Bone-Making Ability of Amniotic Epithelial Cells


Human amniotic epithelial cells (hAECs) develop from the embryo as early as eight days after fertilization. hAECs have been shown to possess remarkable plasticity, or the ability to form many cell types. Mattioli and others showed that AECs from sheep have capacity to differentiate into bone cells (see Cell Biol Int 2012;36:7-19). Therefore, it is possible that these cells could be developed into a source of cells for regenerative treatments.

Steve Shen from the Shanghai Jiao Tong University School of Medicine and his coworkers have examined the ability of cultured hAECs to make bone in culture, and then applied their techniques to an animal system to test the ability of cultured hAECs to restore tooth sockets and facial bone.

To begin, hAECs were isolated from healthy mothers who had undergone childbirth by cesarean section. These cells were cultured in a standard cell culture medium (DMEM/F12 for those who are interested), expanded in culture, and then subjected to flow cytometry to isolate those cells with all the right cell surface markers.

These cells wee then placed into a specialized culture medium designed to convert mesenchymal stem cells into bone-making cells. This osteogenic induction medium was changed every three days for 21 days.  The figure below shows how well the bone-induction worked.  A red stain called alizarin red binds to calcium and therefore stains forming bone matrices rather well.  As you can see in the panel labeled “G” below, the hAECs grown in the osteogenic medium stain red rather well, which shows that they are making bone.

Characterization of hAECs in vitro. (A, B): hAECs at passages 0 and 1 displayed a cobblestone-like morphology. (C): Some hAECs changed into fibroblast-like cells after 7 days of osteoblastic culture. (D): hAECs showed significant morphological changes and settled on superimposed layers after 21 days of osteoblastic culture. (E): Cell proliferation of hAECs at passage 1 was significantly higher than at passages 0 and 5 from day 4 to day 12; hAECs at passage 5 displayed the lowest proliferation rate from day 6 to day 14 (☆, p < .05). (F): Flow cytometry analysis showed that hAECs expressed CD44, CD90, CD105, and SSEA-4 and did not express CD34, CD45, and HLA-DR. Values represent the percentages of all assessed cells positively stained by the indicated antigens (bottom of each graph). Nonspecific fluorescence was determined as the blank control using isotype-matched monoclonal antibodies (PE blank, FITC blank). (G): Representative images of microscopic and general photographs for ALP and ARS staining in osteogenic and control groups indicated the osteogenic differentiation of hAECs in vitro. Scale bar = 200 μm. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; FITC, fluorescein isothiocyanate; hAEC, human amniotic epithelial cell; OD, optical density; p, passage; PE, phycoerythrin.
Characterization of hAECs in vitro. (A, B): hAECs at passages 0 and 1 displayed a cobblestone-like morphology. (C): Some hAECs changed into fibroblast-like cells after 7 days of osteoblastic culture. (D): hAECs showed significant morphological changes and settled on superimposed layers after 21 days of osteoblastic culture. (E): Cell proliferation of hAECs at passage 1 was significantly higher than at passages 0 and 5 from day 4 to day 12; hAECs at passage 5 displayed the lowest proliferation rate from day 6 to day 14 (☆, p < .05). (F): Flow cytometry analysis showed that hAECs expressed CD44, CD90, CD105, and SSEA-4 and did not express CD34, CD45, and HLA-DR. Values represent the percentages of all assessed cells positively stained by the indicated antigens (bottom of each graph). Nonspecific fluorescence was determined as the blank control using isotype-matched monoclonal antibodies (PE blank, FITC blank). (G): Representative images of microscopic and general photographs for ALP and ARS staining in osteogenic and control groups indicated the osteogenic differentiation of hAECs in vitro. Scale bar = 200 μm. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; FITC, fluorescein isothiocyanate; hAEC, human amniotic epithelial cell; OD, optical density; p, passage; PE, phycoerythrin.

When hAECs were subjected to gene expression assays, it was clear that the cells grown in the osteogenic medium expressed a whole host of bone-specific genes.  Therefore, the formation of bone was not a fluke, since these cells not only concentrated calcium, but also increased their expression of bone-specific genes (these include osterix, Runx2, Alkaline phosphatase, Collagen I, and osteoprotegerin).

