Fisher John P. e.a. (ed.) Tissue Engineering
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1-4 Tissue Engineering
1.3.1 In Vitro Assays for the Osteogenic and Chondrogenic Lineages
We have established an in vitro assay for the differentiation of human and animal MSCs into bone and
cartilage phenotypes [20–23]. For osteogenesis, the inclusion of dexamethasone (dex), ascorbate, and
eventually β-glycerophosphate (a phosphate donor) causes the MSCs to enter the osteogenic lineage,
upregulate alkaline phosphatase activity (two- to ten-fold), secrete various bone proteins, and eventually,
organize calcium apatite deposition within type I collagen fibrils comparable to that observed in in vivo
osteoid.
Likewise, we have established the in vitro conditions for causing MSCs to differentiate into chondrocytes
[21,22]. Simply, MSCs are pelleted to the bottomof15-mlconical plastic tubes, and the medium is changed
to a chemically defined medium containing TGF-β at a concentration of 2–10 ng/ml. The pelleted cells
come off the bottom of the tube and form a sphere of cells that differentiate into chondrocytes that
fabricate type II collagen- and aggrecan-rich ECM. Both of these in vitro assays and the porous ceramic
assay in vivo have been tested by diluting the human or rat marrow-derived MSCs with dermal fibroblasts.
In these experiments, the human dermal fibroblasts were shown to not differentiate into bone or cartilage
phenotypes. The results of these assays are that MSC preparations can have 25–50% non-MSCs without
experiencing a major loss of osteo- or chondrogenic tissue formation [51]. In the context of Tissue
Engineering logics, these observations could mean that the implanted MSCs could be “contaminated” by
host-derived fibroblasts, but would still retain their capacity to regenerate osteochondral tissue.
1.4 MSCs and Hematopoietic Support
The marrow stroma is a highly specialized connective tissue that provides different microenvironmental
niches for the different lineage pathways of hematopoiesis. Each of the different lineage pathways of
hematopoiesis requires a different combination of cytokines and growth factors. The marrow stromacytes
not only fabricate these unique physical niches of connective tissue, but they secrete specific bioactive
molecules for the initiation and control of these lineage pathways. The MSC differentiation sequence
into these different hematopoietic lineage support phenotypes has yet to be described. However, just as
complex multicellular Dexter cultures are able to support in vitro hematopoiesis, so can homogeneous
populations of MSCs incubated in Dexter medium provide support for hematopoiesis. Because of these
very specific hematopoietic-support functions, the term “marrow stromal cells” should be reserved for
these highly differentiated marrow connective tissue cells that facilitate hematopoietic differentiation.
It is important to stress that the differentiation of MSCs into marrow stroma or osteoblasts involves
a time-dependent sequence of differentiation steps (the lineage) with each step involving the up- and
down-regulation of many genes. For example, we have compared the cytokine secretion into the medium
in 24 h by human MSCs cultured in growth medium, in osteogenic medium (dex, ascorbate), and in
stroma-generating medium (Dexter conditions or in the presence of IL-1α). The cytokine quantity is very
different for these three culture conditions [28]. Also of considerable importance was the realization that
the constitutive levels of these cytokines are different for each donor batch of cells under standard growth
or differentiation conditions. These measured differences emphasize the influence of the genotype on
the observed differentiation/lineage pathways and multigenic expression profiles of each MSC donor or
recipient or both.
1.4.1 Muscle, Tendon, and Fat
Both in vitro and in vivo, microenvironments control the differentiation and expressional profile of the
MSCs and their differentiated descendants. Without going into all of the details, MSCs have been shown
to differentiate into skeletal muscle, into Achilles or patella tendon tissue, and into adipocytes as reviewed
earlier in this chapter. The cell culture conditions or in vivo tissue sites were different for each of the
phenotypes observed. Importantly, the introduction of MSCs into specific tissue locations was followed
by using markers or molecular probes [53,54]. For example, normal rodent marrow-derived MSCs form
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Fundamentals of Stem Cell Tissue Engineering 1-5
Proliferation
Commitment
Lineage
progression
Differentiation
Maturation
Osteogenesis
Transitory
osteoblast
Osteoblast
Osteocyte
BONE
CARTILAGE
MUSCLE
MARROW
TENDON/
LIGAMENT
CONNECTIVE
TISSUE
Hypertrophic
chondrocyte
Myotube
Stromal
cells
T/L
fibroblast
Adipocytes,
dermal, and
other cells
Mesenchymal tissue
Bone marrow/periosteum
THE MESENGENIC PROCESS
Mesenchymal stem cell (MSC)
MSC prollferation
Chondrogenesis Myogenesis Marrow stroma
Tendogenesis/
ligamentagenesis
Transitory
fibroblast
Transitory
stromal cell
Myoblast
Myoblast fusion
Chondrocyte
Transitory
chondrocyte
Unique
micro-niche
Other
FIGURE 1.2 The mesengenic process in which transdifferentiation is depicted by horizontal arrows. For example,
adipocytes can transdifferentiate into osteoblasts.
