1Joint Department of Biomedical Engineering at University of North Carolina at Chapel Hill and North Carolina State University




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Development and application of naturally renewable scaffold materials for bone tissue engineering

Seth D. McCullen1, Ariel D. Hanson1, Lucian A. Lucia2, & Elizabeth G. Loboa1*

1Joint Department of Biomedical Engineering at University of North Carolina at Chapel Hill and North Carolina State University

2Department of Wood and Paper Science at North Carolina State University

*Corresponding author: Elizabeth G. Loboa, Ph.D.

2142 Burlington Laboratories, Campus Box 7115

North Carolina State University, Raleigh, NC, 27695-7115

Email: egloboa@ncsu.edu Phone: 919-513-4015 Fax: 919-513-3814


1.0 Introduction

The progression of regenerative medicine has largely been catapulted by the implementation of tissue engineering based therapies with the hope of providing a replacement for organ transplantation. Tissue engineering therapies can be defined where a population of progenitor or stem cells are directed by their surrounding milieu to differentiate into a desired tissue. This differentiation process is regulated by both their physical (eg, scaffolds, mechanical loads) and chemical (eg, inductive soluble factors) environments. In practice, tissue engineering approaches include the assembly of cells on a temporary scaffold resembling the tissue’s natural extracellular matrix in vivo. The function of this scaffold is to provide the appropriate template for cellular organization while maintaining necessary physical and mechanical properties for the seeded cells to differentiate/mature. The scaffold also achieves the desired physical integrity for a specific defect site by promoting the cells to deposit their own natural extracellular matrix within the scaffold prior to implantation of the cell-seeded construct at a tissue defect site. Of the four tissue types in the human body, connective tissue has seen the most prolific advances in tissue engineering due to its primary function being rooted in mechanical stability and ambulatory function (1, 2). Within the realm of connective tissue, bone tissue engineering has emerged with the most clinical success(3, 4, 4-7, 7-9). Currently, bone is the most transplanted tissue, second only to blood transfusions, with approximately 500,000 cases occurring annually in the United States.(10)


Bone tissue engineering is aimed at developing implantable substitutes to replace the use of autograft and allograft treatments. At present, autografts and allografts are most commonly used for bone grafting. Autografts are ideal based on their high acceptance rate within the body and ability to become integrated into the skeletal system by being osteoinductive, osteoconductive, and having osteogenic properties (11). Osteoinductive refers to the graft’s ability to attract surrounding mesenchymal stem cells into the area of repair that can then become a source of osteoblasts, while osteoconductive refers to the facilitation of vascularization and the orientation of haversian canal systems (10). Osteogenic potential implies that osteoprogenitor cells are present in the graft itself (10). However, autografts have also been associated with multiple problems including donor site morbidity (12-14), chronic pain, nerve damage, infection, fracture, pelvic instability, hematoma, and tumor transplantation(15). Allografts negate these concerns but have their own limitations such as carrying the risk of causing an immune response in the host, transferring diseases to the host (16), storing and transplanting of the allograft, and/or weakening of the allograft’s biological and mechanical properties during the storage and transplant process that would have made it an ideal replacement for bone constructs (17). The limitations of autografts and allografts have led to the use of tissue engineered constructs for bone grafts. Scaffolds are a key component for developing a tissue engineered bone construct for implantation into a critical bone defect. Ideally, a scaffold should have the following characteristics for successful implantation: 1) be biocompatible and bioresorbable with a controlled degradation rate to match cell/tissue growth in vivo; 2) have mechanical properties capable of withstanding the mechanical loads experienced in the physiological environment during cell matrix maturation; 3) be three-dimensional and allow for adequate diffusion for cell growth, nutrient delivery, and waste removal; and, 4) have suitable surface chemistry for cell attachment, proliferation, and differentiation(18). In order to accommodate all of these qualities, a diverse portfolio of materials, fabrication techniques, and modifications have been implemented over the years to achieve successful skeletal integration for use in clinical applications.


Recently, investigators have focused on the use of natural or renewable materials as a scaffolding choice over synthetically derived options. The main driving force for the use of naturally renewable materials is that these materials are highly biocompatible, biodegradable, offer chemical functionality (which is desirable for cell processes such as attachment, migration, and differentiation), and provide a cheap and replenishing source of material. Thus, renewable scaffolding materials can be defined as materials that can be obtained from natural resources including plant, fungal, animal, or bacterial derivation. Typically, these materials are some form of secondary product and must undergo some chemical treatment and sterilization process before end use. Two reviews by Mano et al. and Malafaya et al. have addressed the overall status of these types of materials in tissue engineering, hence, this chapter will specifically focus on their use in bone tissue engineering applications with in vitro or in vivo examples (19, 20).