Additionally, those hAECs grown in osteogenic medium stopped forming sheets of cells and began to grow as individual cells.  This is an important transformation for the synthesis of bone because bone-making cells secrete a bone-specific protein matrix upon which calcium phosphate-based crystals deposit to form bone.  If the cells were organized in sheets, then bone deposition would not be possible.  When isolated from amniotic membranes, hAECs grow as sheets of cells that are known as epithelia.  In order to become bone-making cells, the hAECs must undergo an “epithelial-to-mesenchymal transformation” or EMT.  This requires turning on new genes and turning off others.  Gene expression assays of hAECs grown in the osteogenic medium, show extensive evidence of EMT.  For example, a protein called E-cadherin is essential for cells growing in sheets, because it helps cells stick to each other.  However, in hAECs grown in the osteogenic medium, E-cadherin expression was quite low.  Also, a protein called vimentin is highly expressed during EMT, and the hAECs grown in osteogenic medium showed high expression of vimentin.  Thus, these hAECs were undergoing all the necessary changes in order to become bone-making cells,, making all the right genes, and made bone in culture to boot.

This is certainly interesting, but can these hAECs repair bone in a living animal?  Shen’s group tried that very experiment.  The hAECs that were grown in the osteogenic medium were loaded on tricalcium phosphate scaffolds and implanted into rodents with tooth socket lesions.  Control animals were implanted with tricalcium phosphate scaffolds without cells.  The scaffold with cells significantly increased bone formation in the rodents, and showed much more infilling with mineralized tissue.  There was also extensive evidence of the formation of new vasculature and wandering cells called macrophages that are important for the degradation of the implanted scaffold required for new bone formation.  Tissue samples were examined 4 to 8 weeks after the implants were placed.

In vivo healing process in alveolar defect at 4 and 8 weeks postoperatively. (A): Representative three-dimensional micro-computed tomography (CT) reconstruction images of hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 1 mm. (B): Micro-CT parameters acquired among β-TCP scaffold in vitro, hAECs+β-TCP scaffold in vivo, and β-TCP scaffold alone in vivo at 4 and 8 weeks postoperatively (☆, p < .05). (C): Hematoxylin and eosin staining of the rat alveolar defect at 4 and 8 weeks postoperatively revealed more active new bone formation in the EXP group than in the CTR group (×50 and ×200 magnification). Alveolar defect treated with hAECs+β-TCP scaffold exhibited a more mature lamellae-bone formation at 8 weeks postoperatively. Scale bar = 200 μm. (D): ANA-positive cells, visible as green fluorescence in the nuclei, were observed within the newly deposited OCN, and OPN-positive bone tissue, visible as red fluorescence, in the EXP group at 4 weeks postoperatively, indicating a mature osteoblastic function of these hAEC-derived cells. Scale bar = 50 μm. (E): Representative images of immunohistochemical staining of sections with anti-VEGF antibody and anti-CD68 antibody in hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 200 μm. (F): The histomorphometric quantification of the relative new bone area, VEGF-positive area, and CD68-positive area showed that more bone tissue regeneration was observed in the EXP group than in the CTR group at 4 and 8 weeks postoperatively. The positive signal of VEGF and CD68 in the EXP group was much weaker at 4 weeks postoperatively and became more intense at 8 weeks postoperatively compared with the CTR group (☆, p < .05). Abbreviations: ANA, anti-nuclear antibody; BV/TV, bone volume/tissue volume ratio; BMD, bone mineralization density; hAECs, human amniotic epithelial cells; OPN, osteopontin; post op, postoperatively; SMI, structure model index; Tb.Th., trabecular thickness; Tb.N., trabecular number; Tb.Sp., trabecular separation; β-TCP, β-tricalcium phosphate; VEGF, vascular endothelial growth factor.
In vivo healing process in alveolar defect at 4 and 8 weeks postoperatively. (A): Representative three-dimensional micro-computed tomography (CT) reconstruction images of hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 1 mm. (B): Micro-CT parameters acquired among β-TCP scaffold in vitro, hAECs+β-TCP scaffold in vivo, and β-TCP scaffold alone in vivo at 4 and 8 weeks postoperatively (☆, p < .05). (C): Hematoxylin and eosin staining of the rat alveolar defect at 4 and 8 weeks postoperatively revealed more active new bone formation in the EXP group than in the CTR group (×50 and ×200 magnification). Alveolar defect treated with hAECs+β-TCP scaffold exhibited a more mature lamellae-bone formation at 8 weeks postoperatively. Scale bar = 200 μm. (D): ANA-positive cells, visible as green fluorescence in the nuclei, were observed within the newly deposited OCN, and OPN-positive bone tissue, visible as red fluorescence, in the EXP group at 4 weeks postoperatively, indicating a mature osteoblastic function of these hAEC-derived cells. Scale bar = 50 μm. (E): Representative images of immunohistochemical staining of sections with anti-VEGF antibody and anti-CD68 antibody in hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 200 μm. (F): The histomorphometric quantification of the relative new bone area, VEGF-positive area, and CD68-positive area showed that more bone tissue regeneration was observed in the EXP group than in the CTR group at 4 and 8 weeks postoperatively. The positive signal of VEGF and CD68 in the EXP group was much weaker at 4 weeks postoperatively and became more intense at 8 weeks postoperatively compared with the CTR group (☆, p < .05). Abbreviations: ANA, anti-nuclear antibody; BV/TV, bone volume/tissue volume ratio; BMD, bone mineralization density; hAECs, human amniotic epithelial cells; OPN, osteopontin; post op, postoperatively; SMI, structure model index; Tb.Th., trabecular thickness; Tb.N., trabecular number; Tb.Sp., trabecular separation; β-TCP, β-tricalcium phosphate; VEGF, vascular endothelial growth factor.