dystrophin-positive skeletal muscle in the limbs of mdx mice that are dystrophin-negative [25]. Likewise,
reparative neo-tissue formed in rabbit tendon defects when autologous MSCs were introduced in suitable
scaffolds [30,31].
The isolation of MSCs from fat provides an intriguing question of the origin of these progenitor cells
[34,55]: Are these MSCs an intrinsic occupant of fat, are they associated with the blood vessels, or are
they dedifferentiated adipocytes? Both marrow- and fat-derived MSCs differentiate into adipocytes and
the other phenotypes of the Mesengenic Lineage (Figure 1.1). These observations raise the question of
phenotypic plasticity and whether differentiated cells dedifferentiate into a stem cell and then rediffer-
entiate into an alternate phenotype or whether the differentiated cells transdifferentiate directly into a
different mesenchymal phenotype (as pictured in Figure 1.2). The current data indicate that cells can
transdifferentiate along certain permissible routes without backing up to the stem cell status.
1.4.2 A New Fundamental Role for MSCs
Previously, all of the preclinical models used MSCs to provide the cells that fabricate the specialized,
differentiated tissues. I propose that MSCs could be used in a different clinical context: that MSCs could
provide a trophic influence to structure a reparative microenvironment. The precedent for cells secreting
cytokines/growth factors that have regulator effects is well established in regenerative biology. For example,
Singer long ago established the trophic effect of nerves on amphibian limb regeneration [56]. He showed
that a certain quantum of nerves in the limb stump is required for successful limb regeneration; the nerves
delivered neural trophic factors and the electrical or neural transmitter release was not associated with
this trophic effect as evidenced by switching the ratios of different nerves into the limb stump.
In this regard, the bone marrow stroma derived from MSCs provides very specific instructional niches
for various lineage pathways of hematopoiesis as discussed earlier. The question arises, “Can MSCs
structure reparative microenvironments without differentiating into specific differentiated cells or tissue?”
The answer appears to be “yes!” In the cases of cardiac infarct (ischemia) and stroke (brain ischemia),
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1-6 Tissue Engineering
bone marrow-derived MSCs when injected into the ischemic zones do not massively differentiate into
cardiac myocytes or neural cells. Rather, the MSCs appear to establish a trophic influence that is antifibrotic
(antiscar tissue) and angiogenic [57–60]. The lack of the formation of extensive fibrotic, scar tissue and
the rapid revascularization of the affected tissue zones result in clinically improved tissues.
These new findings suggest to me a new Tissue Engineering paradigm: the site-specific delivery of
trophic cells (cells that secrete specific cytokines/growth factors) that structure reparative microenviron-
ments. Already well established is the concept of genetically engineering the insertion of a specific gene,
for example, transfection with a BMP-containing insert, to be synthesized by an implanted cell to stimulate
bone regeneration at that implantation site [61,62]. In this context, why not implant to a specific location a
normal cell that has a powerful trophic activity that will cause the implantation site to support restorative
therapy? Likewise, the trophic effect may not stimulate repair, but rather, it may inhibit a debilitating
effect, such as fibrosis, so that the slower, more natural host-mediated regenerative events can take place.
In some cases, as mentioned earlier, MSCs can provide such a trophic effect.
