June 15, 2015 (Vol. 35, No. 12)

Richard A. A. Stein M.D., Ph.D.

Regenerative Medicine Is Solidly Based On Decellularized Fabrics and Matrices

For millennia, the idea of regenerating tissues and organs inspired myths and legends, but today it is becoming a reality. Thanks to advances in molecular biology and biotechnology, certain bioengineered organs are already in clinical use.

And a broader array of bioengineered organs will soon be available, now that regenerative medicine is emerging as a vibrant field at the nexus of multiple disciplines.

“For years, tissue engineers have been developing synthetic three-dimensional scaffolds that can be repopulated with cells to regrow organs,” says Peter C. Johnson, M.D., co-editor-in-chief of Tissue Engineering. One of the ideal types of scaffolds is obtained from a solid organ that has been decellularized. Such an organ can be used as a framework for a new organ. The framework is repopulated with cells, and the new organ resembles the original organ.

This strategy has been explored in the research setting, and it has started attracting the interest of developers around the world who want to translate it into the clinic and generate a variety of organs. “Learning about decellularized scaffolds is ideally positioned to provide insight into rebuilding adult organs in an engineered fashion,” Dr. Johnson remarks. “I see this as a learning phase in the greater scheme of tissue engineering.”

A learning phase is definitely on the agenda. New challenges are bound to arise simply because regenerative medicine is interdisciplinary in nature—it is positioned at the interface of life sciences, engineering, and clinical medicine. Also regenerative medicine poses unique technological and regulatory challenges.

In a recent initiative that fueled the publication of a set of strategic directions in tissue engineering, Dr. Johnson and colleagues queried worldwide leaders in the field to identify and prioritize the best strategies for optimizing research and development. The results of this undertaking emphasize some of the technical and regulatory hurdles, and define four major initiatives that are imperative for advancing the field:

  1. Developing enabling tools.
  2. Formulating biomaterial scaffolds
  3. Focusing on the cellular response
  4. Promoting commercialization

“Commercialization challenges are enormous,” notes Dr. Johnson. “We tried to identify gaps that tissue engineers have in this respect.” One aspect that tissue engineering shares with other complex biomedical fields is that even after technical challenges are overcome, regulatory roadblocks might persist and delay the clinical implementation of the products. “We are only now starting to encourage tissue engineers to have this aspect in mind from the beginning when building the technology,” explains Dr. Johnson.

Characterizing the Matrix

“We showed that we can reliably quantify extracellular matrix proteins,” says Kirk C. Hansen, Ph.D., associate professor of biochemistry and molecular genetics, University of Colorado, Denver. “One of our most recent findings is that very large percentages of these proteins are just being discarded when standard methods are used.”

The objective of decellularization for tissue engineering is to remove all cellular components that contain antigenic epitopes, while preserving the extracellular matrix of the organ, with minimal damage or disruption. This process can be performed using physical, chemical, or enzymatic approaches.

A major drawback during decellularization, whether for whole organs or for matrices that are already being used therapeutically, is the paucity of tools that can reliably quantitate their composition to obtain proteomic compositional information. “To a great extent, this happens because standard proteomic methods rely on the ability to solubilize the sample,” comments Dr. Hansen.

Collagen is a major structural component of the extracellular matrix in tissues, and its quantitation is essential for developing decellularization protocols and for characterizing the scaffolds. One of the most widely used approaches to quantitate collagen in tissues has relied on measuring hydroxyproline, an amino acid that constitutes as much as 10–13% of various collagen types.

“The hydroxyproline assay is a very crude method for characterizing extracellular scaffolds,” notes Dr. Hansen. The abundance and activities of prolyl hydroxylases, the enzymes that generate these post-translational modifications, fluctuates with the tissue type and changes during disease states, and several noncollagen proteins have been found to contain this modification.

With current methods for extracellular matrix enrichment, some of the functionally important proteins are partitioned to an insoluble pellet that is discarded in the final analysis. This includes glycoproteins and proteoglycans that are essential determinants of the viscoelastic properties of the matrices, and contribute to organ architecture and function. Furthermore, decellularizarion was shown to differentially change the abundance of proteins in the extracellular matrix, making comparisons between native matrixes and decellularized scaffolds challenging.