hAECs are typically not recognized by the immune system as foreign and they also have anti-scarring capabilities.  Not only can they effectively form bone in culture, but they can also repair an alveolar defect in a rodent model.  Clearly these cells show promise for clinical applications.  However, before they can be used in the clinic more has to be known about their culture conditions, how many cells are required for transplantation, the best cell dose, at what passages they need to be used, and so on.  Thus, this paper represents a good start for hAECs, but they have to be better understood before they can come to the clinic.

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Direct Reprogramming Cells with Recombinant Proteins


In my opinion, for what it’s worth, we will probably see direct reprogramming take a prominent place in regenerative medicine in the future. It will not be in the near future, but as direct reprogramming becomes better understood and more feasible, it will probably become a central part of the discussion of regenerative medical strategies.

Direct reprogramming, which is also known as lineage conversion, uses cell type-specific transcription factors to convert a mature, adult cell into a different type of mature, adult cell. The cell does not pass through a pluripotent intermediate, and becomes a wholly different type of cell.

Of course, forcing the expression of lineage-specific transcription factors in cells requires that they be treated with recombinant viruses or other such tools. These genetic manipulations present problems for regenerative medicine, since such viruses can cause mutations or cause the introduced genes to be constantly activated, both of which can cause cells to die to grow uncontrollably. Genetically engineering cells needs to be done in a “kinder and gentler” way (to quote George HW Bush).

To that end Dennis Clegg and his colleagues from the Center for Stem Cell Biology and Engineering at UC Santa Barbara have used specially designed proteins to directly cultured retinal pigmented epithelial cells to neurons.

Newly discovered C-end rule (CendR) cell- and tissue-penetrating peptides have a arginine-rich sequence at the end of proteins that allows them to bind particular cell receptors and be internalized into the cell. These CendR peptides bind to the NRP-1 protein and are internalized. Several laboratories have used CendR peptides to increase the efficacy of anti-cancer drugs in experimental cases (see Alberici L, et al (2013) Cancer Res 73:804–812; Sugahara KN, et al. (2010) Science 328:1031–1035; Sugahara KN, et al. (2009) Cancer Cell 16:510–520; and Roth L,, et al. (2012) Oncogene 31:3754–3763).

By tacking a CendR peptide to the end of the Sox2 protein, Clegg and others were able to convert retinal pigmented epithelial (RPEs) cells to neurons. The Sox2 protein is highly expressed in neural progenitor cells. Other studies have shown that Sox2 can reprogram mouse and human fibroblasts to neural stem cells (Ring KL, et al. (2012) Cell Stem Cell 11:100–109). Thus, Sox2 should do the trick.

Making cultured RPE cells from embryonic stem cells is relatively easy to do. Therefore, Clegg and his coworkers made cultured RPEs and then treated them with viruses that expressed Sox2. The cultured RPEs showed conversion to neurons and the expression of neuron-specific genes.

Since they had established that Sox2 could convert RPEs to neurons, they tried recombinant Sox2 protein with the CendR peptide RPARPAR at the end of the protein. After 60 days in culture, the cells expressed a host of neuron-specific genes, and were capable of taking up a dye that only active neurons can take up (FM1-43).

