In this final part of our four-part series on regenerative medicine we digest reports of advances in engineering new tissues and organs, including methodologies and specific organs/tissues. We began the series by noting there is a “gold rush” of research and venture capital into regenerative medicine. In showing examples of nuggets unearthed so far, this issue perhaps best explains why.

Tissue Self-Assembly and Sculpting

Cells coated with sticky bits of DNA have self-assembled into functional three-dimensional microstructures that could become the building blocks of precision-engineered multicellular tissues. This “bottom-up” method of tissue engineering is in contrast to the “top-down” method of seeding cells on a scaffold (the method used by Dr. Anthony Atala to create whole bladders.) The new method, still in its development infancy, has the potential to better recreate tissue complexity, or at least to contribute to other methods such as “bioprinting”—printing out an organ in 3D using an ink-jet printer adapted to spray cells rather than ink.

In another approach to self-assembled complex tissue-like structures, early last year a bacteriophage was used to create scaffolds that support the growth and organization of nerve cells. Bacteriophages are viruses that infect bacteria but not animal cells. Genetically engineered bacteriophages have already been approved by the FDA as an antibacterial food preservative for lunch meats such as bologna.

The “M13” bacteriophage used to create nerve structures is similar in form to the protein fibers that make up the body’s natural cellular matrices.

Another experimental approach to scaffolds, one that has the potential to provide a “controlled microarchitecture” for engineered tissue, is to encase the regenerative cells in polyethylene glycol (PEG), a polymer commonly applied in medicine. The PEG casing forms a cube around each cell, which can be arranged like Lego blocks in specific shapes, without need for any special equipment.

The method was used to build tubes that could function as capillaries.

For bone damage, a variation on the scaffold approach, to be licensed to commercial developers by the end of the year, is to wrap the damaged bone or missing bone area in real or artificial periosteum (the outer surface of all bones.) The periosteum wrap contains (or can be seeded with, if artificial) bone stem cells that proliferate and create new bone.

A real (not artificial) periosteal graft was tested on a disabled patient who needed surgery to lengthen one of her legs. The patient saw new bone growth one month after surgery. The artificial periosteum, seeded with pieces of periosteum taken from the patient’s surrounding bone, was successfully tested on sheep.

The method can’t fix large gaps in bone, but according to one expert it “will have a significant effect for healing fractures.”

Another approach uses a scaffold made from flexible, soluble fibers containing growth factors which, under controlled release, encourage regenerating tissue to connect with existing healthy tissue. The scaffold can be sculpted into any shape. When the tissue is established, the fibers dissolve. The technology could be used to regenerate not only bone but also muscle, arteries, nerves, and skin and might one day be used in cosmetic and not just reconstructive surgery.

As of last Fall, in vitro results on bone were encouraging, as were unpublished results from animal models.

Yet another potential bioscaffold approach uses soluble, porous glass that stimulates bone regeneration by eluting calcium, silicon, and other mineralizing elements into adjacent body fluids as it slowly dissolves. The mineralization of their environment stimulates stem cells to turn into bone cells. The elution can be controlled practically down to the atomic level.

A glass/polymer hybrid stronger than the glass alone will be developed next. Human clinical trials are about five years out.

Extracorporeal Tissue Engineering

The problem of microarchitecture—in particular, how to build a vasculature inside an engineered solid organ—has been addressed in one project by using tissue temporarily removed from the patient as a scaffold. Since the tissue comes with vasculature already intact, all that needs to be done is to keep it fed with oxygen and nutrients and seed it with stem cells. The stem cells are then induced to turn into healthy, specialized cells that produce proteins missing in people with conditions such as hemophilia or diabetes. Once the stem cells become established in the tissue, it is put back in the patient and reconnected to the circulatory system.

Because the technique uses the patient’s own tissue, it avoids the rejection and complication risk associated with the use of artificial or donor scaffolding materials.

The good news: In many patients, the re-implanted tissue became nearly indistinguishable from surrounding tissue within 28 days. The bad news: The patients were mice. But eventually it should be possible to “supply the synthetic function of an organ by stimulating the cells to form insulin-producing pancreas cells or albumin-producing liver cells,” as one of the researchers responsible put it.

The researchers are already trying to use the technique to deliver blood-clotting components (missing in hemophiliacs) but they concede not only that much remains to be done before the technique could be used to generate whole organs, but also that other methods could reach that goal first.

Extracorporeal Organ Repair

Patients with end-stage respiratory diseases such as emphysema and cystic fibrosis can have their donated lungs reconditioned “while-u-wait” for the transplant in the OR. A technology first used in a patient in late 2008 keeps a pair of human lungs “alive” for up to 12 hours while surgeons assess their function and repair them if necessary and possible.

