New stem cell studies at the University of Maryland Dental School demonstrate that surgeons could one day routinely use strong, mold-able, and inject-able pastes to regenerate needed bone tissue to repair broken bones, fractures, genetic defects, even combat bone wounds.

The Dental School’s Huakun Xu, PhD, MS; Michael Weir, PhD, MS; and Ryan Zhao, MD, PhD, presented their findings today at the World Stem Cell Summit at the Baltimore Convention Center before hundreds of stem cell experts from 25 countries.

The Dental School presentation showed that human stem cells seeded in a tissue engineering scaffolding exhibited “excellent attachment and osteogenic differentiation,” which is the process of laying down new bone material.

The researchers said the new findings buoy hopes that an inject-able paste of stem cells will be available one day to fill any shape of cavity from bone defects, breaks or wounds by regenerating needed bone tissue.

In test tube studies, stem cells from bone marrow, when placed into an inject-able scaffold of calcium phosphate and chitosan, started growing and forming minerals needed for new bone tissue.

Xu, an associate professor, is the principal investigator of a $230,000 grant from the Maryland Stem Cell Research Fund for “Stem Cell Delivery via Inject-able, Nano-apatite Scaffolds for Bone Engineering,” and a $1.84 million grant from the National Institute of Dental and Craniofacial Research.

The Dental School researchers have so far tested four scaffolding materials for gripping and holding the stem cells. “Which of the materials will be used in a commercial product really depends on where you want to place the material, whether in the jaw bone, the cranium or other bones,” said Weir, a research assistant professor.

Weir said, “Ultimately we want this to be an inject-able paste so we can put it into voids that are not square, rectangular or circular, that they are irregular shapes that need to be filled. The paste will include the cells.”

Xu added that such a product could also be used in periodontal bone repair, mandibular and maxillary ridge augmentation, reconstruction of frontal sinus and craniofacial skeletal defects, and other stress-bearing orthopedic applications. After a tumor removal or traffic accident, there may be a need to repair the damage or void left. It will beneficial, he said, to have a paste that can be shaped easily to achieve a high degree of aesthetics. After shaping, the paste hardens to form a solid scaffold full of pores and channels and still containing stem cells throughout, still living and growing to form new bone. Eventually the scaffold material degrades and is replaced entirely by new bone tissue grown from the stem cells.

The researchers found that a significant number of the cells were alive after a few weeks in the scaffolding material. They then discovered that the cells were differentiating into osteoblasts, essentially turning into bone cells. (From Greek words for bone, an osteoblast cell is responsible for bone formation.)

After staining the scaffold, the researchers found the osteoblasts forming “a lot” of the mineral, which then forms the bone after only 21 days, said Weir. In a subsequent experiment, the cells survived even better when mixed in a gel of the scaffolding material.

The researchers have recorded similar success with umbilical cord-derived stem cells, which “appear to be more potent in terms of growth and transforming into osteoblasts on the scaffold than the cells from bone marrow,” said Xu. It is likely that the umbilical cord cells are more vital because they are younger than stem cells obtained from the adult bone marrow and in theory will act more quickly to repair wounds or bone defects.

Xu explained: “When a 16-year-old breaks a bone, it usually takes a few weeks to heal. In a 60-year-old, it likely takes a few months. Umbilical cord stem cells are only 9 months old and hence are fast in healing.” Xu said human umbilical cord stem cells have the promise to be a superior alternative to bone marrow-derived stem cells, the latter requiring an invasive procedure to harvest. For combat medics, the umbilical cord derived stem cells could potentially be on the shelf and used in the field without causing immunuorejection, said Xu.

Xu said that after a literature search, he believes his laboratory is the first to investigate the seeding of umbilical cord-derived stem cells in injectable and load-bearing scaffolding for bone tissue engineering.

“We are excited about the promise of encapsulating umbilical cord stem cells in an injectable scaffold for stem cell delivery and bone regeneration.” Xu said. “Our research is still in an early stage. We will perform more systematic investigations and animal studies. If indeed human umbilical cord stem cells delivered using injectable scaffolds are more effective in bone regeneration than the commonly studied bone marrow stem cells, it will broadly impact the field of stem cell-based regenerative medicine.”

With a simple, new technique, orthopaedic surgeons can now regenerate damaged cartilage in injured joints with stems cells harvested from their patients’ own blood.

