molecularunderpinnings of their osteogenic differentiation, analyze the effect of harvest techniques onosteogenic differentiation in vitro and in vivo, and finally detail future clinical correlates.

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DefinitionsCaplan et al. originally described mesenchymal cells when observing bone formation afterbone marrow cells were transplanted to a heterotopic location. These cells were named“mesenchymal” stem cells because of the belief that they could differentiate into skeletalmuscle, smooth muscle fat, cartilage, connective tissues, tendon and bone.7 Within bonemarrow, mesenchymal cells are located in the stromal compartment and are unique from thehematopoietic compartment. Mesenchymal stem cells (MSCs) harvested from the marrowcompartment of bone, have been used for tissue engineering, including the study of boneformation for the spine. 8 MSC harvest, however, requires aspiration from the iliac crestwhich only yields 10–40 ml of marrow or from bone marrow biopsies, both of which can bepainful and yield low numbers.9 As an alternative to marrow derived MSCs, laboratorieshave identified a similar multi-lineage mesenchymal progenitor cells from adipose tissue. Awide variety of terms have been used to describe the multipotent cells derived from whiteadipose tissues that adhere to plastic. Such descriptions include preadipocytes, adiposederived mesenchymal cells, adipose derived adult stem cells, processed lipoaspirate cells,human adipose-derived adherent stromal cells and adipose derived stromal cells (ASC).10 Atthe Second Annual International Fat Applied Technology Society meeting, scientistsreached a consensus to refer to these cells as ASCs which is how they will be referredthroughout this review.11 It is crucial to differentiate ASCs from the stromal vascularfraction which refers to the minimally processed cells that have not yet been exposed toplastic. These, ASCs are both mesenchymal stem cells (in that they are a postnatal progenitorof mesoderm derived cell types) as well as stromal cells (as they are fibroblastic cell type invitro and likely derived from adipose connective tissue).12

Characterizing ASCs based on surface proteinsPrevious studies have demonstrated similar phenotypes between human MSCs and ASCs aswell as similar adhesion and receptor profiles.13 Numerous investigators have describedASC surface antigen expression profiles, but a definitive surface antigen profile thatcompletely defines ASCs and allows prospective isolation has been lacking. Cultured ASCsare STRO-1 negative, however, cultured bone marrow derived MSCs (BMSCs) are reportedas 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 thissurface antigen. Such discrepancies underlie the need to indicate the cell passage,proliferative stage and patient profile. Despite inconsistencies, scientists generally defineASCs as those cells that express the surface receptor molecules CD44 (hyaluronate) andCD90, as well as integrin β1 (CD29), endoglin (CD105) and integrin α4 (CD49) and to notto express the hematopoietic markers CD45, CD34 and cKit (CD117).2 Despite certain cellsurface panels being used to define ASCs, there has been observed a significant amount ofdrift when looking at ASC markers immediately after harvest compared to later passagecells making it difficult to define a “pure population.” 2

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

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Following harvest, adipose specimens should first be washed, undergo enzymatic digestionfollowed by neutralization, straining and plating. The specimen that is plated is the stromalvascular fraction (SVF) and those adherent cells are the ASCs. On average, 15cc of startinglipoaspirate can be plated onto 1x 10cm cell culture plates which three days later, can besplit to three 10cm plates. The total number of cells from adipose harvest has been describedas 308,849 per ml of lipoaspirate or 1.5 × 1010 from 1000ml of lipoaspirate after twopassages.11 At a plating density of 1.85 × 103 cells/cm2, ASCs reach confluence by day13.14

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

Differences in hASCs based on tissue location, and preparationFat distribution between men and women and within each gender demonstrate significantheterogeneity. Adipose tissue from subcutaneous locations, or depots, have different bloodsupplies, cytokine signaling and gene expression profiles leading to differences inosteogenic capacity. 15 Stimulated by the rising incidence of obesity and the need for a morethorough understanding of adipose biology, a growing number of studies have investigatedthe differences between subcutaneous and visceral fat depots with visceral fat depots havinga greater osteogenic potential.15 The potential breakthroughs in the use of stromal cells haveprompted some individuals to store their own tissues in a fee-for-service fashion andallowed the formation of biotechnology companies specializing in stromal cell storage. Themost prevalent example is the cryostorage of umbilical cord derived blood. The process ofcell freezing and the long-term storage under such conditions inevitably alters cellularprocesses and characteristics.16 In vitro cellular parameters, including proliferation, osteo-and adipogenic differentiation have also been assessed after the freeze-thaw processdemonstrating a decreased capacity to proliferate and differentiate after cryostorage. 17, 18

Data from our laboratory confirm that the freezing process of hASCs, has deleterious effectson the ability of these cells to undergo osteogenic differentiation even if only in cryostoragefor 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 osteogenicreconstruction.