Reprogramming human fetal RPE (hfRPE) cells to neurons using recombinant SOX2 proteins. (A): Efficiency of hfRPE cells to be reprogrammed to neuron-like cells after recombinant proteins was added to the media every 24 hours for 30 days. (B): Efficiency of hfRPE cells to be reprogrammed by adding SOX2-RPARPAR recombinant protein every 48 hours for different time courses. (C): Representative images of hfRPE (fRPE1914) cells during reprogramming to neuron-like cells after 30, 40, and 50 days in culture with SOX2-RPARPAR protein. Scale bars = 100 μm. (D): Representative images of hfRPE (fRPE1914) cells reprogrammed to neuron-like cells expressing neuronal markers, but not an RPE marker (PAX6), using SOX2-RPARPAR protein. Scale bars = 50 μm. Abbreviations: D, days; RPE, retinal pigmented epithelial cells.
Reprogramming human fetal RPE (hfRPE) cells to neurons using recombinant SOX2 proteins. (A): Efficiency of hfRPE cells to be reprogrammed to neuron-like cells after recombinant proteins was added to the media every 24 hours for 30 days. (B): Efficiency of hfRPE cells to be reprogrammed by adding SOX2-RPARPAR recombinant protein every 48 hours for different time courses. (C): Representative images of hfRPE (fRPE1914) cells during reprogramming to neuron-like cells after 30, 40, and 50 days in culture with SOX2-RPARPAR protein. Scale bars = 100 μm. (D): Representative images of hfRPE (fRPE1914) cells reprogrammed to neuron-like cells expressing neuronal markers, but not an RPE marker (PAX6), using SOX2-RPARPAR protein. Scale bars = 50 μm. Abbreviations: D, days; RPE, retinal pigmented epithelial cells.

The efficiency for this experiment was lousy (0.3%) as opposed to the efficiency for the use of recombinant viruses (11%). Nevertheless, this experiment shows that it is possible to directly reprogram cells without using recombinant viruses.

2014 in review


The WordPress.com stats helper monkeys prepared a 2014 annual report for this blog.

Here’s an excerpt:

Madison Square Garden can seat 20,000 people for a concert. This blog was viewed about 67,000 times in 2014. If it were a concert at Madison Square Garden, it would take about 3 sold-out performances for that many people to see it.

Click here to see the complete report.

A New Way to Extract Bone-Making Cells From Fat


With the holiday season upon us, many of us tend to put on a few extra pounds of fat. However, fat also is a reservoir of mesenchymal stem cells (MSCs) that can make cartilage, bone or even more fat.

A new study by scientists at Brown University demonstrate the efficacy of a new method for extracting potential bone-making cells from human fat. To perfect their technique, the Brown University team, led by senior author Eric Darling, developed a fluorescent tag that identified any cell that expressed the ALPL (alkaline phosphatase liver/bone/kidney) gene. This probe is a fluorescent, oligodeoxynucleotide molecular beacon probe specific for ALPL mRNA.  This probe hybridizes to the ALPL mRNA and fluoresces strongly as a result of the hydridization.  Fluorescence-activated cell sorting ten isolates the ALPL-expressing cells from the non-expression cells effectively and easily.

Molecular beacon probes are single-stranded nucleic acid molecules that have a fluorescent dye attached to one end and a quenching molecule that prevents the dye from fluorescing at the other end.  A short, complementary sequence at either end of the probe causes the probe to self-hybridize, thus bringing the glowing molecule and its inhibitor close together.  Under these conditions, the probe does not glow.  However, if the probe hybridizes to a specific sequence, then the glowing molecule and its inhibitor are far apart and the glowing molecule fluoresces.

Molecular Beacons hybridize to their specific target sequence causing the hairpin-loop structure to open and separate the 5’ end reporter from the 3’ end quencher. As the quencher is no longer in proximity to the reporter, fluorescence emission takes place. The measured fluorescence signal is directly proportional to the amount of target DNA.
Molecular Beacons hybridize to their specific target sequence causing the hairpin-loop structure to open and separate the 5’ end reporter from the 3’ end quencher. As the quencher is no longer in proximity to the reporter, fluorescence emission takes place. The measured fluorescence signal is directly proportional to the amount of target DNA.

The level of ALPL gene expression strongly correlates with the ability of cells to form bone. According to Darling and his team, their technique more than doubled the quantity of bone-making cells that could be extracted from fat.  Brown University has applied for a patent on this method.

