Tissue Engineering with Mesenchymal Stem Cells

Transplant surgery is a leading therapy to treat organ failure or tissue loss. In 2001 in the United States alone, despite 24,076 lifesaving organ transplants, 6,439 people died waiting for a transplant, leaving 84,798 registrations on the waiting list for an organ at the end of the year [1]. It is clear that organ transplant alone is not a viable solution to treating organ failure and tissue loss. For some defects, autologous transplants, where a patient’s own tissue is taken from another site of the body for repair or reconstruction, are performed. This method eliminates immune response and related complications, but it introduces the possibility of donor-site morbidity. The replacement of damaged or defective tissue with newly regenerated tissue grown from the patient’s cells is an ideal solution and is the motivation for tissue engineering. As stated by Langer and Vacanti in 1993, “Tissue engineering is an interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function” [2].
There are three main approaches originally described for tissue engineering [2], [3], which are continually being refined. These strategies are as follows: 1) to guide tissue regeneration using engineered matrices alone; 2) to inject autologous, allogeneic, or xenogeneic cells alone; and/or 3) to create constructs of cells placed on or within matrices (Figure 1). The first method typically involves implanting a scaffold at the site of interest and allowing cells to migrate in from surrounding tissues to populate the scaffold. The second method has the advantage of minimal surgical invasion; in addition, cells may be manipulated by recombinant gene technologies or clonal expansion prior to injection or infusion. In the third method, constructs of cells seeded in biodegradable scaffolds designed to function as synthetic extracellular matrices are implanted at the repair site in the body. In practice, organ-specific cells are often seeded into the scaffold ex vivo prior to transplantation, and with time the cells synthesize a new extracellular matrix as the scaffold degrades simultaneously and eventually produce a new, properly functioning tissue.
This article focuses on the second and third above-mentioned tissue engineering therapies and presents the potential use of autologous stem cells in place of allogeneic or xenogeneic tissue-specific cells, with an emphasis on adult mesenchymal stem cells. Scaffolds, synthetic extracellular matrices to support cell growth and tissue development, are covered with respect to specific material requirements, scaffold types, and fabrication techniques. Stem cells, unspecialized cells that possess significant self-renewal ability and have the potential to become cells with specialized functions, are discussed in greater detail with an emphasis on mesenchymal stem cells (MSCs). Tissue engineering studies with scaffolds seeded with MSCs are reviewed with emphasis on cartilage and bone tissue engineering from our group.

The extracellular matrix (ECM) consists of tissue-specific macromolecules secreted by the cell to form a complex network that functions as a supporting framework to yield the form and shape of the tissue. The ECM provides an organized environment within which migratory cells can move and interact with one another and stationary cells are anchored. Cell-matrix interactions also serve to initiate and mediate responses that regulate cell growth, migration, differentiation, survival, tissue organization, and matrix remodeling. In connective tissues, the matrix bears most of the mechanical stress to which the tissue is subjected. The role of the tissue engineering scaffold is to serve as a synthetic ECM to support cell growth and tissue development. Clearly, the ECM is a complex and dynamic structure. As such, the scaffold should at minimum satisfy certain requirements. It should be three-dimensional and highly porous with an interconnected pore network to facilitate cell/tissue growth and diffusional transport of nutrients and waste. The scaffold material should be biodegradable or bioresorbable with controlled degradation rate to match tissue development rate; rapid breakdown of the scaffold will not provide enough structural support to a new tissue, while the other extreme may impede tissue growth and development. The material should also have suitable surface chemistry for cell attachment, proliferation, and differentiation. Furthermore, the mechanical properties of the scaffold should match those of the tissues at the site of implantation. Finally, the scaffold defines the shape and size of the regenerated tissue and thus needs to be easily fabricated in a variety of shapes and sizes.
Tissue engineering scaffolds have been fabricated with a variety of synthetic and natural materials. Synthetic materials range from aliphatic polyesters [4]-[6] to poly(ethylene oxide) [7] to hydroxyapatite [8] (Figure 2). On the other hand, collagen [9] and alginate [10], [11] are examples of some of the natural materials that are commonly used (Figure 3). Numerous methods have been employed in fabricating scaffolds [12], [13],many adapted from well-established materials science techniques. Fabrication techniques include textile technologies, solvent casting and particulate leaching, phase separation, melt processing, emulsion diffusion or freeze-drying, fluid gassing, and solid free-form fabrication. These and other methods, used independently or in concert, have created a variety of scaffolds, many of which are porous sponges, woven or nonwoven structures, and hydrogels.

