Laboratory for Bioengineering and Tissue Regeneration

 

The LABRET (Laboratory for Bioengineering and Tissue Regeneration) group, headed by Dr. José Becerra, has always been interested in understanding the phenomenon of tissue regeneration in living organisms. At first, they focused their research on epimorphic regeneration, a phenomenon occurring only in lower vertebrates and some non-vertebrate organisms. Then, they gradually moved to the study of tissue regeneration in mammals, in particular de skeletal system, and soon started to apply their knowledge and expertise to the field of regenerative medicine and tissue engineering. From 1998 the group’s research activity is focused on the search of novel therapeutic solutions based on the use of mesenchymal stem cells, active biomolecules and natural and synthetic scaffolds oriented to the obtaining of constructs to be applied to bone and cartilage defects.

Tissue Regeneration

Bone tissue loses its natural self-healing capacity with age, and becomes prone to fractures that no longer heal. Bone diseases are a major concern for healthcare systems because they are common in every period of life. Skeletal reconstructive surgery is needed in infants to correct skeletal malformations, and in adults to treat severe back pain (spinal fusion), nonunion fractures, trauma due to accidents, bone loss as a consequence of infections or cancer, etc. In orthopaedic surgery, autologous bone grafts are considered the gold standard for the repair of defects, but the small amount of available tissue and the risk of morbidity limit their use. On the other hand, allogeneic grafts, sourced from bone banks, can cause rejection and transmission of certain diseases. Tissue engineering (TE) has emerged as a promising alternative to bone grafts by developing in vitro constructs made up of cells, biomolecules and biocompatible scaffolds.

Biomaterials

Bone defects can be the result of trauma, disease or congenital malformations. Traditionally, the clinical solution has involved the use of resorbable biomaterials, call bone substitutes, to fill the defect while new bone is regenerated. Current bone substitutes are still far from representing a definite solution, because they offer the surrounding bone no regeneration-inducing signal, and they not always match their resorption rate with the rate at which native bone tissue is repairing itself.

On the other hand, when the bone was unable to regenerate due to the age or the disease of the patient, the missing bone or joint is replaced by a prosthesis, typically made of some type if inert metal. Although prostheses allow functional recovery, they fail in the long term due to poor osseointegration, i.e., poor structural and biochemical connection between the prosthesis and the surrounding bone.

Bone tissue engineering seeks the creation of ex vivo bone tissue that can be used to reconstruct bone defects. For this purpose, novel biomaterials are needed that can mimic the role played by the extracellular matrix of healthy bone. This means they not only have to be biocompatible and resorbable, like currently used bone substitutes. They also need to be able to mimic the bony architecture, with its high porosity and mechanical strength, and its ability to host and interact with biological elements that will stimulate bone repair. These elements are osteinductive signals (growth factors, peptides, etc) and stem cells. A biomaterial decorated with stem cells and appropriate osteoinductive signals would be able of stimulating bone regeneration in those situations where bone has lost its self-healing capacity.

Our group works with material scientists to develop novel biomaterials based on either organic (collagen, fibrin, PRP) or inorganic (ceramics, bioglasses) origin. We test their biocompatibility and osteoconductive properties in vitro and in vivo, and then evaluate their potential to be combined with stem cells and/or signaling molecules in order to create a complex, tissue-engineering product that can be used to stimulate bone regeneration.

We also seek for novel solutions in those applications where the use of a prosthesis is inevitable. We are designing new types of prosthetic implants that can be customized to the patients’ specific needs, and we are combining metal additive manufacturing with the elements of tissue engineering (resorbable biomaterials, osteoinductive molecules and stem cells) in order to improve prosthesis osseointegration, so that they no longer fail.

Modified bioactive molecules

The most promising molecules for bone repair are the bone morphogenetic proteins (BMPs), which have been shown to act on osteocompetent cells and to induce bone formation. BMPs are EMA- and FDA-approved in Europe and in the United States, respectively, for the treatment of spinal disc diseases and open tibial fractures. For being effective, BMPs must be combined with an adequate matrix, which is used to retain the proteins at the site of application and to support the migration of osteoprogenitor cells to the implant. In spite of having low affinity to collagen, recombinant human BMPs are currently delivered on absorbable collagen sponges. As a consequence, most of the BMP is washed away after its application. This not only has economic consequences as high doses (milligrams) of rhBMP-2 or rhBMP-7 (OP-1) must be used to achieve bone repair, but might also be potentially dangerous because of the undesired side effects such amounts of BMP may cause.

For surpassing these disadvantages, biomimetic peptides derived from extracellular matrix proteins are widely used to promote the adhesion, survival and differentiation of osteoblastic cells. In the area of active biomolecules the group has developed different molecular tools directed to selectively induce bone formation. Our expertise is oriented to adding to different growth factors and biomimetic peptides an additional collagen-binding decapeptide (CBD) derived from the von Willebrand factor which enhances their affinity to collagen type I. The binding of these improved osteogenic factors to collagen has been demonstrated to be very stable during a prolonged period of time. The application of such factors to absorbable collagen sponges provides a controlled delivery system with a high osteoinductive capacity. So far, our group has produced and patented a collagen-targeted rhBMP-2 (rhBMP2-CBD) and a collagen-targeted RGD biomimetic peptide (CBD-RGD), and is pursuing the characterization of several other novel molecules.

Mesenchymal stem cells

Stem cells are one of the body's own mechanisms used to repair bone and all other tissues. In adult stages, MSCs reside within the connective tissue of most organs contributing to their maintenance. MSCs can be induced in vitro to differentiate into various mesenchymal tissues such as bone, cartilage, muscle, tendon, adipose tissue and hematopoiesis-supporting stroma. Their self-renewal ability, together with their differentiation potential, make MSCs promising candidates for therapeutic applications in tissue engineering and tissue repair.

In elder and diseased patients a sufficient population of MSC may not be available, as the amount of these cells decrease with age and disease.. For that reason, stem cells need to be included in the tissue-engineering constructs that are meant to regenerate bone defects. No use is provided by biomaterials and signaling molecules if not enough native cells are there to populate the biomaterial and respond to these signals. Stem cells can be obtained from a variety of adult tissues, being the bone marrow and the fat the most commonly used. Our group works with these cells, but also seeks for new sources of stem cells in locations that do not precise an invasive surgical intervention in order to isolate them. Perinatal stem cells, coming from umbilical cord and placenta, are now being subject to intensive research. Induced pluripotent stem cells (iPSC) are also under investigation to evaluate their potential use in bone tissue engineering.

Experimental procedures

To evaluate the tissue engineering products, our group has developed several in vitro and in vivo techniques that are specific to the field of bone tissue engineering. Among in vitro techniques, we have developed 3D MSC cultures with several specificities (3D collagen gel cultures, micromasses, etc.). Among the in vivo techniques, we have also developed animal models for ectopic bone formation, cranial, maxillary and intrasegmental defects in rats, bone and chondro-orthotopic implants in rabbits and spinal fusion in rat and sheep.

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