In culture, ALPL-expressing cells produced, on average, twice as much bony matrix as other cells. ALPL-expressing cells also were better at making cartilage or even fat. Even though other groups have isolated cells on the basis of gene expression, none of the available published techniques, to date, have used gene expression-based isolation to enrich for cell populations of a specific tissue, such as bone.

The lead author of this work, Hetal Marble, said that targeting gene expression rather than cell surface proteins is a relatively new paradigm for cell isolation and purification. Because using cell surface proteins always requires accepting certain assumptions about the cell types that are being isolated, gene expression-based isolation is a more pragmatic way to approach the problem. Consider this: cells that are in certain tissues express certain genes characteristic of that tissue. Therefore, using those gene expression patterns to isolate such cells leaves doubt that the cells you will isolate are in the process of differentiating into the desired tissue. Using cell surface proteins, however, is less precise because particular cell populations rarely are the only cell types that express that protein.

“Approaches like this allow us to isolate all the cells that are capable of doing what we want, whether they fit the archetype of what a stem cell is or not,” said Marble. “The paradigm shift is thinking about populations that are able to achieve am end point rather than isolating populations that fit a strictly defined archetype.”

In this experiment, Darling’s group used a special culture medium designed to crank up bone-specific gene expression in sensitive cells. This so-called “priming step” took four days in culture before the ALPL gene expression levels were high enough to properly detect it. However, in the future, Darling believes that by targeting a gene whose levels of expression increase earlier in during the process of differentiation, he and his team should be able to dispense with the four-day delay. This way, their technique would be applicable in the operating room, since surgeons could isolate fat from the patient with a bone break and prime it (or not), and then isolate the bone-making cells from the fat, which they would use to treat their patient’ fracture.

“If you can take the patient into the OR, isolate a bunch of their cells, sort them, and put them back in that’s ideally where we’d like to go with this,” said Darling. “Theoretically we could do this with other genes that might upregulate very quickly or are innately expressed.”

A “SMARTer” Way to Isolate Mesenchymal Stem Cells


The Singapore-MIT Alliance for Research and Technology or SMART employs a team of engineers and life scientists to design technologies that address problems in science and medicine. In particular, a SMART team has devised a new technique to identify mesenchymal stem cells from bone marrow cells on the basis of cell size, cell stiffness, and the deformation of the nucleus.

Mesenchymal stem cells (MSCs) constitute less than one percent of the total cells in bone marrow. Therefore, isolating these cells from the morass of cells that are in the bone marrow is somewhat of a challenge. Most of the procedures for isolating MSCs from bone marrow utilize cell surface proteins found on the surfaces of MSCs, but there are no few cell surface proteins that are unique to only MSCs. Therefore, such isolation procedures tend to be tedious and not completely efficient. Because MSCs can differentiate into cells that produce bone, cartilage, fat, or muscle, they have proven invaluable for tissue repair therapies.

This new study by the SMART team has identified three physical characteristics of MSCs that can distinguish them from other immature cells found in the bone marrow. These physical characteristics should help them invent devices that could rapidly isolate MSCs, and facilitate the isolation of sufficient numbers of stem cells to treat patients.

Presently there are no sure-fire ways to quickly and efficiently separate MSCs from bone marrow cells that have already begun to differentiate into other cell types, but share the same molecules on the cell surface. This caveat may explain why experimental results vary among labs, and why stem-cell treatments now in clinical trials are not as effective as they could be, said Krystyn Van Vliet, an MIT associate professor of materials science and engineering and biological engineering and a senior author of the paper, that appeared in the Proceedings of the National Academy of Sciences.

“Some of the cells that you’re putting in and calling stem cells are producing a beneficial therapeutic outcome, but many of the cells that you’re putting in are not,” Van Vliet said. “Our approach provides a way to purify or highly enrich for the stem cells in that population. You can now find the needles in the haystack and use them for human therapy.”

In bone marrow, MSCs exist alongside other immature cells, such as osteogenic cells, which have already begun the developmental path toward becoming cartilage- or bone-producing cells. Currently, researchers try to isolate MSCs based on protein markers found on the cell surfaces, but these markers are not specific to MSCs. Therefore isolation techniques that rely on cell surface proteins can also co-isolate other types of immature cells that are more differentiated.

“Conventional cell-surface markers are frequently used to isolate different types of stem cells from the human bone marrow, but they lack sufficient ‘resolution’ to distinguish between subpopulations of mesenchymal stromal cells with distinct functions,” Lee said.