Osteogenic Differentiation In VitroOsteogenesis is defined by a series of events which starts with a commitment to anosteogenic lineage by mesenchymal cells. Subsequently, these cells proliferate anddemonstrate an up regulation of osteoblast-specific genes and mineralization. Afterattachment, ASCs can be treated with osteogenic differentiation medium (ODM) containingDMEM, 10% FBS, 100μg/mL ascorbic acid, 10mM β-glycerophosphate, and 100 U/mLpenicillin/streptomycin. Retinoic acid can be supplemented to augment mASCdifferentiation but is not necessary for hASCs.

Multiple signaling pathways have been demonstrated to participate in the differentiation ofan osteoblast progenitor to a committed osteoblast including TGFB/BMP, Wnt/B-Catenin,Notch, Fibroblast growth factor and Hedgehog. BMPs in particular have been demonstratedto play a significant role in osteoblast differentiation and osteogenesis, most notablyBMP-2,4 and 7. BMP is a member of the TGF-B superfamily and initiates its signalingcascade through BMPR receptors types I and II. These activated receptor kinasessubsequently phosphorylate transcription factors Smad 1,5 and 8.20 These phosphorylatedSmads 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, regulateproliferation of osteoprogenitors.21 Investigators have demonstrated that inhibition of theseId 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 majorisoforms 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 ofthe osteoblast lineage and is considered a key transcriptional activator of osteogenicdifferentiation and is seen two to three days prior to osteogenesis.24 Binding sites forRunx-2 are present in genes whose gene products play a role in extracellular matrixmineralization such as collagen type I and osteopontin (OPN) as well as genes involved cellgrowth, 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 showevidence of Runx-2 expression.25 Transfection of Runx-2 in fibroblasts stimulatesexpression of osteoblast specific genes such as Osteocalcin whereas inhibition of Runx-2during development leads to the absence of osteoblasts and has a severe phenotype ofcleidocranial dysplasia that is associated with perinatal death.25

Specific to mesenchymal stem cells, studies have shown that Runx2 remains present duringmitosis and may induce lineage differentiation of multipotent cells.23 It is felt that aftermesenchymal cell differentiation down an osteoblastic pheonotype, Runx2 promotesdeparture from the cell cycle and decreases proliferation but increases osteogeneicdifferentiation.

Osx is a zinc-finger containing transcription factor that is expressed in osteoblasts duringmouse bone development. Up regulation of Osx has been demonstrated to stimulate theosteogenic differentiation of ES cells in vitro and a retroviral transduction of Osx intomarrow derived MSCs has been shown to increase their osteoblastic markers such asalkaline phosphatase, osteocalcin and osteopontin.26 Osx-null mice, similar to Runx-2 nullmice have no cortical or trabelcular bone. 5 Osx, however, appears to play a role moredownstream as Osx-null mice had normal cartilage and normal Runx-2 expression whereasRunx-2 null mice display delayed chondrocyte maturation and decreased Osx expression.5More recent studies using Osx knockdown demonstrated significantly impaired osteogenesisduring osteogenic differentiation of fetal bone cells.27

To assess the osteogenic differentiation of ASCs, RNA of these osteogenic genes can beanalyzed at different time points. Specific gene markers of early ostoeogenesis include,RUNX-2 and Alkaline Phosphatase (ALP), intermediate markers include, OPN and lateosteogenesis is best determined by Osteocalcin (OCN) expression. Alkaline phosphatase andAlizarin Red stains also allow assessment of early osteogenic differentiation and terminalbone mineralization respectively.