1.1 Natural renewable materials for bone tissue engineering

The primary role of the extracellular matrix (ECM) is to endow tissues with their specific mechanical and physicochemical properties while providing a platform for cell attachment and migration. The ECM exerts a regulatory role in promoting or maintaining cellular differentiation and phenotype expressions through its composition, structure, and morphology. For bone tissue engineering, three groups of naturally renewable materials are commonly used: polysaccharides, fibrous proteins, inorganic materials, and any combination thereof. Polysaccharides are composed of repeating sugar rings linked by oxygen bonds, and in their natural state they function as membranes, participate in cellular communication, and can act as sequestering agents. On the molecular level, the tailoring of polysaccharides and their function can be controlled by their molecular weight, stereochemistry, primary sequence, and chemical reactivity. Polysaccharides can be derived from a number of resources with the most common forms including cellulose, hyaluronan, chitosan, dextran, alginates, and starches, to name a few. The main difference between these materials is the location of the linking glucosidic bonds between rings, the relative position of this linkage either being equatorial (β) or axial (α), and the presence of different pendant groups on each ring . Polysaccharides can be classified into four broad categories and include the ribbon, hollow helix, crumpled, and loosely jointed families (21).


Fibrous proteins are materials that are formed by repeating amino acid sequences and possess four levels of organization. These materials are the major structural components of tissues by providing high mechanical strength and resiliency. The mechanical integrity of proteins is preserved by the various levels of organization of its molecular and macroscopic arrangement which include its: 1) primary structure or the sequence of amino acids, 2) secondary structure or conformation of the chain, 3) tertiary structure or polypeptide chain arrangement, and 4) quaternary structure or configuration of multiple polypeptide chains. Fibrous proteins display one of the following conformations or secondary structures: α-helix, β-sheet, triple helix, and random coil. The most popular fibrous proteins used as scaffolding materials include collagen, silk, keratin, and fibrin. Collagen is usually derived from mammalian sources, primarily from bovine and human origin, and its functional unit is arranged in a triple helix where three collagen molecules are intertwined. These molecules are known as tropocollagen and are approximately 300 nm in length and 1.5 nm in diameter (19, 22). Type I Collagen is largely used in bone tissue engineering due to its natural occurrence and large quantity in bone; thus numerous collagen based systems have been developed as a starting point for bone tissue scaffolds. Silk is another fibrous protein that is produced by spiders and silkworms. This protein is composed of β-sheet structures that allow the tight packing of stacked sheets of hydrogen bonded anti-parallel chains and account for its high tensile modulus and elasticity(23, 24). Keratin is another protein that displays either an α-helix or β-sheet structure (depending on source) along with cysteine residues to create disulfide bridges for enhanced stability and strength (25). Fibrin is the polymerized form of fibrinogen after it has been crosslinked with thrombin, and is known for both its coagulation effects in blood and as an extracellular matrix substitute (26).

Inorganic materials include demineralized bone matrix (DBM), hydroxyapaptite, nacre, and coral. DBM can come from allograft and xenograft sources and is also known as decalcified cortical bone. To reduce host rejection and foreign body response, it is processed until it only retains a highly porous collagenous structure (10). Hydroxyapatite is the natural inorganic component of bone and is typically incorporated as a filler material in composite systems. Nacre is a calcified structure that forms the inner layer of some sea shells. Coral is a marine invertebrate that consists of CaCO3. Coral has a porous structure with an interconnected network of pores. To be used in vivo it undergoes a partial hydrothermal exchange process that converts carbonate to phosphate (27). These materials resemble the natural architecture and porosity of bone and are adequate scaffold materials based on this striking similarity.


This remainder of this chapter will present a brief background on the anatomy and function of bone, highlighting the extracellular matrix components, physical properties, overall architecture, and the osteodifferentiation of progenitor cell populations featuring mesenchymal stem cells. This will be followed by specific examples of investigators implementing naturally renewable materials for bone tissue engineering, discussing the successes and limitations with each example.