1.4.3 The Use of MSCs Today and Tomorrow
The current use of MSCs is facilitated by their capacity to be expanded in culture. The introduction of these
cells back in vivo is facilitated by site-specific scaffolds. In some cases, the MSCs can be jump-started down
a lineage pathway by exposing them in culture to a specific bioactive agent (dex for bone [23,49], TGF-β
for cartilage [20,21], etc.). In the long-term, there will be a way to mobilize the body’s own MSCs, direct
them to a site, expand them at the site, and signal them into a lineage pathway including modulation of the
phenotypic expression of the cells to produce the appropriate tissues for that repair site (articular cartilage
at the knee and auricular cartilage in the ear). There are at least two unknowns in this process. First, we
do not know what mobilizes MSCs from marrow or other depots, although there are data that suggest
such mobilization takes place following injury [34,35,63]. Second, we do not know how such mobilized
MSCs dock into specific tissues. Consistent with this lack of knowledge is our inability to obtain high
engraftment percentages for systemically administered, culture-expanded (marked) MSCs [64]. At best,
only 2 to 3% of systemically administered MSCs home back to bone marrow with most of the infused
cells lodging in the lung (probably a size issue) and liver (probably a fibronectin issue).
1.5 Cell Targeting
To systematically learn more about control of engraftment of systemically or locally introduced reparative
cells, we have developed protocols which facilitate the targeting of cells to specific molecular docking sites.
The approach is to insert specific targeting molecules into the cell membranes. The first approach involved
palmitoylating protein G and painting this “primer” coat on cells, since the hydrophobic fatty acid tail of
palmitic acid will quantitatively insert into the cell’s plasma membrane lipid bilayer core. This positions
the protein to orient out of the cell’s surface. Protein G binds strongly to the Fc-region of antibodies.
Thus, we can put different paints (antibodies) on the primer coat on the cell’s surface. Moreover, if we had
specific addressing or docking peptides, we could construct a fusion protein with such addresses in series
with the Fc-region of antibodies in a molecularly reconstructed fusion protein as pictured in Figure 1.3.
In a proof of this technology concept, Dennis et al. [65] have created a deep cartilage defect in the
condyle of rabbits. A library of antibodies exists against various epitopes in the ECM of deep articular
cartilage as opposed to the surface of the condylar cartilage. With these antibodies, cultured rabbit or
human chondrocytes were shown to home to the defect and subsequently fabricate cartilage ECM in an
organ culture test system. The painting moieties were shown to not affect the viability or replication of
the painted cells nor the differentiation capacity of painted MSCs. The long-term goal is the injection of
painted chondrogenic cells into the synovial space to direct their docking to cartilage defects and, then, to
facilitate the repair/regeneration of the cartilage.
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Fundamentals of Stem Cell Tissue Engineering 1-7
Any peptide or protein
Protein A or G PRIMER
PAINT
Palmitate anchor
Cell membrane
Fc
g1
SYSTEMIC CELL TARGETING
FIGURE 1.3 Cell targeting technology involves palmitic acid that is derivatized with Protein A or G (PRIMER). The
fatty acid hydrophobic tail quantitatively inserts into the lipid bilayer of the cell membrane. An antibody or fusion
protein with the Fc domain at one end is referred to as the Paint.
I could imagine using the painting technology to insert tissue-specific address or targeting molecules
to implanted scaffold docking sites as a basis for various systemically or locally supplied reparative cells,
for example, stem cells for a variety of tissues. The docking of these cells could improve tissue performance
or repair localized defects. Enhanced engraftment of stem cells, either by mobilizing intrinsic popula-
tions or by injecting extrinsically expanded cells, should provide useful Tissue Engineering strategies for
tomorrow. Clearly, a lot of work needs to be done before this long-term goal will be realized.
Acknowledgments
The research was supported by grants from NIH, DOE, and the State of Ohio. I thank my colleagues at
Case Western Reserve University and elsewhere for providing the experimental basis for this chapter.
References
[1] Nye, H.L. et al., Regeneration of the urodele limb: a review, Dev. Dyn., 226, 280–294, 2003.
[2] Bell, E., Living tissue formed in vitro and accepted as skin-equivalent tissue of full thickness, Science,
211, 1052–1054, 1981.
[3] Boyce, S.T., Cultured skin for wound closure, in Skin Substitute Production by Tissue Engineering:
Clinical and Fundamental Applications, chap. 4, Rouabhila, M. (Ed.), Landes Bioscience, Austin,
1997.
[4] Hardin-Young, J. et al., Approaches to transplanting engineered cells and tissues, in Principles of
Tissue Engineering, chap. 23, Guilak, F., Butler, D.L., Goldstein, S.A., and Mooney, D.J. (Eds.), 2nd
ed. Academic Press, 2000.
