Fat-derived cells Enhance the Bone-Forming Capacity of Hypertrophic Cartilage Matrices


Treating particular bone defects or injuries present a substantial challenges for clinicians. The method of choice usually involves the use of an “autologous” bone graft (“autologous” simply means that the graft comes from the patient’s own bone). However, autologous bone grafts have plenty of limitations. For example, if a patient has a large enough bone defect, there is no way the orthopedist and take bone from a donor site without causing a good deal of risk to the donor site. Even with small bone grafts, so-called “donor site morbidity” remains a risk. Having said that, plenty of patients have had autologous bone grafts that have worked well, but larger bone injuries or defects are not treatable with autologous bone grafts.

The answer: bone substitute materials. Bone substitute materials include tricalcium phosphate, hydroxyapatite, cement, ceramics, bioglass, hydrogels, polylactides, PMMA or poly(methy methacrylate) and other acrylates,, and a cadre of commercially available granules, blocks, pastes, cements, and membranes. Some of these materials are experimental, but others do work, even if do not work every time. The main problem with bone substitute materials is that, well, they are not bone, and, therefore lack the intrinsic ability to induce the growth of new bone (so-called osteoinductive potential) and their ability to integrate into new bone is also a problem at times.

We must admit that a good deal of progress has been made in this area and it’s a good thing too. Many of our fabulous men and women-at-arms have returned home with severe injuries from explosives and wounds from large-caliber weapons that have shattered their bones. These courageous men and women have been the recipient of these technologies. However, the clinician is sometimes left asking herself, “can we do better?”

A new paper from the laboratories of Ivan Martin and Claude Jaquiery from the University Hospital of Basel suggests that we can. This paper appeared in Stem Cells Translational Medicine and describes the use of a hypertrophic cartilage matrix that was seeded with cells derived from the stromal vascular faction of fat to not only make bone in the laboratory, but to also heal skull defects in laboratory animals. While this work benefitted laboratory animals, it was performed with human cells and materials, which suggests that this technique, if it can be efficiently and cheaply scaled up, might be usable in human patients.
The two lead authors of this paper, Atanas Todorov and Matthias Kreutz and their colleagues made hypertrophic cartilage matrices from human bone marrow mesenchymal stem cells (from human donors) that were induced to make cartilage. Fortunately, protocols have been very well worked out and making cartilage plugs with chondrocytes that are enlarged (hypertrophic) is something that has been successfully done in many laboratories. After growing the mesenchymal stem cells in culture, half a million cells were induced to form cartilage with dexamethasone, ascorbic acid 2-phosphate, and the growth factor TGF-beta1. After three weeks, the cartilage plugs were subjected to hypertrophic medium, which causes the cartilage cells to enlarge.

Chondrocyte enlargement is a prolegomena to the formation of bone and during development, many of our long bones (femur, humerus, fibula, radius, etc.), initially form as cartilage exemplars that are replaced by bone as the chondrocytes enlarge. Ossification begins when a hollow cylinder forms in the center of the bone (known as the periosteal collar). The underlying chondrocytes degenerate and die, thus releasing the matrix upon which calcium phosphate crystals accrete. The primary ossification center commences with the calcification of the central shaft of the bone and erosion of the matrix by the invasion of a blood vessel. The blood vessels bring osteoprogenitor cells that differentiate into osteoblasts and begin to deposit the bone matrix.

Next, Todorov and his crew isolated the stromal vascular fraction from fat that was donated by 12 volunteers who had fat taken from them by means of liposuction. The fat is then minced, digested with enzymes, centrifuged, filtered and then counted. This remaining fraction is called the stromal vascular fraction or SVF, and it consists of a pastiche of blood vessel-forming cells, mesenchymal stem cells, and bone-forming cells (and probably a few other cells types too). These SVF cells were seeded onto the hypertrophic cartilage plugs and used for the experiments in this paper.

First, the SVF-seeded plugs were used to grow bone in laboratory rodents. The cartilage plugs were implanted into the backs for nude mice. Different cartilage plugs were used that had been seeded with gradually increasing number of SVF cells. The implanted plugs definitely made ectopic bone, but the amount of bone they made was directly proportional to the number of SVF cells with which they had been seeded. Staining experimental also showed that some of the newly-grown bone came from the implanted SVF cells.

Ectopic bone formation. Grafts based on devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction cells from adipose tissue and implanted subcutaneously in nude mice. (A): Representative hematoxylin and eosin, Masson-Tri-Chrome, and Safranin-O (Saf-O) staining and in situ hybridization for human ALU sequences (dark blue = positive) after 12 weeks in vivo. Saf-O stainings are blue-green because of lack of glycosaminoglycans and counterstaining with fast green. Osteoid matrix and bone marrow are visible. Scale bars = 200 µm. (B): Stainings for metalloproteinase (MMP)13 and MMP9, as well as for the N-terminal neoepitope at the major MMP cleavage site (DIPEN) after 12 weeks in vivo (red/pink = positive). Scale bars = 50 µm. +, osteoid matrix; ⋆, bone marrow. Abbreviations: ALU, Arthrobacter luteus; H&E, hematoxylin and eosin; Masson, Masson’s trichrome; MMP, metalloproteinase; Saf-O, Safranin-O; SVF, stromal vascular fraction.
Ectopic bone formation. Grafts based on devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction cells from adipose tissue and implanted subcutaneously in nude mice. (A): Representative hematoxylin and eosin, Masson-Tri-Chrome, and Safranin-O (Saf-O) staining and in situ hybridization for human ALU sequences (dark blue = positive) after 12 weeks in vivo. Saf-O stainings are blue-green because of lack of glycosaminoglycans and counterstaining with fast green. Osteoid matrix and bone marrow are visible. Scale bars = 200 µm. (B): Stainings for metalloproteinase (MMP)13 and MMP9, as well as for the N-terminal neoepitope at the major MMP cleavage site (DIPEN) after 12 weeks in vivo (red/pink = positive). Scale bars = 50 µm. +, osteoid matrix; ⋆, bone marrow. Abbreviations: ALU, Arthrobacter luteus; H&E, hematoxylin and eosin; Masson, Masson’s trichrome; MMP, metalloproteinase; Saf-O, Safranin-O; SVF, stromal vascular fraction.