Stem Cells
Proliferative capacity of many adult tissue-specific cells is very limited, making their expansion in vitro difficult in preparation for scaffold seeding. Long-term in vitro cultivation also reduces their functional quality. Thus, attention has started shifting from seeding scaffolds with tissue-specific cells to the use of stem cells or progenitor cells. Stem cells are unspecialized cells that possess significant self-renewal ability and have the potential to be induced to become cells with specialized functions. Three unique properties of all stem cells are as follows: 1) they are capable of dividing and renewing themselves for long periods; 2) they are unspecialized; and 3) they can give rise to specialized cell types [14].


Fig. 1. Three main approaches to tissue engineering. Strategy 1: To guide tissue regeneration using engineered matrices alone. Strategy 2: To inject autogenic, allogeneic, or xenogeneic cells alone. Strategy 3: To create constructs of cells placed on or within matrices.

A hierarchy exists among stem cells that is defined by a capacity to self-renew and also a differentiation potential (Figure
4). A stem cell can be considered totipotent, pluripotent, or multipotent [15]. A totipotent stem cell can form all cells/tissues that contribute to the formation of an organism (e.g., the fertilized egg or zygote); a pluripotent stem cell can form most (but not all) cells/tissues of an organism (e.g. embryonic stem cells and embryonic germ layers); and a multipotent stem cell can form a small number of cells/tissues that are usually restricted to a particular germ layer origin (e.g., bone marrow stromal or mesenchymal stem cells). Recently, the concept of transdifferentiation, referring to the ability of differentiated cells to assume another lineage, is actively being investigated.


Fig. 2. Scanning electron microscopy image of human bone-marrow-derived mesenchymal stem cells seeded on nano-spun poly(ε-caprolactone) fibrous scaffold. Unpublished data, courtesy of W.J. Li and R.S. Tuan.


Fig. 3. Optical microscopy micrograph of human bone marrow-derived mesenchymal stem cells seeded in collagen gel. Magnification: 1000×. Unpublished data.

Stem cells are unspecialized cells that possess significant self-renewal ability and have the potential to be induced to become cells with specialized functions.

Embryonic stem cells have the capacity to give rise to differentiated cell types that are derived from the three primary
germ layers of the embryo: endoderm, mesoderm, and ectoderm. Under specific culture conditions, embryonic stem
cells are capable of long-term self-renewal without differentiating. However, legal and ethical issues surrounding the use of embryonic stem cells have turned much attention to the use of adult stem cells. An adult stem cell is an undifferentiated (unspecialized) cell that resides in a differentiated (specialized) tissue, with the capacity for self-renewal and specialization to yield multiple cell types, including those specific from the original tissue. Relative to embryonic stem cells, adult stem cells have lower proliferation capacity and reduced tumorigenicity and thus are preferable for therapeutic purposes because they are considered safer for implantation.