[5] Brisco, D.M. et al., The allogeneic response to cultured human skin equivalent in the hu-PBL-SCID
mouse model of skin rejection, Transplantation, 67, 1590–1599, 1999.
[6] Caplan, A.I., Biomaterials and bone repair, BIOMAT, 87, 15–24, 1988.
[7] Caplan, A.I., Cell delivery and tissue regeneration, J. Control. Release, 11, 157–165, 1989.
[8] Caplan, A.I., Mesenchymal stem cells, J. Orthop. Res., 9, 641–650, 1991.
[9] Caplan, A.I. et al., Cell-based technologies for cartilage repair, in Biology and Biomechanics of
the Traumatized Synovial Joint: The Knee as a Model, Finerman, G.A.M. and Noyes, F.R. (Eds.),
American Academy of Orthopaedic Surgeons, Rosemont, pp. 111–122, 1992.
[10] Haynesworth, S.E., Baber, M.A., and Caplan, A.I., Cell surface antigens on human marrow-derived
mesenchymal cells are detected by monoclonal antibodies, Bone, 13, 69–80, 1992.
[11] Caplan, A.I., The mesengenic process, Clin. Plast. Surg., 21, 429–435, 1994.
mikos: “9026_c001” — 2007/4/9 — 15:50 — page8—#8
1-8 Tissue Engineering
[12] Jargiello, D.M. and Caplan, A.I., The fluid flow dynamics in the developing chick wing, in Limb
Development and Regeneration, Part A, Fallon, J.F. and Caplan, A.I. (Eds.), Alan R. Liss, Inc.,
New York, pp. 143–154, 1983.
[13] Caplan, A.I., Cartilage, Sci. Am., 251, 84–94, 1984.
[14] Caplan, A.I., The molecular controlof muscle and cartilage development, in 39th AnnualSymposium
of the Society for Developmental Biology, Subtelney, S. and Abbott, U. (Eds.), Alan R. Liss, Inc.,
New York, pp. 37–68, 1981.
[15] Friedenstein, A.J., Petrakova, K.V., Kurolesova, A.I., and Frolova, G.P., Heterotopic of bone marrow.
Analysis of precursor cells for osteogenic and hematopoietic tissues, Transplantation, 6, 230–247,
1968.
[16] Owen, M., Lineage of osteogenic cells and their relationship to the stromal system, in Bone and
Mineral Research, chap. 1, Peck, W.A. (Ed.), Elsevier Science, Oxford, 1985.
[17] Haynesworth, S.E. et al., Characterization of cells with osteogenic potential from human marrow,
Bone, 13, 81–88, 1992.
[18] Haynesworth, S.E., Goldberg, V.M., and Caplan, A.I., Diminution of the number of mesenchymal
stem cells as a cause for skeletal aging, in Musculoskeletal Soft-Tissue Aging: Impact on Mobility,
Section 1, chap. 7, Buckwalter, J.A., Goldberg, V.M., and Woo, S.L.-Y. (Eds.), American Academy of
Orthopaedic Surgeons, Publishers, pp. 79–87, 1994.
[19] Lennon D.L. et al., Human and animal mesenchymal progenitor cells from bone marrow: iden-
tification of serum for optimal selection and proliferation, In Vitro Cell Dev. Biol., 32, 602–611,
1996.
[20] Johnstone, B. et al., In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells,
Exp. Cell Res., 238, 265–272, 1998.
[21] Yoo, J.U. et al., The chondrogenic potential of human bone-marrow-derived mesenchymal
progenitor cells, J. Bone Joint Surg., 80, 1745–1757, 1998.
[22] Dennis, J.E. and Caplan, A.I., Differentiation potential of conditionally immortalized mesenchymal
progenitor cells from adult marrow of a H-2Kb-tsA58 transgenic mouse, J. Cell. Physiol., 167,
523–538, 1996.
[23] Jaiswal, N., Haynesworth, S.E., Caplan, A.I., and Bruder, S.P., Osteogenic differentiation of pur-
ified, culture-expanded human mesenchymal stem cells in vitro, J. Cell Biochem., 64, 295–312,
1997.
[24] Wakitani, S., Saito, T., and Caplan, A.I., Myogenic cells derived from rat bone marrow mesenchymal
stem cells exposed to 5-azacytidine, Muscle Nerve, 18, 1417–1426, 1995.