To provide a more complete characterization of the proteins that are part of the extracellular matrix, Dr. Hansen and colleagues developed a proteomics approach to extract and quantitate extracellular matrix proteins from tissues. This strategy relies on isotope-labeled peptides and captures both soluble and insoluble protein fractions.

In a study that focused on extracellular matrix and intracellular proteins that are difficult to remove and analyze, investigators from Dr. Hansen’s group designed two quantification concatamers (QconCATs). These are synthetic genes that allow the simultaneous detection and absolute quantitation of multiple proteins in a low-cost and highly sensitive assay.

Using QconCATs, the investigators generated 74 tryptic peptides, and they were able to quantitate 56 proteins from decellularized and native rat lung matrices. Structural collagens emerged as the most abundant protein in the acellular extracellular matrix, and most of them were contained in the insoluble fraction. A comparative quantitative analysis of the abundance of 49 proteins revealed that the basement membrane and some of the collagens were more abundant in the native lung than in the decellularized scaffold, indicating that decellularization removed not only cells but, in addition, part of the extracellular matrix.

More recently, Dr. Hansen and colleagues expanded the list of peptides that quantitate scaffold proteins. “We have six concatamers that cover over 200 peptides in about 100 more proteins,” reports Dr. Hansen. “We are further expanding our coverage to probe the vast majority of the extracellular matrix with multiple peptides per protein.”

One of the advantages of this approach is the ability to capture proteins in a polymeric state. In these studies, the chemical digestion protocol relied on cyanogen bromide, a chemical that hydrolyzes proteins at the C-terminus peptide bond of methionine residues. However, certain proteins are more refractory to digestion, and improving protein hydrolysis is an ongoing technical focus in Dr. Hansen’s lab. “There are some proteins, such as elastin, that do not have methionine, and for which this method should theoretically not work,” cautions Dr. Hansen.

The original research that opened this avenue in Dr. Hansen’s lab has been focusing on dissecting the tumor microenvironment. “At the end of the day, we develop methods to characterize the tumor microenvironment,” informs Dr. Hansen. “While this has been a spinoff, it will have as much utility for the tissue engineering community as for the tumor microenvironment community.”

Decellularized pig lung, encased in a silicone “pleura,” inside a tissue engineering bioreactor that provides breathing movements and a flow of nutrients to growing lung tissue. [Yale University]

Refining Decellularization Protocols

“This methodology will become incredibly important in many aspects of regenerative medicine,” says Laura E. Niklason, M.D., Ph.D., professor of anesthesiology and biomedical engineering, Yale University School of Engineering and Applied Science. “In the long term, it will help us perform quality control and benchmark how good our matrices are when compared to a native matrix.”

Comparatively characterizing matrix proteins in native and decellularized organs provides the possibility of quantitating protein loss at different steps during matrix preparation. “This will enable us to objectively tune decellularization regimens to make the matrices more like the native ones,” adds Dr. Niklason. “Over time, this will be transformative for the field.”

A major research effort in Dr. Niklason’s group involves characterizing the conditions that are used for preparing decellularized scaffolds. Extracellular matrix components are a key determinant of the cell–cell and cell–matrix interactions that are established once the scaffold is repopulated with cells, and these conditions are, therefore, a fundamental determinant of the functionality of the future organ.

Studies from Dr. Niklason’s lab revealed that different detergent-based decellularization protocols generate lung scaffolds with varying degrees of collagen retention. Certain extracellular proteins, such as laminins and fibronectins, are particularly important in providing an environment for the differentiation of the cells that repopulate a scaffold, while other components, including collagens and elastin, provide tissue architecture, resistance, and elasticity.

In an analysis that examined the effect of pH during decellularization on the extracellular matrix, Dr. Niklason and colleagues identified this parameter as a critical determinant of the preservation of multiple components of the lung extracellular matrix. A comparison of decellularization protocols using pH ranges from 8 to 12 revealed that elastin, laminin, and fibronectin retention are greater at pH values closer to neutral, but with these mild conditions, more DNA was retained in the scaffold, indicating the presence of residual cellular debris.

A recurring challenge in many areas of regenerative medicine revolves around the effective reconstitution of the microvasculature. “When an organ is decellularized, cells from the microvasculature are removed, and the basal membrane around them is left intact,” explains Dr. Niklason. Reconstituting microvessels is crucial, particularly in the case of the lung. Close apposition between the alveolar air sac endothelium, the microvascular endothelium, and their shared basal membrane allows gas exchanges to take place.

