Tissue Engineering Bone

The State of the Art of Tissue Engineering – Bone Content

1. Introduction………………………………………………….……………………………………………3

2. Bone Grafts and Bone Graft Substitutes…….……………………………………………4

3. Bone Biology……………………………………………………………………………………………..4

4. Tissue engineering…………………………………………………………………………………..5
 4.1. Ideal Scaffold
 4.2. Growth factors
 4.3. Stem cells
5. Future remarks/Conclusion………………………………………………………………………7

6. Appendix…………………………………………………………………………………………………..8 1. Introductions
In the U.S. and E.U alone, bone fractures results in more than 2.4 million surgical procedures a year. Currently, bone grafting procedures are used in the healing and the repairing of bone defects. Autologous bone grafting, although effective as a clinical gold standard for bone repair, is gravely challenged by the availability of enough donor tissue supply and problems associated with donor site morbidity. Efforts to deal with these problems and restrictions have led to development of new biomaterials and other therapies, among which is tissue engineering holds immense potential.

The approaches to tissue engineering are illustrated in (Fig 1). The main idea is to make replacement tissues; two approaches can be used, depending on the target tissues.

In approach A, a small number of cells can be harvested from the patient using a biopsy, and then are cultured in vitro. The cells can be cultivated within a three dimensional scaffold in the presence of suitable growth and differentiation factors, an with the right conditions, the signals secrete several matrix materials to create a living tissue that can be used as replacement tissue that can be replaced back into the target site of the tissue. The scaffold should be biodegradable to avoid certain risks that can take place with any foreign material in the human body.

Approach B, on the other hand, acquires scaffold materials loaded with or without suitable growth and differentiation factors to be implanted into the target site; the scaffold materials will guide growth in situ with the help of suitable growth and differentiation factors secreted by the host or by the release of loaded factors .

In essence, three elements are central in tissue engineering:
I. Stem or precursor cells;
II. An appropriate scaffold and;
III. Growth factors.

The developments and limitations of each of these areas will be addressed in turn and their impact within bone tissue engineering.

2. Bone Grafts and Bone Graft Substitutes
At the moment the preferred option for bone repair is the use of autografts. With a success rate of between 80%-90%, autografts are considered as the gold standard of bone grafts. The high rate of success can be attributed to the make-up of autografts; tissue harvested for an injury from a remote site of the patient. The process of harvesting the tissue can lead to complications independent of the initial injury since the tissue harvest can result in infections, and possibly mechanical weakening of the donor site. These complications are jointly known as donor-site morbidity and are seen in nearly a fifth of all procedures. There are also constraints on the amount of tissue that can be harvested from the site, presenting limitations in supply of harvested tissue.

Allograft, bone taken from somebody else’s body, could be an alternative. However, the rate of graft incorporation is lower than with the autograft. Allograft bone introduces the likelihood of immune rejection and even the chance of an infection in the recipient’s body after the transplantation.

Hence it is clearly seen that an adequate bone replacement is yet to be found and it is at the same time urgently needed for full recovery of the patients. A possible way around this problem may be in tissue engineering, which is discussed below. But firstly an understanding to bone biology is very important and vital step tissue engineering.

3. Brief insight into bone biology
Bone tissue in the adult skeleton at a macroscopic level consists of two types of tissues: trabecular (around 20% of the total skeleton) and cortical bone (around 80% of the total skeleton). Bone together with cartilage, make up the human skeletal system. Bone is a dynamic, highly vascular, constantly changing, specialised type of mineralised connective tissue which serves the following functions:

1. bone provides mechanical support and is the site of muscle attachment for movement;
2. bone protects the internal organs and surrounds bone marrow;
3. Additionally the skeleton serves as a mineral reservoir, such as calcium, phosphate, and other ions.

Bone develops by osteogenesis, the process of ossification, starting out as a highly specialized form of connective tissue. Two main players in the formation of bone are the bone cells called osteoblasts (bone-forming) and the osteoclasts (bone-resorbing). During the process of ossification, osteoblasts secrete type I collagen, in addition to many non-collagenous proteins secreted extracellular matrix may initially be amorphous and non-crystalline, but it gradually transforms into more crystalline forms. Mineralization is a process of bone creation encouraged by osteoblasts and is thought to be initiated by the matrix vesicles of osteoblasts to create an environment for the concentration of calcium and phosphate, allowing crystallisation. Collagen serves as a template and may also commence and spread mineralisation independent of the matrix vesicles.

4. Tissue Engineering
Tissue Engineering is “an interdisciplinary field of research that applies the principles of engineering and the life and science s towards the development f biological substitutes that restore, maintain, or improve tissue function. ” So by developing a scaffold that is biocompatible and biodegradable and allows cells to attach, proliferate, and migrate throughout its structure, the damaged tissue can be completely replacing the scaffold initially implanted. This method holds great promise as a bone repair model provided that the nature and structure of bone is considered in the scaffold design. For bone tissue engineering, one may look to autografts to form a selective requirement for a successful scaffold. An autografts finds it success through several attributes:

• Osteoconductivity: the quality of a porous interconnected structure that permits new cells to attach, proliferate, and migrate through the structure, and also allows for nutrients/waste exchange and new vessel penetration.
• Osteoinductivity: possessing the necessary protein and growth factors that induce mesenchymal stem cells and other cells toward the osteoblast lineage.
• Osteogenicity: the osteoblasts that are at the site of bone formation are able to produce minerals to solidify the collagen matrix that forms the substrate for the new bone.
• Mechanical match: having similar mechanical properties to the tissue of the implant site to prevent mechanical mismatch which can lead to stress shielding and bone resorption.
• Biocompatibility: the lack of immunogenic response.