Adipocyte Differentiation of ASCsThere has been increasing interest in examining the interdependency between adipogenesisand osteogenesis as it is thought there was an inverse relationship between adipocytes andosteoblasts 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, butspontaneously differentiated into osteoblasts. Furthermore, these ES had restoration of theiradipogenic potential with reintroduction of the Ppar γ gene.29 Subsequent findings indicatethat 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 andosteogenesis while suppressing Ppar γ and adipogenesis.28 Specific mutations in Ppar γallowed scientists to demonstrate that young heterozygous Ppar γ +/− mice had increasedbone mass and elevated mRNA of Runx2 and Osx.30 These studies into adipogenesis andosteogenesis indicate that Ppar γ down-regulation may be a future target in order to enhancethe osteogenic capability of ASCs.

In vivo modelsTo adequately translate in vitro findings to the clinical realm, robust in vivo data must beobtained to demonstrate the osteogenic capacity of ASCs. Long bone skeletal defect modelsoffer the benefit of using bones that are under load bearing stress. Many groups use femoraldefects as well as tibial defects as a load bearing bone.31 The ideal model should representthat clinical condition of the bone injury or defect. Thus, if interested in treating long bonenon-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.

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 thetibia thus tolerating a larger defect and allowing for the placement of an external fixatordevice. Several groups have used this defect model to seed ASCs on an osteoconductivescaffold. Peterson et al demonstrated the use of ASCs genetically modified to over-expressBMP-2 to heal a femoral critical sized defect, though this same study showed that BMP-2alone on a scaffold also allowed for significant healing.32 Other laboratories attempting toenhance osteogenesis using ASCs seeded on a BMP-2 releasing scaffold failed todemonstrate improved healing over using the BMP-2 laden scaffold alone, however, theauthors failed to demonstrate viability of the ASCs in vivo.33 Thus, it is known that BMP-2stimulates surrounding tissues, however, more robust data is needed to demonstrate thatBMP-2 also augments the osteogenic potential of implanted ASCs. Human ASCs promotedfracture 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 bonemodel offers a reliable, easily replicated and easily followed defect model (Figure 1). Thismodel 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 treatmentused. When a hydroxyappatite coated PLGA scaffold is seeded with 150,000 human ormouse ASCs, we have observed over 80% healing at 8 weeks.6 In comparison, those treatedwith a PLGA scaffold alone healed less than 20% proving the osseous healing is likely dueto the implanted ASCs.

When using human cells, nude or athymic animals should be used to decrease theinflammatory response which may confound results. The pro-inflammatory environment is asignificant feature in bone repair and is initiated by the innate immune system. Similar toinjuries to other tissues, an increase in intereukin-1 and 6 as well as tumor necrosis factoralpha is seen during bone remodeling.34 Injured bone repair requires the participation ofboth the immune and hematopoeitic niche of immune marrow cells as well as osteogenicprecursor 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, theyonly 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 theperiosteum.

ImagingAlthough histology and histomorphometry remain the basis of in vivo analysis of pathwaysand defining tissues at a specific time point, such methods are less able to demonstratephysiologic whole body effects.35 Furthermore, such hostologic methods require chemicalfixation and examination of tissues under nondynamic conditions.35 Imaging techniques,such as Live micro-computed tomography (microCT), however, allow scientists to seriallyfollow osseous healing without sacrificing the animal and 3-D reconstructions allows forhigh resolution images. Unlike histology, imaging techniques allow the repetitive study ofthe same animal model allowing a dynamic analysis.35 Simultaneously, cells can be taggedwith reporters allowing studies of the implanted cells to continue while osteogenesis occurs.Along with osseous healing, scientists and surgeons must demonstrate the viability of theASCs in vivo to prove that the donor ASCs participate in healing of the recipient defectrather than just secreting cytokines to stimulate the surrounding host tissues. One way todemonstrate viability is to stably transduce ASCs with a lentivirus carrying triple fusionreporter genes, including firefly luciferase (Fluc), red fluorescence protein (RFP) and herpessimplex virus truncated thymidine kinase (HSV-ttk) genes.35 Stably expressed ASCs canthen be purified by fluorescence activated cell sorting based on the RFP expression (Figure1). Bioluminesence allows monitoring in vivo whereas the RFP or GFP allows for markingof these cells during histologic examination to determine if the host or implanted cells arelocated in the region of osteoid formation.