The researchers set out to find biophysical markers for multipotency (the ability to differentiate into several different cell types). They hypothesized that cell size might be a factor, since fetal bone marrow stem cells, which tend to have a higher percentage of MSCs, are usually small in diameter.

Jongyoon Han, an MIT professor of electrical engineering and biological engineering, had previously invented a device that captures circulating tumor cells based on their size. The SMART team used Han’s machine to isolate bone marrow cells based on size and discovered that none of the larger cells were multipotent, but not all of the smaller cells were multipotent. Therefore, size alone in insufficient to distinguish MSCs.

After measuring several other physical traits, the SMART team observed that two other physical characteristics could be combined with cell size to completely distinguish MSCs from other stem cells: stiffness of the cell, and the degree of fluctuation in the cell’s nuclear membrane.

“You don’t need more than these three, but you also can’t use fewer than these three,” Van Vliet said. “We now have a triplet of characteristics that identifies populations of cells that are going to be multipotent versus populations of cells that are only going to be able to become bone or cartilage cells.”

These features seem to correspond to what is already known about stem cells, Van Vliet said. In contrast to cells that have already committed to their final fate, immature cells have genetic material that moves around inside the nucleus, producing more fluctuations of the nuclear cell membrane. Stem cells also have a less rigid internal cytoskeletal structure than those of highly differentiated cells, which makes them seem less stiff.

The researchers then tested the regenerative abilities of MSCs isolated on the basis of these three characteristics in mice. They found that immature MSCs could help repair both muscle and bone injuries, but cells identified as osteogenic stromal cells were able to repair bone but not muscle.

“We have provided the first demonstration that subpopulations of mesenchymal stromal cells can be identified and highly enriched for bone growth and muscle repair,” Lee said. “We envision that this approach would also be important in the selection and purification of bone marrow-derived stem cells for tissue repair in human patients suffering from a range of tissue-degenerative diseases.”

“This is potentially a big step forward in establishing a marker-free way of identifying mesenchymal stem cells with maximum differentiation capacity,” said Jochen Guck, a professor of cellular machines at the Dresden University of Technology. “Biophysical markers have long been discussed and sought as an alternative to antibody labeling. What sets this work apart from others is that it clearly said that no single marker (at least of those tried) alone is predictive enough, but that a combination of them is required.”

The SMART team is now working on high-speed methods for separating MSCs. Creating more pure populations of such cells should lead to more effective stem-cell treatments for tissue injuries, Van Vliet said.

“Instead of putting in 30 percent of the cells that you want, and 70 percent filler, you’re putting in 100 percent of the cells that you want,” she explains. “That should lead to more reliable patient outcomes, because you’re not going to have this variability from batch to batch, or patient to patient, in how many of each cell population are present.”

Van Vliet and Poon also hope to initiate a clinical trial that utilizes the osteogenic cells isolated in this study, which could potentially prove useful for treating bone injuries.

Bone Marrow Stem Cells and Tissue Engineering Give a Woman a New Smile


Massive injuries to the face can cause bone loss and “tooth avulsion.” Medically speaking, avulsion simply refers to the detachment of a body structure from its normal location by means of surgery or trauma. Dental implants and help with lost teeth, but if the facial bone has suffered so much loss that you cannot place implants in them, then you are out of luck. Dental prostheses can help, but these do not always fit very well.

Darnell Kaigler and his group at the University of Michigan Center for Oral Health wanted to help a 45-year-old woman who had lost seven teeth and a good portion of her upper jaw bone (maxilla) as a result of massive trauma to the face. This poor lady had some dentures that did not fit well and a mouth that did not work well.

Bone can be grown from stem cells, but getting those stem cells to survive and do what you want them to do is the challenge of regenerative medicine. Therefore, Dr. Kraigler and his group used a new technique to help this young lady, and their results are reported in the December 2014 issue of the journal Stem Cells Translational Medicine.