[25] Saito, T. et al., Myogenic expression of mesenchymal stem cells within myotubes of mdx mice in vitro
and in vivo, Tissue Eng., 1, 327–344, 1996.
[26] Majumdar, M. et al., Human marrow-derived mesenchymal stem cells (MSCs) express hema-
topoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and
osteogenic lineages. J. Hematother. Stem Cell Res., 9, 841–848, 2001.
[27] Dennis, J.E., Carbillet, J.-P., Caplan, A.I., and Charbord, P., The STRO-1+marrow cell population
is multi-potential, Cells Tissues Organs, 170, 73–82, 2002.
[28] Haynesworth, S.E., Baber, M.A., and Caplan, A.I., Cytokine expression by human marrow-derived
mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1α, J. Cell Physiol., 166,
585–592, 1996.
[29] Dennis, J.E. et al., A quadripotential mesenchymal progenitor cell isolated from the marrow of an
adult mouse, J. Bone Mineral. Res., 14, 1–10, 1999.
[30] Young, R.G. et al., The use of mesenchymal stem cells in Achilles tendon repair, J. Orthop. Res., 16,
406–413, 1998.
[31] Awad, H. et al., Autologous mesenchymal stem cell-mediated repair of tendon, Tissue Eng.,5,
267–277, 1999.
[32] Weimann, J.M. et al., Contribution of transplanted bone marrow cells to Purkinje neurons in
human adult brains, Proc. Natl Acad. Sci. USA, 100, 2088–2093, 2003.
mikos: “9026_c001” — 2007/4/9 — 15:50 — page9—#9
Fundamentals of Stem Cell Tissue Engineering 1-9
[33] Prockop, D.J., Marrow stromal cells as stem cells for nonhematopoetic tissues, Science, 276, 71–74,
1997.
[34] Laflamme, M.A., Myerson, D., Saffitz, J.E., and Murry, C.E., Evidence for cardiomyocyte repop-
ulation by extracardiac progenitors in transplanted human hearts, Circ. Res., 90, 634–640,
2002.
[35] Orlic, D. et al., Mobilized bone marrow cells repair the infracted heart, improving function and
survival, Proc. Natl Acad. Sci. USA, 98, 10344–10349, 2001.
[36] Jackson, K.A. et al., Regeneration of ischemic cardiac muscle and vascular endothelium by adult
stem cells, J. Clin. Invest., 107, 1395–1402, 2001.
[37] Shi, S. and Gronthos, S., Pervascular niche of postnatal mesenchymal stem cells in human bone
marrow dental pulp, J. Bone Mineral Res., 18, 696–704, 2003.
[38] Jiang, Y. et al., Pluripotency of mesenchymal stem cells derived from adult marrow, Nature, 418,
41–49, 2002.
[39] Reyes, M. et al., Purification and ex vivo expansion of postnatal human marrow mesodermal
progenitor cells, Blood, 98, 2615–2625, 2001.
[40] Caplan, A.I., Tissue engineering strategies for the use of mesenchymal stem cells to regenerate
skeletal tissues, in Functional Tissue Engineering, chap. 10, Guilak F., Butler, D.L., Goldstein, S.A.,
and Mooney, D.J. (Eds.), Springer-verlag, New York, 2003.
[41] Caplan, A.I. et al., The principles of cartilage repair/regeneration, Clin. Orthop. Relat. Res., 342,
254–269, 1997.
[42] Caplan, A.I., Embryonic development and the principles of tissue engineering, in Novartis
Foundation: Tissue Engineering of Cartilage and Bone, John Wiley & Sons, London, pp. 17–33,
2003.
[43] Kujawa, M.J. and Caplan, A.I., Hyaluronic acid bonded to cell culture surfaces stimulates
chondrogenesis in stage 24 limb mesenchyme cell cultures, Dev. Biol., 114, 504–518, 1986.
[44] Kujawa, M.J., Carrino, D.A., and Caplan, A.I., Substrate-bonded hyaluronic acid exhibits a size-
dependent stimulation of chondrogenic differentiation of stage 24 limb mesenchymal cells in
culture, Dev. Biol., 114, 519–528, 1986.
[45] Caplan, A.I., Tissue engineering designs for the future: new logics, old molecules, Tissue Eng.,6,
1–8, 2000.
[46] Ponticiello, M.S. et al., Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal
stem cells in cartilage regeneration therapy, in Osiris Therapeutics, John Wiley & Sons, 2000.