Bone Tissue Engineering: ScaffoldsStem cells exist in tightly controlled environmental niches and alterations in thismicroenvironment can dramatically modify their behavior and differentiation capacities.Furthermore, stem cells are often utilized in the setting of disease or injury whereinflammatory signals may be prevalent altering their function. Biomaterial scaffolds canpotentially provide a controlled environment protecting implanted cells from harmfulstimuli. Biomaterial matrices are also used to deliver genetic material and/or inductivebiochemical cues which allow for some degree of developmental control over the deliveredstem cells.

Numerous studies have demonstrated the benefits of utilizing stem cell-scaffold constructsin regenerative medicine. Scaffolds provide a highly modifiable vehicle for inductive factorsas 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 enhancethe osteogenic capability of ASCs. 32 BMP-2, however, is osteoinductive and likelyenhances the osteogenesis of surrounding osteoblasts, periosteal cells and underlying dura incalvarial defects. Thus, further studies must determine if the enhanced osteogenesis seenwith 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 beenapproved and is frequently used in spine surgeries. Original clinical studies in 2002 analyzeda prospective randomized analysis of 279 patients with degenerative lumbar disc diseasewho were treated with either InFUSE, or autogenous iliac bone grafts. The authorsdemonstrate that at 24 months, 94.5% fusion rate in the InFUSE treated group compared to88.7% in the bone graft treated group. Furthermore, mean operative time and blood losswere 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 theInFUSE group had lower complication rates likely related to the lack of a secondary defectcreated by harvesting iliac bone. Interestingly, this study also demonstrated a similar totalcost 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 anddemonstrated successful in vivo genetic manipulation of fibroblasts to induce boneformation in rats.38 Schek et al. demonstrated that bone formation was greater with hydrogeldelivery 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 toenhance the regenerative potential of stem cells.

Bone Tissue Engineering: CellsInvestigations into cell-based skeletal tissue engineering must identify a cell type with theability to generate bone. Shortcomings of cell types often involve their availability, viability,vascularization and elicitation of immune response upon implantation, and directeddifferentiation. Options for skeletal tissue engineering include osteoblasts, embryonic stemcells and other postnatal mesenchymal cells. Though osteoblasts have already undergonefurther differentiation than MSCs down an osteogenic pathway, osteoblasts are limited inavailability and their harvest requires loss of bone from the donor site. Attempts to enhancethe osteogenic capability of osteoblasts using mitogens and cytokines also failed to improveosteogenesis in aged osteoblasts.

The capability of ESCs to differentiate into almost any cell type found in the human bodyhas led to many promising areas of investigation which may yield deeper understanding ofcellular biology and potential cures for diseases. Previous studies have demonstrated theability of ESCs to undergo osteogenic differentiation in vivo and in vitro, however, theirpluripotency 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 clinicalcapacity, also potentially raises the challenging issue of graft-versus-host disease associatedwith allogeneic stem cell transplantation. While investigations continue to reduce donor-hostrejection, concerns regarding xenogeneic contamination also exist. ESCs have beentraditionally cultured in vitro in the presence of mouse embryonic fibroblast (MEF) feederlayers 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-freeconditions. To accomplish this, scientists developed extracellular matrix coated plates fromMEFs and sterilized prior to use.41 Other strategies proposed to avoid animal products andgraft versus host reactions include the use of autologous donor adult stem cells or the morerecently 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 todifferentiate into multiple mesenchymal tissues including bone. These cells have been ofparticular interest to dentists and oral surgeons given the increased ease of harvestingperiosteal cells compared to bone marrow cells during periodontal surgery. Furthermore,studies have demonstrated an improved proliferation rate of periosteal cells compared toMSCs and demonstrate a robust capability to undergo osteogenesis.43 Despite thisproliferative 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 differentiationin vitro and in vivo. MSCs implanted on Matrigel and placed into the lumbar fusion bed ofrats have been shown to result in enhanced fusion compared to animals receiving mixedmarrow stromal cells alone.44 A study following 51 long bones (tibia and femur) undergoingdistraction osteogenesis of long bones, and injection of MSCs along with platelet-richplasma into the callus during both lengthening and consolidation phases, resulted inaccelerated mature bone formation and a reduction in the incidence of complications in thefemur.45 Horwitz and colleagues have also investigated the use of MSCs in the treatment ofosteogenesis imperfecta, a genetic disorder of type I collagen resulting in fragile bones andskeletal deformities.46 While there is no current cure for osteogenesis imperfecta,preliminary reports have shown MSCs to be potentially successful in enhancing boneformation in these patients.46 Thus, the success of marrow derived MSCs can hopefully betranslated to the much more readily available and more easily accessible ASCs.