The paramount requirement for lung regeneration, Dr. Niklason suggests, is actually a set of requirements: repopulate the microvasculature with sufficient numbers of cells; ensure that the cells form tight junctions with each other; and confirm that the endothelium has the correct anticoagulant phenotype. These requirements are also relevant, Dr. Niklason advises, for the regeneration of any other organ.

The Matrix Meets the Bioreactor

“We showed that we can decellularize several human and porcine organs, including heart, lung, kidney, and pancreas,” says Harald C. Ott, M.D., assistant professor of surgery at Harvard Medical School and principal investigator at the Ott Laboratory for Organ Engineering and Regeneration. Dr. Ott and colleagues recently described a pressure-controlled perfusion protocol for decellularizing the rat heart. Detergent removes cellular components while preserving the complex three-dimensional acellular scaffold and the extracellular matrix proteins. The protocol does not rely on enzymatic treatment, and it does not involve freeze-thaw treatments.

During the engineering of bioartificial organs, decellularization is merely an initial step. After a scaffold is generated, subsequent steps involve sterilization, reseeding with the appropriate cells, the formation of a vasculature, and its stimulation and development into a mature organ. These processes are performed in bioreactors, which support development and preserve organ function.

“Our group is working on generating the bioreactors that are essential for growing these organs,” explains Dr. Ott. Recently, Dr. Ott and colleagues have reported a fully automated, computer-controlled, closed-circuit clinical-scale bioreactor that was able to maintain human and porcine lungs for at least 72 hours. This strategy promises to expand the pool of organ donors by helping the long-term ex vivo maintenance and the repair of organs intended for transplantation.

Decellularized porcine heart mounted in a bioreactor. [Bernhard Jank, Ott Laboratory, Harvard Medical School]

Engineered Organs Enter the Clinic

“We started using decellularized solid structures in the early 1990s,” recalls Anthony Atala, M.D., chair of urology at Wake Forest University and director of the Wake Forest Institute for Regenerative Medicine. “It took a long time to get where we are.”

According to Dr. Atala, the bioengineering challenges posed by tissues and organs have four levels of complexity. From simple to complex, these levels correspond to flat structures such as the skin; tubular structures such as blood vessels; hollow organs such as the bladder and the vagina; and solid organs such as the heart, the lung, and the kidney. “We have already developed bioengineered organs from the first three levels of complexity, and we are using them in the clinic,” asserts Dr. Atala.

In the early 1990s, Dr. Atala and colleagues developed approaches to grow muscle and endothelial cells in animal models. “We did not have the technology available to create three-dimensional scaffolds,” notes Dr. Atala. “This is when it occurred to us that we could use detergents to wash the cells away and repopulate the organs, just as we have done for flat structures.”

In a rabbit model, Dr. Atala and colleagues previously reported the successful seeding of autologous corpus cavernosal smooth muscle and endothelial cells on collagen matrices to form functional corpora cavernosa tissue. Stemming from that work, investigators in Dr. Atala’s group used a bioengineering approach to reconstruct the rabbit penile corporal body in its entirety. The structural parameters of the autologous implants were comparable to those of the native tissue, and most male rabbits receiving bilateral implants were able to impregnate females, which gave birth to healthy pups.

Most recently, expanding on in vitro and animal studies conducted for over two decades, Dr. Atala’s group successfully developed, for the first time, a technique to engineer neovaginal organs. The technique involved seeded  biodegradable scaffolds with autologous muscle and epithelial cells.

As shown at an eight-year follow-up after transplantation into women with congenital vaginal aplasia caused by the Mayer-Rokitansky-Küster-Hauser syndrome, engineered autologous vaginal organs presented all the expected structural parameters, including epithelial tissue, matrix, and smooth muscle. A self-administered functional questionnaire revealed a normal range of function in all areas tested, without any long-term complications, opening a promising avenue for reconstructive medicine.

The possibility of generating bioengineered tissues and organs is intimately linked to and dependent on advances in other fields, including stem cell biology, bioengineering, chemistry, and biomaterials. As a new paradigm in translational medicine, regenerative medicine is powerfully positioned to reshape virtually every medical field and bring about transformative changes in healthcare. 

At the Wake Forest Institute for Regenerative Medicine, a 3D printer was used to fabricate the kidney, finger bone, and ear scaffolds shown here.

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