**Bioreactor-Based Bone Tissue Engineering**

Inga Marijanovic, Maja Antunovic, Igor Matic, Marina Panek and Alan Ivkovic

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62546

#### **Abstract**

The aim of this chapter is to describe the main issues of bone tissue engineering. Bone transplants are widely used in orthopedic, plastic and reconstructive surgery. Current technologies like autologous and allogenic transplantation have several disadvantages making them relatively unsatisfactory, like donor site morbidity, chronic pain, and immunogenicity and risk hazard from infectious disease. Therefore, regenerative orthopedics seeks to establish a successful protocol for the healing of severe bone damage using engineered bone grafts. The optimization of protocols for bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreactor, should be able to solve the current limitations in managing bone injuries. We discuss mesenchymal stem cells as the most suitable cell type for bone tissue engineering. They can be isolated from a variety of mesenchymal tissues and can differentiate into osteoblasts when given appropriate mechanical support and osteoinductive signal. Mechanical support can be provided by different cell scaffolds based on natural or synthetic biomaterials, as well as combined composite materials. Three-dimensional support is enabled by bioreactor systems providing several advantages as mechanical loading, homogeneous distribution of cells and adequate nutrients/waste exchange. We also discuss the variety of osteoinductive signals that can be applied in bone tissue engineering. The near future of bone healing and regeneration is closely related to advances in tissue engineering. The optimization of protocols of bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreac‐ tor, should be able to solve the current limitations in managing bone injuries.

**Keywords:** bioreactor, bone, stem cells, differentiation, scaffold

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Tissue engineering is a multidisciplinary field that applies basic concepts and techniques of life science and engineering. It is generally understood as a process of taking human or animal tissues, isolating cells from the tissues, culturing the isolated cells in a supporting material, i.e., scaffold to fabricate cell-scaffold complex, and transplanting the fabricated cell-scaffold complex into human or animal subject. It is applied to fabricate almost every human organ including artificial skin, artificial bone, artificial cartilage, artificial cornea, artificial blood vessels and artificial muscles.

Bone is one of the few organs/tissues capable of spontaneous regeneration rather than simple repair. In other words, after disruption of its structure (fracture), its unique microanatomy and biological properties enable complete structural restoration without the creation of fibrotic scar tissue. However, in certain clinical situations where extensive injury, disease or malformation cause such large defects, it is necessary to resort to bone reconstruction, restoration and/or regeneration by a surgical procedure that replaces missing bone, i.e., by bone grafting. A bone graft is an implanted material that promotes bone healing alone or in combination with other material(s), through osteogenesis, osteoinduction and osteoconduction, in combination or alone [1].

The selection of an ideal bone graft relies on several factors such as tissue viability, defect size, graft size, shape and volume, biomechanical characteristics, graft handling, cost, ethical issues, biological characteristics and associated complications. The materials used in bone grafting can be divided into several major categories, including autografts, allografts, and xenografts. Synthetic and biologically based, tissue-engineered biomaterials and combinations of these substitutes are other options. Altogether, tissue-engineered bone graft requires the optimal selection of cells that are seeded on biomaterial-based scaffolds and exposed to specific biochemical and physical signals known to induce osteogenesis. The development of the successful bone tissue-engineering protocols depends very much on our understanding of bone structure, physiology and development.

Bone is a dynamic biological tissue consisting of metabolically active cells. The cell component of bone consists of the precursor cells (progenitors), osteoblasts, osteoclasts, osteocytes and bone marrow hematopoietic elements. Osteoblasts are metabolically active mature boneforming cells. They secrete osteoid, non-mineralized organic corpuscle that in turn undergoes mineralization process. Osteocytes are mature osteoblasts trapped within the bone matrix. Every osteocyte extends network of cytoplasmic tubules to the blood vessels and other cells. These cells are involved in the control of extracellular calcium. Osteoclasts are large multi‐ nucleated cells that degrade bone. Beside cells, bone is also composed of organic and inorganic elements. Approximately 20% of the weight of bone is water until the weight of dry inorganic bone makes calcium phosphate (65–70%) and the organic matrix of fibrous proteins and collagen (30–35%). Bone formation models in vitro are based on the fact that cell differentiation and function can be modelled according to factors that are important for embryonic develop‐ ment. Stem cells represent the building blocks of our bodies, functioning as the natural units of embryonic generation during development and adult regeneration following tissue damage. Stem cells are undifferentiated cells that can, under certain influence, differentiate into specialized cells and tissues. During development, the potency of stem cells decreases from totipotent stem cells (morula stage), capable of differentiating into all embryonic and extra embryonic tissues, to pluripotent stem cells (PSCs) (blastocyst stage), forming all embryonic tissues, and to multi- or unipotent adult stem cells (ASCs), forming tissues within their germ layer or tissue compartment [2]. Here, we discuss clinically relevant multipotent ASCs found in various adult tissues. Adult stem cells, also called somatic stem cells, in adult organism act as repair system for the body, replenishing adult tissues, prompt tissue homeo‐ stasis throughout life and ensure tissue regeneration following damage and they have great potential in regenerative medicine. Mesenchymal stem cells replenish connective tissues including bone. Therefore, they are the first choice among ASCs for regeneration of bone tissue.

Osteogenic differentiation *in vitro* is induced by ascorbic acid, b-glycerophosphate and dexamethasone. Ascorbic acid is essential for the development of osteoblasts, serves as a cofactor in the synthesis of collagen and stimulates the production of extracellular matrix, proliferation and differentiation of cells. b-Glycerophosphate serves as a source of phosphate for the formation of calcium phosphate *in vitro*. It is also responsible for the formation of threedimensional bone nodules between cells as proof of realized osteoblast phenotype. Dexame‐ thasone (DEX) is composed by a synthetic glucocorticoid, which regulates the expression of osteoblast genes, enhances differentiation *in vitro*, alkaline phosphatase activity and mineral‐ ization of bone. Understanding of osteoblast differentiation provided us the information on key pathways' components and enabled us the induction of differentiation using different recombinant proteins like BMP-2, -6 to -9. Also, mechanical stimulation promotes osteoblast differentiation and induces mineralization of extracellular matrix. Mechanical stimulation can be achieved using steady and dynamic fluid flow in bioreactors. For this purpose, different dynamic culture systems have been developed. These systems improve nutrient delivery to the cells and generate shear stress that promotes cell differentiation into osteoblastic pheno‐ type. Bioreactors for bone engineering applications are broadly classified in few main catego‐ ries, including rotating wall vessels, spinner flasks, perfusion bioreactors and compression systems. In addition to these, combinations of different bioreactors types have been explored in order to better mimic the bone physiological environment *in vitro*, such as for example compression bioreactors with added perfusion [3]. The process of bone tissue engineering in three-dimensional dynamic bioreactor system is a recapitulation of bone healing process *in vivo* in which progenitor cells, due to signals in the microenvironment, are stimulated to differentiate into osteoblasts [4].