Adult stem cells have been reported to be found in many places, including bone marrow, peripheral blood, brain, spinal
cord, dental pulp, blood vessels, skeletal muscle, epithelia of the skin and the digestive system, cornea, retina, liver, bone, and pancreas [14]. The bone marrow, in particular, harbors cells that give rise to the distinct lineages of hematopoietic stem cells as well as nonhematopoietic stem cells that differentiate into mesenchymal tissues. Friedenstein’swork was the first to demonstrate that clonogenic stromal cells, defined as colony-forming units-fibrobastic (CFU-f), are present in the bone marrow and have the ability to form fibroblast colonies from single cells [16]. CFU-f reside in the bone marrow arrested in the Go-period of the cell cycle. In culture, however, they begin proliferating and form colonies composed of marrow stromal fibroblasts. When transplanted into diffusion chambers or ectopically under the kidney capsule, these cells formed bone, cartilage, reticular tissue, adipose tissue, and myelosupportive stroma [17]-[19]. These studies were some of the first to indicate the existence of either multipotent stem cells or at least a mixed population of progenitor cells.
The terms that have been used to describe these cells include bone marrow stromal cells, mesenchymal progenitor cells, mesenchymal stem cells, and others. Bone marrow stromal cells and mesenchymal progenitor cells are often used as more widely encompassing terms to describe a heterogeneous population of lineage committed precursor cells. While the term mesenchymal stem cells is sometimes used similarly, it includes the multipotential stem cells in the adult that precede committed precursor cells. There is not yet conclusive evidence to state that the population is either strictly multipotential stem cells or a heterogeneous population of committed precursor cells. This review will refer to the bone marrow-derived CFU-f as mesenchymal stem cells (MSCs).

MSCs are adult stem cells that readily differentiate along various mesenchymal lineages, including bone, cartilage, muscle, tendon, adipose, and stroma. While bone marrow is a major source of these stem cells, MSCs have also been isolated from various tissues including adipose, periosteum, synovial membrane, muscle, dermis, pericyte, blood, bone marrow, and trabecular bone [20]. The most common techniques used to isolate and culture expand in vitro MSCs include aspiration from bone marrow followed by density gradient fractionation or direct plating [21], [22]. It has been suggested, but not proven, that the cells isolated via the different methods are similar and exhibit the same capacity for osteogenesis, chondrogenesis, and adipogenesis. Unlike hematopoietic stem cells, which can be identified and isolated on the basis of characteristic surface markers, there is not currently a well-established, unique profile of surface markers to identify MSCs [23], [24]. Instead, MSCs are most commonly identified by their tendency to adhere to tissue culture plastic, their expansion potential, and their capacity to undergo all three differentiation pathways of osteogenesis, chondrogenesis, and adipogenesis in culture with appropriate combination of growth factors and supplements.

Tissue Engineering with Adult Mesenchymal Stem Cells
Many different types of scaffolds have been fabricated with material properties particular to the tissue of interest and its native properties. For instance, bone is commonly regenerated in scaffolds that are considered osteoinductive (inducing new bone formation) and osteoconductive (favoring new bone tissue ingrowth). Bioceramics such as hydroxyapatite and tri-calcium phosphate can be manipulated to match the mechanical properties of bone and are generally considered osteoinductive, and with highly porous architecture, they are considered osteoconductive. Porous ceramics of hydroxyapatite and β-tricalcium phosphate loaded with MSCs were shown to be capable of healing critical-sized segmental bone defects not capable of being healed by implantation of the scaffold alone [25]. While MSCs seeded in bioceramics have been shown to regenerate bone in a variety of studies [25]-[27], nonceramic scaffolds have also been shown to support bone regeneration with MSCs [28].


Fig. 4. Traditional view of stem cell hierarchy. The totipotent stem cell can form all cells/tissues that contribute to the formation of an organism; a pluripotent stem cell can form most cells/tissues of an organism; and a multipotent stem cell can form a small number of cells/tissues that are usually restricted to a particular germ-layer origin. Mesenchymal stem cells are characterized as multipotent stem cells.