As of April last year, six more patients had received donor lungs reconditioned in this way and all were said to be doing fine.

This technique could reduce by half the critical shortage of acceptable donor lungs (assuming good long-term survival rates), but it is not the only technique. Another technique, announced last October, uses extracorporeal gene therapy to repair donated lungs deemed too damaged to transplant. This approach is “a long way from prime-time,” a surgeon commented, but it “has the potential to change the way we do things.”

Organ Factories

Meanwhile, scaffold-based tissue engineering is much further advanced and already at the bedside. Dr. Atala told a newspaper that engineered bladder transplants are now common enough that “bladders come in three sizes: small, medium and large.” Remember, these are not bladders taken from organ donors, but bladders grown in what is essentially an organ factory. Production takes about six weeks. Presently, the bladders are grown from bladder cells painted onto a biocompatible scaffold shaped like a bladder, but Atala and his colleagues are turning to bioprinting of tissues and organs including ears, arteries, heart valves, fingers, and toes.

On Their Way

Some other organs and tissues at various stages of development, from experimental to in-the-OR, are described below:

Windpipes and Voice Box

The first tissue-engineered partial windpipe was implanted in a patient in Spain in 2008. Last March, a British boy became the first child to receive a tissue-engineered whole windpipe. In both cases, donor trachea were stripped down to their bare collagen scaffolding then seeded with stem cells from the patient. But in the Spanish case, the stem cells were grown on the scaffold extracorporeally, while in the British case the stem cells were grown intracorporeally after implant.

Eighteen months ago, a British medical task force began looking into the possibility of engineering a whole voice box, including the windpipe and laryngeal muscle. Muscle is said to be difficult to engineer.

Heart Muscle

But they are working on it. At Harvard, a “patch” of heart muscle made of ventricular muscle cells, the type damaged in heart attacks, has been created from mouse embryonic stem cells (ESCs.) Using such tissue as a patch to repair damage may be better than injecting stem cells from bone marrow directly into the heart, which has had mixed success so far. The researchers would also prefer to engineer the tissue using induced pluripotent adult stem cells (iPSCs) instead of ESCs. Then, a skin biopsy from a heart-attack patient would be all it takes to engineer and grow a rejection-less heart patch.

Besides its therapeutic potential, engineered muscle could be used to test drugs, chemicals, and nanomaterials. But before human therapies are possible, three-dimensional versions of the two-dimensional patches so far created must be made. That will require the addition of vasculature to feed the muscle—part of the tissue “microarchitecture.”

Duke researchers have apparently made a similar patch, also from mouse ESCs and also lacking a vasculature.

Penile Tissue

Dr. Atala’s team has engineered erectile tissue of the rabbit penis. Rabbits treated with it had normal sexual function and produced offspring. The hope is that “patients with congenital abnormalities, penile cancer, traumatic injury and some cases of erectile dysfunction will benefit from this technology in the future,” Dr. Atala said. But there is much to do before human penile tissue is engineered and tested in humans.

Nerve Cells

After injecting a neuron-destroying chemical into the subiculum area of the hippocampal system in 48 brain-damaged adult rats, Indian researchers transplanted hippocampal cells, taken from newborn transgenic mice and further cultured in the lab, into the hippocampi of about half the rats. The transplant rats completely recovered their ability to learn, while those without transplants did not.

The transplants appeared to promote the secretion of factors that boost the growth and survival of neuron progenitor cells. This finding could lead to therapies for various brain disorders, though “We are still some way from achieving a new therapy based on these findings,” a researcher cautioned.

Engineered Jaw Joint

The complexity of the temporomandibular joint (TMJ) structure makes it hard to repair with grafts. A part of the TMJ has been made from human iPSCs extracted from bone marrow and seeded into a tissue scaffold formed into the precise shape of the human jaw bone. The precision was achieved by using digital images from the patient.

The engineered TMJ did not include other tissue, such as cartilage, and work is ongoing to add that. plus a blood supply. So there’s a lot of work to do, but engineering the bone to a precise shape is a significant step forward. The same technique could be used to repair other bones in the head and neck, including skull bones and cheek bones, which are similarly difficult to graft.

Sculpted Breasts

Coming sooner to market is a sculpted scaffold designed to stimulate natural breast tissue (basically, fat) regrowth for reconstruction following mastectomy. The scaffold is about to enter phase I trials in Australia, following apparently successful tests in pigs. The scaffold contains a gel made using the patients’ muscle cells to induce fat tissue production. Future scaffolds will be biodegradable, so presumably the current scaffolds need to be removed somehow once the breast has finished regrowing.

