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Synthetic graft

New techniques and biomaterials facilitate the production of parts to replace human bones

Matrices produced with ceramic biomaterials and polymers that induce bone growth

EDUARDO CESARMatrices produced with ceramic biomaterials and polymers that induce bone growthEDUARDO CESAR

Various scientific studies on the development of new techniques and biomaterials to replace parts of human bone, lost due to disease or injury, have been reported worldwide in recent years. In Brazil, researchers at the University of Campinas (Unicamp), the Federal University of Pará (UFPA) and the Federal Institute of Pará (IFPA) recently made two types of synthetic bones, which can be used in grafts for applications in medicine and dentistry. These new biomaterials are made ​​of polymers and hydroxyapatite (HA) mineral nanoparticles primarily, a bioactive ceramic, which induces bone tissue growth and revascularization of the implant area. In another line of research, scientists at São Paulo State University (Unesp) are studying the interactions and integration of artificial biomaterials with the living tissue of patients.

The first experiments in humans using bone grafts made ​​from animal bones took place in the 17th century. In the 19th century, autogenous bone transplants began to be done ​​with material from the patient. Since then, there have been advances in allogeneic experiments, from a donor of the same species, and xenogenous experiments, from a donor of a different species than the recipient, that is, from animals to humans. In this scenario, much is already known about the biocompatibility of bovine bone and biomedical applications, including commercial products approved by the U.S. Food and Drug Administration (FDA), the federal regulatory agency for food and drugs in the United States.

The problem is that all of these techniques have limitations. Although considered the best option for treating bone loss, autografts (autogenous transplant) can not be done in large quantities because there is no way to harvest enough bone from a single part of the body to implant in another part. Furthermore, it requires a second surgery elsewhere in the body. This process thus increases the length of convalescence and the risk of patient infection, in addition to raising costs for the public health system. On the other hand, transplantation between different individuals or species increases the risk of infection or rejection. Therefore there is a need to create synthetic bones for implants. The problem is that they are unlike natural grafts in terms of their structure and composition, and therefore synthetic bones do not always have all the essential elements needed to replace human tissue.

Graphic image of a rat’s jaw produced with açaí polymer

Carmen Tavares Dias/UFPA Graphic image of a rat’s jaw produced with açaí polymerCarmen Tavares Dias/UFPA

Cell migration
Willian Fernando Zambuzzi, a researcher at Unesp’s Department of Chemistry and Biochemistry of the Botucatu Institute of Biosciences says that bone is a specialized connective tissue that is dynamic and capable of repairing minor injuries through its tissue remodeling mechanisms. Large lesions, however, require surgical procedures to help bones rehabilitate and, in most cases, biomaterials are essential to the process and allow migration of the cells responsible for bone tissue production. They can be used to recover small amounts of bone loss due to trauma or disease. “But, in order for them to be suitable for implantation, a series of pre-biological analyses are needed to ascertain their biocompatibility, which refers to the ability of the synthetic piece to promote an appropriate biological response in a given application,” he says.

It is in this context that the work of Sabina da Memória Cardoso de Andrade, a researcher at the Federal Institute of Pará (IFPA), comes into the picture. For her doctorate at Unicamp’s School of Mechanical Engineering (FEM), supervised by Professor Cecilia Amelia Carvalho Zavaglia and Professor Carmen Gilda Barroso Tavares Dias of the Federal University of Pará (UFPA), she developed a bionanocomposite. “The biomaterial was produced by combining two polymers—polyvinyl alcohol (PVAL) and polyurethane (PU)—with hydroxyapatite (HA),” says Andrade.

PVAL is a synthetic polymer that, according to Andrade, is of interest as a biomaterial because of its resistance and biocompatibility, in addition to its ability to absorb shocks. It can be purchased on the open market in both liquid and particle form. PU, on the other hand, is a polymer that spontaneously produces foam during the copolymerization process, is biocompatible, and has antibacterial action. These characteristics are useful in order to obtain a scaffold with good porosity. Scaffold is a key word in this area. “Scaffolds are artificial matrices with a three-dimensional structure that act as guides for the cells when forming new tissue,” says Andrade. “It is important for them to be biocompatible, so as not to harm the host tissue; bioactive, to stimulate bone growth; bioabsorbable so that the body does not reject their presence; have adequate porosity to facilitate the passage of nutrients into the bloodstream; and promote angiogenesis, which is the growth of new blood vessels from existing ones. Thus all these materials—polyurethane, polyvinyl alcohol and hydroxyapatite—promote tissue growth and are then absorbed by the body, without the need for surgery to remove the graft, says Andrade.