[47] Caplan, A.I. and Pechak, D.G., The cellular and molecular embryology of bone formation, in Bone
and Mineral Research, Vol. 5, Peck, W.A. (Ed.), Elsevier, New York, pp. 117–184, 1987.
[48] Caplan, A.I., Bone development, in Cell and Molecular Biology of Vertebrate Hard Tissues, CIBA
Foundation Symposium 136, Wiley, Chichester, pp. 3–21, 1988.
[49] Ohgushi, H. and Caplan, A.I., Stem cell technology and bioceramics: from cell to gene engineering,
J. Biomed. Mater. Res., 48, 1–15, 1999.
[50] Dennis, J.E., Konstantakos, E.K., Arm, D., and Caplan, A.I., In vivo osteogenesis assay: a rapid
method for quantitative analysis, Biomaterials, 19, 1323–1328, 1998.
[51] Lennon, D.P. et al., Dilution of human mesenchymal stem cells with dermal fibroblasts and the
effects on in vitro and in vivo osteogenesis, Dev. Dynam., 219, 50–62, 2000.
[52] Goshima, J., Goldberg, V.M., and Caplan, A.I., The osteogenic potential of culture-expanded rat
marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks, Clin. Orthop. Relat.
Res., 262, 298–311, 1991.
[53] Allay, J.A. et al., LacZ and IL-3 expression in vivo after retroviral transduction of marrow-derived
human osteogenic mesenchymal progenitors, Hum. Gene Ther., 8, 1417–1427, 1997.
[54] Gimble, J.M. et al., The function of adipocytes in the bone marrow stroma: an update, Bone, 19,
421–428, 1996.
[55] Dragoo, J.L. et al., Tissue-engineered cartilage and bone using stem cells from human infrapatellar
fat pads, J. Bone Joint Surg., 85, 740–747, 2003.
mikos: “9026_c001” — 2007/4/9 — 15:50 — page 10 — #10
1-10 Tissue Engineering
[56] Singer, M., Neurothophic control of limb regeneration in the newt, Ann. NY Acad. Sci., 228,
308–322, 1974.
[57] Stamm, C. et al., Autologous bone-marrow stem-cell transplantation for myocardial regeneration,
Lancet, 361, 45–46, 2003.
[58] Shake, J.G. et al., In-vivo mesenchymal stem cell grafting in a swine myocardial infarct model:
molecular and physiologic consequences, Ann. Thorac. Surg., 73, 1919–1926, 2002.
[59] Chen, J. et al., Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells
after cerebral ischemia in rats, J. Neurol. Sci., 189, 49–57, 2001.
[60] Chen, J. et al., Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes
endogenous cell proliferation after stroke in female rat, J. Neurosci. Res., 73, 778–786, 2003.
[61] Lieberman, J.R. et al., The effect of regional gene therapy with bone morphogenetic protein-2
producing bone-marrow cells on the repair of segmental femoral defects in rats, J. Bone Joint Surg.
Am., 81, 905–917, 1999.
[62] Evans, C.H. and Robbins, P.D., Possible orthopaedic applications of gene therapy, J. Bone Joint Surg.
Am., 77, 1103–1114, 1995.
[63] Mahmood, A. et al., Treatment of traumatic brain injury in adult rats with intravenous
administration of human bone marrow stromal cells, Neurosurgery, 53, 697–703, 2003.
[64] Gao, J. et al., The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells
after infusion, Cells Tissues Organs, 169, 12–20, 2001.
[65] Dennis, J.E., Cohen, N., Goldberg, V.M., and Caplan, A.I., Targeted delivery of progenitor cells for
cartilage repair, J. Orthop. Res., 22, 735–741, 2004.