A possibly simpler way to make cells form 3-D tissue structures is via a gel containing phages engineered to carry iron oxide nanoparticles. The nanoparticles get transferred to cells added to the gel, enabling researchers to control the position of the cells with a weak magnet.

It turns out that these “suspended” cells behave more naturally than cells grown on a conventional flat surface. Commercialization of the gel has begun.

Lungs

A functioning lung engineered from fetal lung cells and blood vessel cells grown for a week on the natural scaffold of a donor lung has been successfully transplanted into a live rat, where it exchanged carbon dioxide with oxygen in the blood—just like a normal lung—for two hours. The next step is to increase that time, by improving the engineering based upon the lessons learned.

One of the researchers thought it would “probably be 20 years” before engineered lung transplants for humans are possible. We think he is somewhat irrationally under-exuberant, given the accelerating pace of advances in tissue engineering we cover in this issue.

Liver

Other researchers also used natural structural tissue, this time from a liver, as a scaffold for growing a new organ. And instead of first cleaning the scaffold of all cells before seeding it with progenitor liver cells, they instead devised a way to keep the scaffold’s existing intricate vasculature.

The engineered livers functioned for up to 10 days in culture, and for “several hours” (eight, according to another report) when transplanted into live rats. Like the lung engineers, the liver engineers cautioned that there is a long way to go before human trials are possible, but they were much less pessimistic: five to ten years, they thought.

Eye Tissues

Because the nerves behind photoreceptors (light-switching cells) remain intact in patients with macular degeneration and retinitis pigmentosa, all that is needed to restore vision is new photoreceptors—and a way to connect the cells to the nerves through the scar tissue that forms during the disease progression.

The solution was to attach progenitor cells to an implant made of nanoengineered polymer fibers containing pockets full of scar-dissolving enzymes that slowly migrate out as the fiber degrades. The enzyme dissolves the scar tissue, and the progenitor cells are free to link up with the neurons.

Results in mice have been encouraging. Meanwhile, another research team has transformed iPSCs derived from adult human skins cells into photoreceptors and transplanted them into a mouse retina, where they integrated normally into the surrounding tissue. The team had earlier achieved the same success with embryonic stem cells.

Yet another team has induced bone marrow stem cells to repair damaged retinas in mice. At first, they used a viral vector (a virus to carry the reprogramming genes to the cell) but were later able to achieve the same goal using chemicals that mimicked the body’s natural signaling channels, signaling to the stem cells that they should become retinal cells. After 28 days, treated mice that had previously demonstrated no retinal function responded to light just like normal mice.

Using chemical drugs rather than genetic manipulation opens up a whole new set of therapeutic possibilities. A researcher envisages that ultimately the stem cells could be transformed in this way extracorporeally for transplant into the retina.

Meanwhile in Italy, three quarters of 112 patients blind in one or both eyes, some for several years, as a result of chemical burns regained their vision following implants of healthy stem cells, and have retained it for three to ten years. The cells were taken from the limbus area of their eyes and grown in the lab on a fibrous tissue. After implantation into the damaged area of the eye, the tissue grew into healthy corneal tissue, leaving eyes with normal appearance and color.

While tests of a similar procedure in the US produced only short-term improvements, at least one hospital in India is apparently offering such transplants.

Skin

Skin cells scraped from an undamaged area of a severe second-degree burn victim and sprayed in solution directly onto the burn could replace the need for skin grafts. The method, developed by an Australian surgeon into a kit called ReCell, which has been approved and is already marketed in some countries, is far less invasive and less painful for the patient and cuts the area to be healed in half. The outcome is said to be as good as skin grafts. The US Army is sponsoring a trial of ReCell involving more than 100 patients.

ReCell is not for third-degree (the most severe) burns, though at least one hospital in Australia uses it in conjunction with other treatments for third-degree burns and for treating burn scars. They claim improved outcome and faster healing, and to have eliminated the waiting list of patients for reconstruction surgery to fix the deep scars that accompanied third-degree burns.

What sounds like a much more cumbersome method of applying the scraped-off cells is to spray them onto the skin through one implementation of a bioprinter mounted on a wheeled frame and positioned over the bed of the patient. Cumbersome or not, this device has reportedly worked well in mouse tests.

*  *  *

This marks the end of our four-part series on regenerative medicine, but news of advances arrives daily and at an accelerating pace, so this will hardly be our last word on the topic.

Standard Podcast Play Now | Play in Popup | Download