First, Kraigler and his co-workers extracted bone marrow stem cells from a bone marrow aspiration that was taken from the upper part of the hip bone (the posterior crest of the ilium for those who are interested).  They used a product called ixmyelocel-T from Aastrom Biosciences in Ann Arbor , MI. This product is a patient-specific, expanded multicellular therapy, cell-processing system that selectively expands mesenchymal cells, monocytes and alternatively activated macrophages, up to several hundred times more than the number found in the patient’s bone marrow, while retaining many of the hematopoietic cells collected from only a small sample (50ml) of the patient’s bone marrow. Thus, the healing cells from the bone marrow are grown and made healthy, after which the cells were bagged and frozen for later use.

ixmyelocel-T

Then the patient was readied for the procedure by having the gum tissue cut and lifted as a flap of tissue (under anesthesia, or course). Then four holes were drilled into the bone and setting screws were inserted. This is an important procedure, because implanted stem cells will not survive unless they have blood vessels that can bring them oxygen and nutrients. By drilling these holes, the tissue responds by making new blood vessels. To this exposed surface, the bone marrow-derived stem cells were applied with a tricalcium phosphate (TCP). TCP is a salt that will induce mesenchymal stem cells to form bone. Once the TCP + stem cell mixture was applied to the gum, a collagen membrane was placed over it, and the gum was then sewn shut with sutures.

Cell transplantation procedure. Front view (A) and top view (B) of the initial clinical presentation showing severe hard and soft tissue alveolar ridge defects of the upper jaw. Following elevation of a full-thickness gingival flap, the images show front view (C) and top view (D) of the severely deficient alveolar ridge, clinically measuring a width of only 2–4 mm. Front view (E) and top view (F) of the placement of “tenting” screws in preparation of the bony site to receive the graft. Placement of the β-tricalcium phosphate (seeded with the cells 30 minutes prior to placement at room temperature) into the defect (G), with additional application of the cell suspension following placement of the graft in the recipient site (H). Placement of a resorbable barrier membrane (I) to stabilize and contain the graft within the recipient site, and top view (J) of primary closure of the flap.
Cell transplantation procedure. Front view (A) and top view (B) of the initial clinical presentation showing severe hard and soft tissue alveolar ridge defects of the upper jaw. Following elevation of a full-thickness gingival flap, the images show front view (C) and top view (D) of the severely deficient alveolar ridge, clinically measuring a width of only 2–4 mm. Front view (E) and top view (F) of the placement of “tenting” screws in preparation of the bony site to receive the graft. Placement of the β-tricalcium phosphate (seeded with the cells 30 minutes prior to placement at room temperature) into the defect (G), with additional application of the cell suspension following placement of the graft in the recipient site (H). Placement of a resorbable barrier membrane (I) to stabilize and contain the graft within the recipient site, and top view (J) of primary closure of the flap.

Four months later, the patient underwent a cone-beam computed tomography (CBCT) scan. The bone regrowth can be seen in the figure below.

Cone-beam computed tomography (CBCT) scans. CBCT scans were used to render three-dimensional reconstructions of the anterior segment of the upper jaw and cross-sectional (top view) radiographic images to show volumetric changes of the upper jaw at three time points. (A, B): The initial clinical presentation shows 75% jawbone width deficiency. (C, D): Immediately following cell therapy grafting, there is full restoration of jawbone width. (E, F): Images show 25% resorption of graft at 4 months and overall net 80% regeneration of the original ridge-width deficiency.
Cone-beam computed tomography (CBCT) scans. CBCT scans were used to render three-dimensional reconstructions of the anterior segment of the upper jaw and cross-sectional (top view) radiographic images to show volumetric changes of the upper jaw at three time points. (A, B): The initial clinical presentation shows 75% jawbone width deficiency. (C, D): Immediately following cell therapy grafting, there is full restoration of jawbone width. (E, F): Images show 25% resorption of graft at 4 months and overall net 80% regeneration of the original ridge-width deficiency.

According to the paper, there was an “80% regeneration of the original jawbone.”

Into this newly regenerated bone, permanent dental implants were placed. The results are shown below.

Complete oral rehabilitation. Clinical presentation of the patient prior to initiation of treatment (A) and following completed oral reconstruction (B). (C): Periapical radiographs of oral implants showing osseointegration of implants and stable bone levels at the time of placement, 6 months following placement, and 6 months following functional restoration and biomechanical loading of implants with a dental prosthesis.
Complete oral rehabilitation. Clinical presentation of the patient prior to initiation of treatment (A) and following completed oral reconstruction (B). (C): Periapical radiographs of oral implants showing osseointegration of implants and stable bone levels at the time of placement, 6 months following placement, and 6 months following functional restoration and biomechanical loading of implants with a dental prosthesis.