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2
Growth Factors and
Morphogens: Signals
for Tissue Engineering
A. Hari Reddi
University of California
2.1 Introduction.............................................. 2-1
2.2 Tissue Engineering and Morphogenesis ............... 2-1
2.3 TheBoneMorphogeneticProteins ..................... 2-2
2.4 Growth Factors........................................... 2-3
2.5 BMPs Bind to Extracellular Matrix ..................... 2-3
2.6 Clinical Applications .................................... 2-4
2.7 Challenges and Opportunities .......................... 2-4
Acknowledgments............................................... 2-4
References ....................................................... 2-4
2.1 Introduction
Tissue engineering is the exciting discipline of design and construction of spare parts for the human
body to restore function based on biology and biomedical engineering. The basis of tissue engineering
is the triad of signals for tissue induction, responding stem cells, and the scaffolding of extracellular
matrix. Among the many tissues in the human body bone has the highest power of regeneration and
therefore is a prototype model for tissue engineering based on morphogenesis. Morphogenesis is the
developmental cascade of pattern formation, body plan establishment, and culmination of the adult body
form. The cascade of bone morphogenesis in the embryo is recapitulated by demineralized bone matrix-
induced bone formation. The inductive signals for bone morphogenesis, the bone morphogenetic proteins
(BMPs) were isolated from demineralized bone matrix. BMPsand related cartilage-derived morphogenetic
proteins (CDMPs) initiate cartilage and bone formation. The promotion and maintenance of the initiated
skeleton is regulated by several growth factors. Tissue engineering is the symbiosis of signals (growth
factors and morphogens), stem cells, and scaffolds (extracellular matrix). The rules of architecture for
tissue engineering are a true imitation of principles of developmental biology and morphogenesis.
2.2 Tissue Engineering and Morphogenesis
An understanding of the molecular principles of development and morphogenesis, is a prerequisite for
tissue engineering. We define tissue engineering as the science of design and manufacture of new tissues
2-1
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2-2 Tissue Engineering
for functional restoration of the impaired organs and replacement of lost parts due to disease, trauma,
and tumors [1]. Tissue engineering is based on principles of developmental biology and morphogenesis,
biomedical engineering, and biomechanics.
Morphogenesis is initiated by morphogens. The promotion and maintenance of morphogenesis is
achieved by a variety of growth factors. Generally, morphogens are first identified in fly and frog embryos
by genetic approaches, differential displays, and subtractive hybridization expression cloning. An alternate
biochemical approach of “grind and find” from adult mammalian bone led to the isolation of BMPs, the
premier signals for bone morphogenesis. We now discuss the identification, isolation, and molecular
cloning of BMPs from a natural biomaterial, the demineralized bone matrix.
2.3 The Bone Morphogenetic Proteins
Bone grafts have been used to aid the healing of recalcitrant fractures. Demineralized bone matrix induced
new bone formation. Bone induction by demineralized bone matrix is a sequential cascade [2–4]. The
key steps in this cascade are chemotaxis of progenitor cells, proliferation of progenitor cells, and finally
differentiation first into cartilage and then bone. The demineralized bone matrix is devoid of any living
cells and is a biomaterial that elicits new bone formation. The insoluble collagenous bone matrix binds
plasma fibronectin [3] and promotes the proliferation of cells. Proliferation was maximal on day 3,
chondroblast differentiation was evident on day 5, and chondrocytes were abundant on day 7. Thecartilage
hypotrophied on day 9 with concominant vascular invasion and osteogenesis. On days 10 to 12 maximal
alkaline phosphatase activity, a marker of bone formation, was observed. Hematopoietic differentiation
was observed in the ossicle on day 21. The sequential bone development cascade is reminiscent of bone
morphogenesis in limb.
A systematic study of the biochemical basis of bone induction was initiated. A bioassay for bone
induction was established in vivo in rats. The insoluble demineralized bone matrix was extracted
in 4 M guanidine hydrochloride, a dissociative extractant. About 3% of the proteins were solubil-
ized and the rest was the insoluble residue. The extract and the residue alone were unable to induce
bone formation. However, reconstitution of the extract to residue yielded new bone morphogenesis.
Thus there is collaboration between soluble signals and the insoluble matrix scaffold to yield new
bone formation [5,6]. This key experiment predates the term tissue engineering and demonstrates
the collaboration of soluble signals and insoluble scaffolding as a critical concept in practical tissue
engineering. Collagen appear to be an optimal scaffold [7]. The bone induction is dependent on the
hormonal status including vitamin D [8,9]. Irradiation of the recipient blocked the cellular cascade of
osteogenesis [10].
This bioassay was a critical development in the quest for the purification of the bioactive bone morpho-
gens, the bone morphogenetic proteins [11–15]. There are nearly 15 members of BMPs in the human
genome (Table 2.1). BMPs are dimeric molecules with a single disulfide bond. The mature monomer con-
sists of seven canonical cysteines contributing to three interchain disulfides and one interchain disulfide
bond.