mesenchymal-stem-cells-cultured-PLAFig. 5. Scanning electron microscopy images of mesenchymal stem cells cultured in PLA-alginate amalgam.
(A) PLA scaffold. Bar: 100 μm. (B) PLA scaffold seeded with
bone marrow-derived mesenchymal stem cells. Bar: 10 μm.
(C) PLA scaffold seeded with bone marrow-derived mesenchymal stem cells suspended in alginate hydrogel. Bar: 10 μm. Reprinted with permission from [29].
Cartilage is considerably less vascularized than bone, and it contains cells (chondrocytes) that require maintenance in a rounded or spherical shape to express the correct phenotype. Scaffolds like alginate hydrogels maintain the rounded morphology of chondrocytes and thus are favorable cell carriers for cartilage tissue engineering. Caterson et al. [29] have demonstrated chondrogenic differentiation of MSCs suspended in alginate and loaded into poly-L-lactic acid (PLA) sponges and treated with transforming growth factor (TGF)-β1 (Figure 5). The alginate hydrogel served as a three-dimensional cell carrier while the PLA sponge provided mechanical integrity to the construct. In an alternative approach, high-density cell pellets of human MSCs were press-coated onto PLA scaffolds and the construct cultured in vitro in chondrogenic conditions (Figure 6) [30]. After three weeks, the construct displayed a hyaline cartilage-like morphology, with organized and spatially distinct zones positive for the cartilage ECM markers, collagen type II, and link protein. More recently, our laboratory has fabricated an osteochondral construct by seeding the press-coated scaffold with trabecular bone derived-MSCs that have been precultured in osteogenic medium, followed by culturing in a medium that supported both chondrogenic and osteogenic phenotypes. During the course of long-term in vitro culture, the chondrogenic and osteogenic cells continued to differentiate and maintain their specific phenotypes. The final construct consists of a collagen type II-positive, but collagen type I-negative, hyaline cartilage-like layer adherent to, and overlying, a dense, mineralized bone-like component, and separated by a well-demarcated interface similar to that of native osteochondral tissue of the articulating joint [31].
Cartilage and bone are some of the more commonly studied tissues in tissue engineering involving the seeding of MSCs into three-dimensional scaffolds, but much progress has also been made with muscle, tendon, and other tissues in understanding the effects of culture conditions, scaffold material properties, and other stimuli [20].

While the outlook for stem cells in tissue engineering is promising, there are important issues that need to be addressed from both a biological and engineering standpoint. Fabrication techniques to manufacture scaffolds with controlled material properties to better satisfy the minimum requirements simultaneously are constantly advancing; materials science research is making strides in its quest to create new types of biomimetic scaffolds that actively interact with cells to direct and promote tissue development. At a cellular level, the effect of the scaffold material properties on cell-cell and cell-material interactions is of great importance. Cells will adhere differently to various scaffold materials, triggering different signaling pathways that will affect proliferation, differentiation, and other cell functions.

The high expansion potential and capacity for multilineage differentiation makeMSCs especially attractive for tissue
engineering applications. However, the fundamental nature of these cells still needs to be elucidated. The confusion over terminology with bone marrow stromal cells, mesenchymal progenitor cells, mesenchymal stem cells, etc., is a reflection of the need for thorough characterization of these cells. Are these cells truly multipotential, or simply a heterogeneous population of cells from which certain committed precursor cells are selected to proliferate and differentiate under specific culture conditions? At present, there are no known unique markers for MSCs [23].What is the native function(s) of these stem cells? Are MSCs isolated from different sources functionally similar, and possibly derived from the same origin pool of stem cells? Mesenchymal stem cells isolated from various tissues in the adult body have displayed similar differentiation capacities with respect to chondrogenesis, osteogenesis, and adipogenesis, but the lack of a unique profile of markers to characterize these cells leaves the question open-ended. A question of therapeutic relevance is the potential use of allogeneic MSCs. Preliminary experiments suggest that both human and animal MSCs do not express co-stimulatory antigens and thus appear to be immunoprivileged [32]. The absence of immune rejection of implanted, mismatched MSCs could lead to the establishment of universal donor cells for “off-the-shelf” tissue engineering applications.
Fig. 6. Scanning electron microscopy images of tissue-engineered construct produced by press-coating human mesenchymal stem cells on PLA scaffold block. (a) “Perichondrium-like” and cartilage layers formed by press-coated cell pellet, intermediate zone consisting of cells and ECM, and a cellular layer consisting of scaffold alone. Bar: 150 μm. (b) Cartilage layer between the “perichondrium” and intermediate zone. Bar: 50 μm. (c) Cartilage layer with cells embedded in abundant ECM. Bar: 20 μm. (d) Surface of construct. Bar: 10 μm. Reprinted with permission from [30].
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Tags: Mesenchymal stem cells, TISSUE ENGINEERING