Pardon me, but permit me an unprofessional moment when I say that this is really cool.  Of course, this patient will need to be observed over the next several years to determine the longevity of her bone regeneration, but the initial result is certainly something to be excited about.

Tricalcium phosphate or TCP has been used to induce the bone-making activities of mesenchymal stem cells.  It has also been used in several animal studies as a delivery vehicle for mesenchymal stem cells (for example, see Rai B, et al., Biomaterials 2010, 31:79607970; Krebsbach PH, et al., Transplantation 1997, 63:10591069; Zhou J, et al., Biomaterials 2010, 31:11711179).  TCP also seems to support stem cell proliferation, survival, and differentiation into bone.  Kresbach and others showed that TCP most consistently yielded bone formation when used as a delivery vehicle for mesenchymal stem cells compared to other biomaterials commonly used, such as gelatin sponges and demineralized bone matrix.  However, there are no studies that have ascertained how well stem cells attach to TCP, and this attachment is an important factor in determining how many stem cells reach the site of injury.  This study by Kaigler and his group (A. Rajan and others) showed that a 30-minute incubation of the cells with TCP gave sufficient attachment of the cells to the TCP for clinical use.  The efficiency of this incubation period was also not affected by the temperature.  

The other exciting features of this paper, is that most of the materials used in this study were commercially available.  The bone marrow stem cell isolation technique was pioneered by Dennis JE, and others in their 2007 article in the journal Stem Cells (25:25752582).  Effective commercialization of this technique has shown the efficacy of this procedure for clinical use.  This paper also shows the clinical feasibility of using TCP as a delivery vehicle for mesenchymal stem cell-based bone treatments.

In conclusion, I will quote the authors: “Cell survival and seeding efficiency in the context of tissue engineering and cell-therapy strategies are critical parameters for success that have not been rigorously examined in a clinical context. This study defined optimized conditions for these parameters using an autologous stem cell therapy to successfully treat a patient who had a debilitating craniofacial traumatic deficiency. To our knowledge, there have been no other clinical reports of cell therapy for the treatment of craniofacial trauma defects. This clinical report serves as solid foundation on which to develop more expanded studies using this approach for the treatment of larger numbers of patients with other debilitating conditions (e.g., congenital disorders) to further evaluate efficacy and feasibility.”

Telling a Good Batch of Mesenchymal Stem Cells from a Lousy One


If a clinician isolates mesenchymal stem cells from the fat, bone marrow, or muscles of a patient, how can they tell if these cells will be effective? Short answer – they can’t. How well the cells grow in culture and how they look is the best indicator to date, but these indicators can fool you.

Fortunately, the highly productive laboratory of Darwin Prockop from the Institute of Regenerative Medicine at Texas A & M has discovered that the expression of a gene called TSG-6 can act as an indicator for human bone marrow mesenchymal stem cell quality.

In this paper, Prockop and others examined mice that had suffered damage to the surfaces of their eyes (corneas). To mitigate the inflammation in the eye, Prockop and his colleagues applied bone marrow-derived mesenchymal stem cells, but it was clear that the stem cell batches varied remarkably in their ability to regulate inflammation.

Prockop and his group then examined the genes expressed in the different batches of bone marrow-derived mesenchymal stem cells (MSCs) in order to determine if there was a gene that was consistently expressed in the effective batches as opposed to the ineffective batches.

Fortunately, they hit pay dirt. Reverse Transcriptase-PCR assays of human MSCs for the TSG-6 gene accurately predicted their ability of a specific batch of cells to modulate inflammation during corneal injury, or damage to the body wall (sterile peritonitis), or drug-induced injury to the lung. Thus, if you want implanted MSCs to modulate inflammation, then you want cells that express TSG-6 at a high level.

However, if you want MSCs that make bone, then you do not want cells that express high levels of TSG-6 because the levels of TSG-6 mRNA were negatively correlated with their potential for osteogenic (bone cell) differentiation in culture.

Additionally, when donated MSCs from male and female donors were examined and compared, it was clear that MSCs from female donors more effectively suppressed sterile inflammation, expressed higher levels of TSG-6, and had slightly less osteogenic potential than those from male donors.

Thus, TSG-6 is a marker that can measure the ability of a batch of MSCs to suppress inflammation. It is unclear if this same gene marker equally applies to other types of MSCs, but that will hopefully become clear with further work. Also, markers that correlate with the ability of these cells to do other types of regenerative activities might also result from experiments like these.