AS she took the last steps to the top of Batu Caves, Joanna Hart was exhilarated. It was not something expected she could ever achieve at this point of her life, not when only two years ago, at age 34, she was offered an option usually given to people twice her age: a knee replacement for her left knee.

“Every time I go for X-rays, the radiologist look at the result and go, “‘Gosh, what happened to your knee? Your knee looked like a 70-year-old’s!’ It was very bad,” she described.

The medical history of her left knee was as extensive as it was active. Recurrent dislocations when she was 16 to 19 years old had resulted in a bad knee. “I was doing the high jump, long jump, relay, netball and hockey then. And so when I was 19 my kneecap dislocated – it wouldn’t stay in, it kept coming out,” she explained.

Her doctors recommended surgery to set her kneecap in the correct position to avoid it from dislocating further. However, it ended up giving her a different set of problems. The kneecap was set too high and it was rubbing against her bones.

“Over the years, all the cartilage got worn away,” she said. And as a result of that, bone spurs (osteophytes) started growing where her cartilage had worn off.

“As a midwife, I was very active. And I kept fracturing off those osteophytes and they got stuck in my joint,” she said. “And by then I wouldn’t be able to straighten or bend my leg because it’ll be locked. So, I had to go for surgery – they’ll pull the bit out, sew it up, and off I go again. This kept happening over a period of about 10 years.”

But being physically active in her line of work had kept her knee mobile. It was only when she stopped working that the spurs began building up in her joints again.

“Again, I couldn’t straighten my leg. So, I went to see a surgeon, who looked at the results of my arthroscopy (a minimally invasive surgical procedure to examine or treat a joint), and told me that I needed a total knee replacement,” she said.

As Hart was not keen on the idea, she hesitated – until she found another option that she could accept.

Stem cell repair

What Hart stumbled upon was a minimally invasive procedure, which was in its final stages of research in goats.

Using stem cells from goats, the doctors were able to stimulate cartilage regeneration in the goats’ knees.

“My quality of life was getting lesser by the day because at that point, after all the surgeries, I had to give up athletics, netball and hockey. And then I had to give up jogging and running,” Hart said.

However, what mattered most to her were not the activities she had to give up, but the life she was looking forward to with her children.

“I have two young children and I want to be able to go horse riding and skiing with them. A knee replacement is only going to stop the pain, but it would not make the restrictions any better.”

So, after the completion of animal studies (now accepted for publication in the Arthroscopy: The Journal of Arthroscopic and Related Surgery), Hart proceeded with the surgery. “Now I’m able to jog a little – more like shuffling, really – but I’m moving around a lot more, and I’m going for a skiing holiday this Christmas!” she said with a big grin.

“It’s really simple,” said orthopaedic surgeon Dr Saw Khay Yong, who led the research. “Once the diagnosis of cartilage injury is made, we then start with surgery where the patient has arthroscopy with subchondral drillings into the damaged cartilage areas.

“The stem cells are then harvested one week after surgery. It is a weekly injection into the knee joint starting at one week after surgery, for five consecutive weeks.”

Getting creative with old tools

“Peripheral blood stem cells (PBSCs) have been used by haematologists to treat leukemia patients for the last 20 years and subchondral drilling (the drilling of bone under cartilage layers) is also an established procedure in orthopaedics,” he said.

It all came together when Dr Saw and his colleagues, spurred by the desire to find another alternative to conventional methods of treating damaged cartilage, decided to give stem cells a try.

“If you look at cartilage injuries, currently there are a lot of possible solutions, but the results are inconsistent,” he said.

As some of the current options to treat damaged cartilage (autologous chondrocyte implantations, cartilage transfers and cartilage transplants) may be quite expensive and they often require multiple surgeries, they have never been attractive to him, Dr Saw said.

“So, we started to look into ways we can use stem cells to regenerate our cartilage with the University Veterinary Hospital at Universiti Putra Malaysia,” he added.

Their study in goats started in 2005, where Dr Saw’s team harvested stem cells from goat bone marrow and injected them into the goats’ knees after creating defects (by drilling holes in the cartilage and bone in their joints). When the study was completed in 2007, they proceeded to perform the procedure in humans.

What the procedure does is to accelerate the natural healing process that happens in articular (or hyaline) cartilages in the knee.

“Usually, when you have a partial thickness injury (when the cartilage wear has not exposed the underlying bone), there is no evidence of repair. But when you have a full thickness injury that penetrates into the bone, you can access the bone marrow stem cells within it, which will then initiate repair,” he explained.