1.2 Bone Background

Bone is involved in many diverse roles within the body such as: 1) the protection of vital organs, 2) support and attachment to muscles for locomotion, 3) the generation of red and white blood cells for immunoprotection and oxygenation or other tissues, and, 4) mineral storage and ion homeostasis (28-31). The architecture of bone is representative of the many functions it serves in the body. There are two types of bone that make up the adult skeleton, cortical bone (80%) and cancellous (or trabecular) bone (20%) (30). Cortical bone provides mechanical stability and protection to vital organs and is therefore almost completely solid, having a very low porosity (10%) (28, 30). In comparison, trabecular bone is loosely organized and very porous (50-90%) in order to provide a proper environment for metabolic activity (28, 31). In bone, entire collagen triple helices lie in a parallel, staggered array. 40 nm gaps between the ends of the tropocollagen subunits probably serve as nucleation sites for the deposition of long, hard, fine crystals of the mineral component, which is (approximately) hydroxyapatite, Ca5(PO4)3(OH), with some phosphate (mineralization during endochondral ossification of articular cartilage occurs in a similar fashion). Collagen gives bone its elasticity and contributes to fracture resistance.


Bone replacement has become an important area in tissue engineering. The previously described limitations of autografts and allografts have led researchers to investigate the use of natural scaffold materials, in combination with human mesenchymal stem cells (hMSCs), to provide biocompatible, biodegradable, and mechanically stable bone grafts for critical size defects. The success of bone grafts relies heavily on the architecture of the scaffold, specifically the pore size and porosity, and should be designed to mimic the physical properties of native bone (32, 33). For design purposes, the porosity of native trabecular bone is estimated at >75% (34) and typical pore sizes are approximately 1 mm in diameter (35). Investigators have recently shown a direct correlation between pore size, vascularization, and bone formation (36). Klenke et al. found that scaffolds containing pores ≥ 140 mm had significantly higher ingrowth and bone formation as compared to scaffolds with smaller pores. These findings confirmed results from a separate study demonstrating a relationship between increasing pore size and bone ingrowth, with optimal pore sizes for bone formation ≤ 350 mm (33). Scaffold porosity also plays a key role in bone formation, as demonstrated by Takahashi et al. who reported higher proliferation of mesenchymal stem cells (MSCs) when grown on polyethylene terephthalate fabrics with higher porosities compared to those of lower porosities (37). The increased cell proliferation was attributed to the increased volume allowing for both greater cell migration and increased nutrient and oxygen delivery and exchange.


While large pore sizes and higher porosities have been shown to be beneficial for vascularization and bone formation, they can also result in decreased compressive strength of the scaffold which may then fail under physiological loading (4, 38). Trabecular bone is reported to have a compressive strength of 4-12 MPa and a modulus of 0.02-0.5 GPa (39). For successful repair of critical size bone defects, it is desirable for the bioresorbable scaffold to have similar mechanical properties to the host tissue and retain its physical properties for at least six months (four months in vitro during cell culture and two months in vivo) (18). The strength and stiffness of the scaffold should match that of the host tissue until new tissue has replaced the degrading scaffold matrix.


1.2.1 Progenitor cells for tissue engineering bone

Mesenchymal stem cells (MSCs) are defined as progenitor cells that have the ability to differentiate into tissues of a mesenchymal lineage such as bone, cartilage, adipose, tendon, muscle, ligament and stroma (40). Investigators claim to have isolated these cells from multiple sites including bone marrow (40, 41), umbilical cord blood (42), peripheral blood (43), amniotic fluid (44, 45),land adipose tissue (46-49), although recent studies have found that bone-marrow derived MSCs behave differently than adipose-derived stem cells with respect to growth kinetics and differentiation efficiency (50). It is known that adipose-derived stem cells are known to contain a heterogeneous population of cells. Research has indicated clear biologic distinctions between mesenchymal stem cells derived from multiple tissues and noted site specific differences (51). Nonetheless, MSCs offer tissue engineering a means to fully evaluate biomaterial interactions and afford a cell line capable of undergoing osteogenesis. Typical characterization of mesenchymal stem cells consists of their expression of specific protein markers such as, but not limited to, CD44, CD71, CD90, CD105, CD106, and CD166 (40, 52, 53), and the ability of the MSCs to differentiate down osteogenic (41, 48, 54-62), adipogenic (41, 48, 63, 64), chondrogenic (3, 41, 48, 65-68), fibrogenic (69-71)myogenic (48, 57, 72), and neuronal (41, 46, 57, 73)lineages. The majority of applications using stem cells for tissue or organ replacement typically use bone marrow-derived mesenchymal stem cells (BMMSCs) (55, 66, 70-72, 74-76). Researchers have also begun to extensively investigate other sources of mesenchymal stem cells, including from adipose tissue. In contrast to bone marrow, adipose tissue provides an abundant and easily obtainable source of cells (77) adipose derived adult stem cells (ASCs) exhibit somewhat similar capacity for expansion, growth kinetics, and differentiation as BMMSCs depending on the source and site of tissue (41).

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