Bone Tissue Engineering
The previous section talked about the pressing need for bone substitutes. Bone Tissue Engineering is an emerging interdisciplinary field that seeks to address the needs by applying the principles of biology and engineering to the development of viable substitutes that restore and maintain the function of human bone tissues. This form of therapy differs from standard drug therapy or permanent implants in that the engineered bone becomes integrated within the patient, affording a potentially permanent and specific cure of the disease state.
There are many approaches to bone tissue engineering, but all involve one or more of the following key ingredients: harvested cells, recombinant signaling molecules, and three-dimensional (3D) matrices. One popular approach, depicted in the figure below, involves seeding highly porous biodegradable matrices (or scaffolds), in the shape of the desired bone, with cells and signaling molecules (e.g., protein growth factors), then culturing and implanting the scaffolds into the defect to induce and direct the growth of new bone. The goal is for the cells to attach to the scaffold, multiply, differentiate (i.e., transform from a nonspecific or primitive state into cells exhibiting the bone-specific functions), and organize into normal, healthy bone as the scaffold degrades. The signaling molecules can be adhered to the scaffold or incorporated directly into the scaffold material.
Figure 1. Scaffold-guided tissue regeneration.
In order to understand this approach, one must first understand why you can’t just harvest some cells, such as osteoblasts, then culture them to create a whole bone as depicted below:
Figure 2. Simple culture techniques can’t be used to grow organized tissue. Why?
Conventional cell culturing involves growing cells in an artificial environment where they can thrive and replicate to form larger colonies of cells for applications such as diagnostic testing. These colonies, however, do not become organized into tissues or organs that could then be implanted back into the patient. Cell colonies need external cues or signals to grow into functional 3D tissues or organs. In the body, cells are constantly bombarded with mechanical, electrical, structural, and chemical cues that signal the cells about what they should be doing. If these signals are not properly received or processed due to disease or trauma, then the cells dedifferentiate (i.e., become nonspecific cell types), become disorganized, and eventually die.
The structural cues involve the interaction of cells with their extracellular matrix (ECM). The ECM is that part of our body which gives it form and shape. For example, bone is made up of an ECM composed of a composite fibrous network of collagen encased within a hard matrix of calcium/phosphorous (as described in the tutorial on bone structure). Bone cells (osteoblasts, osteoclasts, osteocytes) exist in a symbiotic relationship with the ECM, first creating it, then remodeling it, and in turn being regulated by it. The physical communication between cells and ECM directly and indirectly impacts cell shape and function, and these signals are all necessary cues for normal cellular activity.
Cell actions and their responses to various environmental cues, including mechanical, electrical, structural and chemical, are mediated by protein based molecules loosely referred to as growth factors (see the tutorial on signaling molecules). The cellular regulatory actions of growth factors in bone include migration of cells from one site to another, morphogenesis from one cell type to another, and mitogenesis or cellular proliferation. Growth factors are produced both locally by bone cells and systemically from other sites. In mediating extracellular communications, growth factors act directly on the very bone cell that produced them (autocrine effect), act on neighboring cells surrounding the growth factor producing cell (paracrine effect), relay a single growth factor communication signal received by one cell to neighboring cells due to direct cell to cell interaction (juxtacrine effect), and act on cells distant from the site of growth factor production by traveling through the blood stream (endocrine effect). Within the local bone environment growth factors reside in the interstitial fluid, on the cell surface, and in the ECM. These growth factors are not only important for growth, development, and day-to-day maintenance of bone tissues, but are mobilized during times of bone remodeling and injury.
Tissue engineering techniques, such as depicted in Figure 1, thus involve mimicking the natural milieu by placing the cells and growth factors in synthetic scaffolds that act as temporary ECMs. However, there are numerous variations of this approach depending on:
- the source of the cells; i.e., autologous (donated by the patient), allogenic (donated by another person), xenograph (from an animal)
- whether or not a scaffold is even used; i.e., direct injection of cells and/or signaling molecule into the defect site may be appropriate for damaged tissue confined to a small region. Larger regions, however, will probably need the matrix as a structural cue
- whether the scaffolds seeded with cells are cultured before surgery, or the cells are seeded into the matrix and immediately implanted at the time of surgery
- whether or not cells are even used; i.e., just use signaling molecules
No single approach or dosage of cells and growth factors will satisfy all clinical needs; the best ‘recipe’ will depend upon the particular application and the relative health status of the patient. For example in bone repair, an older diabetic patient or a smoker heals differently than a young, healthy child, so each would need a different dosing of cells and growth factors. Therapies that use a patient’s own cells are safest from an immunologic point of view, however these methods may not always be practical. For example, many surgeons and insurance carriers are not enthusiastic about performing two operations (i.e., one to harvest the cells, and another, weeks later, to implant the scaffold) because of the additional costs, time, and quality control issues. Even when harvesting a patient’s own cells for immediate implantation there are two surgical sites, i.e., the implantation site and the harvesting site. In these cases, there may be donor site morbidity, including infection and chronic pain, as well as additional surgical costs. Finally, a very sick or elderly patient may not have enough virile cells, even if expanded ex vivo (outside the body), to cause the defective tissue to heal. For all these reasons, there is significant interest in having an off-the-shelf supply of donor cells. These cells would be expanded ex vivo and immortalized. Fetal or neonatal cells are extremely useful for this purpose since they are naturally non-immunogenic and are a rich source for stem cells; this approach, however, is an extremely controversial ethical issue.
Another approach will be “ex vivo gene therapy” consisting of isolation of relevant determined stem cells or committed progenitors from mature adults or from animals, expansion of them ex vivo, transfection of them and selection of transfected cells ex vivo, and then reintroduction of the cells in vivo. Genetic engineering, however, has numerous hurdles to overcome to make this approach reliable, practical, safe, and generally accepted.
Instead of administering growth factors directly, it is also possible to use genes that encode those molecules. The genes are part of a plasmid, a circular piece of DNA constructed for this purpose. The surrounding cells take up the DNA and treat it as their own. They turn into tiny factories, churning out the factors coded for by the plasmid. Because the inserted DNA is free-floating, rather than incorporated into the cells’ own DNA, it eventually degrades and the product ceases to be synthesized.What are some of the challenges ahead for bone tissue engineering?
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Perhaps the biggest challenge for all of tissue engineering is how to insure angiogenesis in a timely fashion within the scaffold construct; cells without a blood supply will die, and mass infection will occur.
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New biomaterials are needed that cause minimal foreign body response and that degrade in a completely predictable fashion.
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A basic understanding of the spatial and temporal distributions of cells and growth factors required for osteogenesis, subject to particular disease states, must still be determined; i.e., a complete bone tissue engineering knowledge base remains to be developed. To achieve this will require better experimental and analysis tools including more realistic in vitro models, better ways to non-invasively image developing tissue in vivo, suitable computational models that capture this vast, multidimensional array of information, and advanced data-mining techniques to extract salient information.
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Readily available, safe, off-the-shelf supplies of osteogenic cells.
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Advanced manufacturing systems are required that can fabricate complex scaffolds with spatially controlled distributions of materials, microstructures, cells and growth factors.
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Design systems that encapsulate the tissue engineering knowledge-base and that understand the constraints of the manufacturing processes must be created to aid the next generation ‘tissue engineer’ in designing and manufacturing their products. Advanced CAD/CAM systems are required to create today’s complex automobiles, aircraft, and electronic products; why would anyone think that it would require anything less to design something as complex as human tissue