By creating full thickness injuries by drilling holes in the bones where cartilage has worn out, you can create an environment where the cartilage can start to heal. And, to aid the process, doctors provide the building blocks: stem cells and hyaluronic acid (a chemical present in cartilage).

But how do the stem cells know where to go? Dr Saw explains: “When you drill the bone, it forms a blood clot. And when that happens, injured cells send out homing signals that attract stem cells from the bone marrow. After that, physiotherapy will provide the environment for the cells to grow into cartilage cells.

“And in this procedure, we provide the stem cells through injections,” he adds.

For the young and active

Although two-year results of the procedure in his patients are encouraging, Dr Saw is not recommending it for everyone.

As it takes a lot of physiotherapy and time – about two years – to achieve best results, he reckons that this might not be the best alternative for the elderly.

Former Miss Malaysia and model Betty Anne Brohier, 43, would attest to the challenges one has to face during recovery. A torn (and later removed) left meniscus (cartilage in the knee joint) when she was in her teens had stopped her from participating in sports but her job as a model has kept her on her feet (and heels) most of the time.

“It used to be quite painful but I thought it was fine. But throughout the years the pain became worse and it affected both knees,” she said.

Her left knee was on the verge of ”collapsing” when she finally agreed to undergo the procedure. “After the surgery, I stayed in the hospital for one week. Following that, for about six months, I used to go for physiotherapy three to four hours every day,” she said.

The road to recovery was long as she needed to learn how to walk and use her leg again. It took her six weeks after surgery to be able to move her leg. Another five months was spent moving around in crutches.

“No pain, no gain, I guess,” she pointed out.

It is widely accepted that stem cells are involved in tissue regeneration. It is also widely accepted that (in most organs) stem cells are vanishingly rare. So: if there doesn’t happen to be a stem cell adjacent to a site of damage, how can stem cells be involved in the process of tissue repair?

One possible answer: There might be more stem cells than we think, because we’ve been missing them for some reason. This possibility (”both”) is strongly supported by the recent findings of Zuba-Surma et al., who have discovered a population of tiny pluripotent cells (termed, appropriately, very small embryonic-like, or VSELs) scattered throughout the body.

Very small embryonic-like stem cells in adult tissues—Potential implications for aging

Recently our group identified in murine bone marrow (BM) and human cord blood (CB), a rare population of very small embryonic-like (VSEL) stem cells. We hypothesize that these cells are deposited during embryonic development in BM as a mobile pool of circulating pluripotent stem cells (PSC) that play a pivotal role in postnatal tissue turnover both of non-hematopoietic and hematopoietic tissues.(cont.)

During in vitro co-cultures with murine myoblastic C2C12 cells, VSELs form spheres that contain primitive stem cells. Cells isolated from these spheres may give rise to cells from all three germ layers when plated in tissue specific media. The number of murine VSELs and their ability to form spheres decreases with the age and is reduced in short-living murine strains. Thus, developmental deposition of VSELs in adult tissues may potentially play an underappreciated role in regulating the rejuvenation of senescent organs. We envision that the regenerative potential of these cells could be harnessed to decelerate aging processes.

Note that both VSEL number and potency diminish with age, consistent with the decrease in proliferative and regenerative capacity that we see in older animals. (And recall that diminishing stem cell potency is just one side of the story: over the course of aging, tissue microenvironments themselves grow more hostile to stem cell growth and function).

The small size of the VSELs, along with their dispersal throughout the body, might explain why they’d been missed up until now. It makes sense that cells devoted to long-term storage of regenerative potential would be very little: other than surviving and maintaining the ability to respond to proliferative signals, they wouldn’t really have much in the way of functional requirements, and wouldn’t need much more than a nucleus, a membrane, and extremely vigilant signal-transduction pathways — the latter ready to awaken the dormant cell when it’s time to turn into a proper stem cell, divide, and differentiate. In a sense, then, these VSELs are not so much progenitors as “progenitor progenitors”, the of regenerative capacity lying silent until they are needed.

(Extending this admitted over-interpretation — small size, after all, does not mean low metabolism, but I’m reasoning by analogy to spores and other very small totipotent cellular forms — another advantage of keeping stem cells metabolically inactive is that they would be less likely to suffer mutations or other damage that could convert them into cancer stem cells.)

Required skepticism: VSELs are both brand new and (so far as I can tell) idiosyncratic to a single group’s work. Before we get too worked up about this, I’d like to see the work reproduced by other labs and in other systems. Hopefully that sort of confirmation is already underway.