These characteristics are the key to a successful bone tissue engineering implant and no one material to date can satisfy each of these requirements. Therefore, for an ideal bone tissue engineering scaffold, it is a reasonable strategy maybe to combine two or more materials into a composite.

 4.1. Ideal Scaffold
As indicated from (Table 1), neither polymer nor ceramic alone possess all the necessary components of an ideal bone tissue engineered scaffold, but by combining them into one material an ideal composite material can be developed (see Fig. 2).

However within both polymers and ceramics there are several choices. Some of the more extensively studied polymers for bone tissue engineering composite scaffolds include polyesters like polylactide, polyglycolide, collagen, chitosan, starch-based polymers, and polymethylmethacrylates (PMMA). The ceramics used in composites include calcium sulphate, bioactive glass, and calcium phosphate. Pore size is also very important issue because, if the pores employed are too small, pore occlusion by the cells will happen. It is accepted that a bone tissue engineering purposes, pore size of scaffolds should be within the 200-900 µm range.

The next step after the development of an adequate porous structure is the choice of a reliable source of cells that allows their isolation and expansion into high numbers. In fact, an ideal cell source should be easily expandable to high passages, non-immunogenic and have a protein expression pattern to the tissue to be regenerated, which require stem cells and growth factors.

 4.2 Growth Factors
Growth factor effects are concentration-dependent and are exerted through their receptors on the cell surface. A secreted growth factor may bind to matrix molecules, carrier molecules, or binding proteins to regulate its activity and stabilization. Growth factors can associate with specific binding proteins that limit access to their receptors to control the bio-availability of the growth factor (e.g. TGF-β, BMPs). For tissue engineering applications it is vital to control the concentration and physical placement and sequestration of a growth factor. Growth factors can also be immobilised to engineered matrices to localise delivery, but spatial patterning remains a challenge.

 4.3 Stem cells
This technique uses autologous osteoblast or osteoprogenic cells. The harvested osteoblast and osteoprogenic cells are cultured in vitro to expand the population of the cells in order to get enough cells for the transplant. Then the cultured cells are seeded in biomaterial scaffold and transplanted. After transplantation, the cells may start to proliferate, secrete a matrix, and release growth factors as the scaffold vascularises and slowly degrades.

Several cell sources have been identified as osteogenic cells. These are embryonic cells, mesenchymal stem cells, osteoprogenitor cells from bone marrow. It has been shown that bone marrow cells can maintain their osteogenic capacity when implanted subcutaneously by showing new bone formation.

The first and most obvious choice because of their non-immunogenicity is the isolation osteoblasts from biopsies taken from the patients (autologous cells), followed by limited expansion in vitro. However this methodology has several limitations: it is time consuming, limiting in this way the number of cells available to be seeded on the scaffolds.

It is in this context that stem cell biology appears as the most valid and more promising solution. Since its beginnings, stem cell research has gone along way, and although a considerable number of questions are yet to be answered, they can be presented as an alternative to the above described approaches. Stem cells are undifferentiated cells with a high proliferated capability, being able of self-renewal, multi-lineage differentiation and therefore the regeneration of tissues.

5. Future remarks/Conclusion

Tissue engineering may offer a new dimension of therapeutic care for patients. The design and development of the tissue engineered therapeutics must be guided by basic, fundamental biological pathways targeting specific clinical applications.

Scaffolds materials play a critical role in providing mechanical stability to tissue engineered constructs prior to synthesis of a new extracellular matrix by the cells. Therefore, it is desirable to have materials which match the mechanical properties of the tissue. For bone scaffolds, in tissue engineering the scaffold needs to have high stiffness and high strength. Though considerable efforts have been made to develop high strength, high stiffness yet biodegradable materials, no satisfactory material are available yet.

6. Appendix

Figure 1: Tissue Engineering approaches in regeneration of tissue of organs
Source: Biomaterials and Tissue Engineering, D. Shi (Ed.), Springer. (2004)

Table 1
Parameters for a Successful Tissue Engineered Scaffold as Indicated by Autografts
Autograft Quality Polymers Ceramics Polymers Biocompatiblity Yes Yes Yes
Osteoconductivity Yes Yes Yes
Osteoinductivity No (but can add factors) No (but can add factors) Yes (with delivered factors)
Osteogenicity No Yes Yes
Osteointegrity No Yes Yes
Mechanical Match Yes No Yes
Neither polymers nor ceramics can accomplish all parameters alone, but this can be achieved when formed into composites. Source: Peter X. Ma, Jennifer Elisseeff, Scaffolding in Tissue Engineering, CRC Taylor & Francis (2006).

Figure 2: Neither the polymer nor the calcium phosphate alone possesses each of the necessary parameters for the successful scaffold, but a composite of a polymer nor can ceramic together take advantage of the benefits of each material while minimising the shortcomings.
Source: Peter X. Ma, Jennifer Elisseeff, Scaffolding in Tissue Engineering, CRC Taylor & Francis (2006).