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

To achieve clinical translation of hASCs, ideally surgeons would harvest the cells, seedthem on an osteoinductive scaffold and reconstruct the skeletal injury/defect in the samepatient without leaving the operating room. Similar cell saving and re-use devices such asthe Cell SaverR, are used in trauma and complicated surgeries in which blood loss getscycled through this FDA approved machine and the erythrocytes can be transfused back intothe 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-2and ASC loaded scaffold back into the patient’s skeletal defect in one step, without leavingthe operating room. Interestingly, ASCs have been shown to up-regulate osteogenic genes inresponse to shear stress making them capable of responding to a mechanical load.48 Suchability to undergo osteogenesis without in vitro differentiation and responsiveness tomechanical stress makes ASCs an appealing cell source for skeletal tissue engineering.

Clinical correlatesCase report studies have examined the potential use of hASCs to heal skeletal defects in thehuman patient. Defects of facial bones and the cranium49 have been demonstrated to heal orstimulate healing with the use of ASCs. Though promising, current studies are limited asmany countries have not yet approved the use of ASCs. Furthermore, the methods of ASCusage have varied dramatically and have included combination with bone grafts, the use ofvarious osteoconductive scaffolds as well as recombinant proteins. Mesimaki et al. used a

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microvascular flap with hASCs, beta-tricalcium phosphate and BMP-2 to heal a large defectin the maxilla, reporting promising outcomes up to 8 months post-operatively. 50 Thoughencouraging, these early studies offer only level 4 and 5 data and lack significant power tohelp tailor clinical practice. Larger scale studies including prospective, randomized controltrials, must verify these findings, and to determine the optimum cell delivery method andcytokine stimuli for hASC driven osteogenesis.

ConclusionsASCs are a readily available, multipotent, abundant cell type with the capability to undergorobust osteogenesis. The fact that they can undergo osteogenic differentiation so rapidly invitro makes them an exciting candidate for in vivo studies. Even more importantly, theirability to undergo osteogenic differentiation without any stimulation when placed on anosteoconductive scaffold in vivo make ASCs a promising candidate for skeletal tissueengineering. Further studies of the mechanism of osteogenic differentiation and ways toimprove ASC osteodifferentiation by eliminating the heterogeneity and stimulating the BMPpathway offer potential for future studies.

AcknowledgmentsWe would like to thank the following people for their outstanding work in our laboratory: Emily R. Nelson, B.S.,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 Researchgrant 1 R21 DE019274-01, 1 RC2 DE020771-01, the Oak Foundation and Hagey Laboratory for PediatricRegenerative Medicine to M.T.L. B.L was supported by the National Institutes of Health, National Institute ofArthritis and Musculoskeletal and Skin Diseases grant 1F32AR057302-02. National Endowment of Plastic Surgery

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Autogenous Skeletal Repair via Paracrine Hedgehog Signaling with Calvarial Osteoblasts. StemCells Dev. 2010

54. Kai T, Shao-qing G, Geng-ting D. In vivo evaluation of bone marrow stromal-derived osteoblasts-porous calcium phosphate ceramic composites as bone graft substitute for lumbar intervertebralspinal fusion. Spine (Phila Pa 1976). 2003; 28:1653–1658. [PubMed: 12897487]

55. Mizuno H, Hyakusoku H. Mesengenic potential and future clinical perspective of human processedlipoaspirate cells. J Nippon Med Sch. 2003; 70:300–306.

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

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nih public access medicine stem cells adipose derived stromal cells for skeletal … b, stem cells....

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