BMPs stimulate chondrogenesis in limb bud mesodermal cells [16]. BMP 2 stimulates osteoblast
maturation [17]. BMPs are chemotactic for human monocytes [18]. In addition to initiating chon-
drogenesis BMPs maintain proteoglycan biosynthesis in bovine articular cartilage explants [19,20].
Recombinant human growth/differentiation factors (GDF-5) stimulate chondrogenesis during limb
development [21].
BMPs interact with BMP receptors I and II on the cell surface/membrane [1,22]. BMP receptors are
serine/threonine kinases. The intracellular substrates for these kinases called Smads function as relays to
activate the transcriptional machinery [23]. The three functional classes are (1) receptor-regulated Smads,
namely, Smads 1, 5, and 8, (2) the common partner Smad — 4, and (3) the inhibitory Smads 6 and 7.
There are Smad-dependent and independent pathways for activation of BMP signaling including new
bone formation.
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Growth Factors and Morphogens 2-3
TABLE 2.1 The BMP Family in Mammals
a
BMP subfamily Generic name BMP designation
BMP 2/4 BMP-2A BMP-2
BMP-2B BMP-4
BMP-3 Osteogenin BMP-3
Growth/differentiation factor-10 (GDF-10) BMP-3B
OP-1/ BMP-7 BMP-5 BMP-5
Vegetal related-1 (Vgr-1) BMP-6
Osteogenic protein-1 (OP-1) BMP-7
Osteogenic protein-1 (OP-2) BMP-8
Osteogenic protein-1 (OP-3) BMP-8B
Growth/differentiation factor-2 (GDF-2) BMP-9
BMP-10 BMP-10
Growth/differentiation factor-11 (GDF-11) BMP-11
GDF-5,6,7 Growth/differentiation factor-7 (GDF-7) or
cartilage-derived morphogenetic protein-3 (CDMP-3)
BMP-12
Growth/differentiation factor-6 (GDF-6) or
cartilage-derived morphogenetic protein-2 (CDMP-2)
BMP-13
Growth/differentiation factor-5 (GDF-5) or
cartilage-derived morphogenetic protein-1 (CDMP-1)
BMP-14
BMP-15 BMP-15
a
BMP-1 is not a BMP family member with seven canonical cysteines. It is a procollagen-C proteinase
related to Drosophila Tolloid.
2.4 Growth Factors
Growth factors are proteins with profound influence on proliferation and growth of cells. Growth factors
stimulate the differentiation of progenitor/stem cells. The growth factors include many subgroups and
such as Insulin like growth factors (IGFs), fibroblast growth factors (FGFs), and platelet-derived growth
factors (PDGFs).
Insulin like growth factors are polypeptides related to Insulin. There are two members, IGF-I and
IGF-II. Liver is the predominant site of IGF-I synthesis, and is stimulated by pituitary growth hormone.
IGF biological activity is modulated by IGF-binding proteins (IGFBP). There are six different IGFBPs.
IGFs promote extracellular matrix biosynthesis by osteoblasts.
Fibroblast growth factors (FGFs) are proteins with multiple members. FGFs are mitogens for endothelial
cells. Along with Vascular Endothelial Growth Factors (VEGFs), FGFs are critical for bone formation. It
is well known that vascular invasion is a prerequisite for endochodral bone formation.
Platelet-derived growth factors come in three isoforms, namely, AA, AB, and BB. They are primarily
produced by platelets in blood. Various isoforms stimulate bone formation.
2.5 BMPs Bind to Extracellular Matrix
The critical role of extracellular matrix in morphogenesis of many tissues during development is well
known. The extracellular matrix is a supramolecular assembly of collagens, proteoglycans, and gly-
coproteins. The collagens are tissue specific and the proteoglycans include chondroitin sulfate, dermatan
sulfate, heparan sulfate/heparin, and keratan sufate. Recombinant BMP 4 and BMP 7 bind to heparan
sulfate/heparin, collagen IV of the basement membrane [24]. Thebinding of a soluble morphogen to insol-
uble extracellular matrix renders the morphogen to act locally and protects it from proteolytic degradation
and therefore extends its biological half-life. Thus, extracellular matrix scaffolding is an efficient delivery
system for tissue engineering. Growth factors such as FGFs bind to heparan sulfate. An emerging concept
for tissue engineering is the tethering of signals to scaffolds to restrict their diffusion.