Polyurethane used in bioceramics

EDUARDO CESAR Polyurethane used in bioceramicsEDUARDO CESAR

Professor Zavaglia says that the scaffolds can be prepared by using conventional methods, such as by mixing a soluble salt into a polymeric matrix, which is then washed away, leaving pores behind. “More modern techniques, however, employ rapid prototyping or 3D printing,” she says. “Thus, scaffolds can be obtained in the desired quantity and with control over the average pore size and interconnectivity between the pores.”

Laboratory tests with rats have shown that the new biocomposite developed by Andrade has the necessary properties for bone growth. These properties include such things as excellent blood compatibility, bactericidal action, greater shock absorption and resistance to stresses caused by chewing. “The synthetic bone placed in the skullcap of the experimental animals promoted cell growth, indicating signs of integration into the bone structure after 30 days of implantation,” Andrade says. “The test results for growth of fibroblasts (cells that make up connective tissue, which synthesize collagen and elastin proteins) were considered excellent from day one after implantation, as indicated by the spreading of the cellular tissue.” According to Andrade, tissue regeneration in that location was present within seven days, and within 14 days the implanted material was already fully invaded by cells, including the areas between pores and micropores.

Andrade is confident that the bionanocomposite she developed has advantages over similar ones. “While collagen is already present in some biomaterials, the material of our research promotes growth of this protein when implanted in a living organism,” she says. “In addition, the compressive strength of our product is considered high, 69-110 MPa (megapascals), greater than that of a human femur, for example, which is 33 MPa. This characteristic is very important for bone grafts.” Another advantage is that due to the antibacterial action of polyurethane, drugs were not used in animal tests and yet the test animals showed no signs of inflammation or infection.

In another line of research, Professor Dias, of UFPA, is working to develop polymers from açaí seeds (Euterpe oleracea). She began this project during her postdoctoral studies at Unicamp, under the supervision of Professor Zavaglia. “Going to Campinas I envisioned a potential market with high added value for a molecular formula polyurethane known for its compatibility with living tissue,” says Professor Dias. “With the help of graduate student Dagoberto José dos Santos, we synthesized a new açaí pre-polymer at Unicamp’s School of Chemical Engineering (FEQ),” says Professor Dias. After polymerization with and without hydroxyapatite, it was characterized by another graduate student, Laís Pellizzer Gabriel. The two master’s candidates have been advised by Professor Rubens Maciel Filho, of Unicamp. The work is entitled Polyurethane based on acaí for bio-manufacturing of medical devices (see Pesquisa FAPESP Issue No. 196). All researchers are part of the Bio-manufacturing Institute (Biofabris), one of the National Institutes of Science and Technology (INCTs), based at the School of Chemical Engineering at Unicamp.

Methodological alternatives
The synthetic bone made with a vegetable polymer is not yet ready for use. According to Professor Dias, before a material can be used in implants, its stability after growth of the host tissue must be assessed. “We have already prepared a jaw of Rattus norvegicus albino, for in vivo studies,” she says. “In addition, various açaí polyurethanes are being evaluated at Northeastern University in Boston under the supervision of Professor Thomas Webster.”

It can be said that Zavaglia, Andrade and Dias are working at the cutting edge of biomaterial engineering, which includes making synthetic bones. But there is another aspect to their work, which is understanding how the living bone tissue of the recipient interacts with and is integrated into the biomaterial. That’s where the work of Zambuzzi, who has been engaged in research in this area since his undergraduate days under a FAPESP grant at the Dental School of the University of São Paulo (USP) in Bauru, comes into play. His focus is on the molecular aspects that regulate the interaction between living cells and biomaterials. “Our group is developing methodological alternatives to understanding this interaction and how these alternatives can be applied to bone tissue bioengineering,” he says. “This will allow us to replace or at least reduce the use of experimental animals. Accordingly, we are working to develop a database of different biomaterials, which we call OsteoBLAST.”

As a result of his research, in 2011 Zambuzzi was invited to join, as principal researcher, an international consortium coordinated by Professor Anna Teti of the University of Aquila, based in Italy. This consortium includes scientists from two groups in the Netherlands, one from India and two from the United States, one of which is Columbia University.