In the second experiment, Todorov and Kreutz used these SVF-seeded cartilage plugs to repair skull lesions in rats. Once again, the quantity of bone produced was directly proportional to the number of SVFs seeded into the cartilage matrices prior to implantation. In both experiments, the density of SVF cells positively correlates with the bone-forming cells in the grafts and the percentage of SVF-derived blood vessel-forming cells correlates with the amount of deposited mineralized matrix.

Bone repair capacity. Devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction (SVF) cells from adipose tissue and implanted in rat calvarial defects. (A): Mineralized volume quantified by microtomography (n = 9 grafts assessed per group). (B): Bone area assessed in histological sections, expressed as percentage of total defect area (n = at least 24 sections assessed per group). ∗∗∗∗, p < .0001. (C, D): Representative three-dimensional microtomography reconstructions (left) and hematoxylin/eosin (H&E) staining (right) of the calvarial defects filled with devitalized grafts, implanted without (C) or with (D) activation by SVF cells after 4 weeks. Dotted circles indicate the defect borders (4 mm diameter). Scale bars = 500 µm. (E): Microtomography (left) and H&E staining (middle and right) displaying the bridging between hypertrophic matrix and bone of the calvarium, or the fusion of single pellets (right) in activated grafts. White bar = 850 µm; black bars = 500 µm. Dotted lines indicate the edge of the calvarium. (F): In situ hybridization for Arthrobacter luteus sequences showing the presence of human cells (dark blue, positive) in the explants. Scale bar = 200 µm. Abbreviations: C, calvarium; dev, fibrin gel without stromal vascular fraction; dev + SVF, fibrin gel with stromal vascular fraction; P, hypertrophic matrix; SVF, stromal vascular fraction.
Bone repair capacity. Devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction (SVF) cells from adipose tissue and implanted in rat calvarial defects. (A): Mineralized volume quantified by microtomography (n = 9 grafts assessed per group). (B): Bone area assessed in histological sections, expressed as percentage of total defect area (n = at least 24 sections assessed per group). ∗∗∗∗, p < .0001. (C, D): Representative three-dimensional microtomography reconstructions (left) and hematoxylin/eosin (H&E) staining (right) of the calvarial defects filled with devitalized grafts, implanted without (C) or with (D) activation by SVF cells after 4 weeks. Dotted circles indicate the defect borders (4 mm diameter). Scale bars = 500 µm. (E): Microtomography (left) and H&E staining (middle and right) displaying the bridging between hypertrophic matrix and bone of the calvarium, or the fusion of single pellets (right) in activated grafts. White bar = 850 µm; black bars = 500 µm. Dotted lines indicate the edge of the calvarium. (F): In situ hybridization for Arthrobacter luteus sequences showing the presence of human cells (dark blue, positive) in the explants. Scale bar = 200 µm. Abbreviations: C, calvarium; dev, fibrin gel without stromal vascular fraction; dev + SVF, fibrin gel with stromal vascular fraction; P, hypertrophic matrix; SVF, stromal vascular fraction.

This is not the first time scientists have proposed the use of cartilage plugs to induce the formation of new bone. Van der Stok and others and Bahney and colleagues were able to repair segmental bone defects in laboratory rodents. Is this technique transferable to human patients? Possibly. Hypertrophic cartilage is relatively easy to make and it is completely conceivable that hypertrophic cartilage wedges can be sold as “off-the-shelf” products for bone treatments. SVF can also be derived from the patient or can be derived from donors.

Furthermore, the protocols in this paper all used human cells and grew the products in immunodeficient rats and mice. Therefore, in addition to scaling this process up, we have a potentially useful protocol that might very well be adaptable to the clinic.

The efficacy of this technique must be confirmed in larger animal model system before human trials can be considered. Hopefully human trials are in the future for this fascinating technique.

New Study Validates Cellular Bone Allograft Technology


In a study in laboratory rats that had defects in their femurs (upper leg bone) showed new bone formation after they were treated with adipose-derived mesenchymal stromal cells that had been seeded on demineralized bone matrix and implanted.  Implanted stem cells were detected for up to 84 days in areas of new formation and had differentiated within the bony repair tissue.

According to AlloSource, the procedures used similar technology to the company’s AlloStem Cellular Bone Allograft process.

Surgical oncologist Nicole Ehrhart of Colorado State University presented these data at the State of Spine Surgery annual symposium and at the Korean American Spine Society meeting.

AlloStem is partially demineralized allograft bone combined with adipose-derived mesenchymal stem cells.  AlloStem is suitable for general bone grafting applications, and is similar to autograft bone because it provides the three key properties necessary for bone formation: osteoconduction, osteoinduction and osteogenesis.

Ehrhart’s study has been accepted for publication in Journal of Biomaterials and Tissue Engineering.

AlloSource provides 200 types of precise cartilage, cellular, bone, skin and soft-tissue allografts.

Key Molecules Tha Control Stem Cell Fate Identified


Adult stem cells, such as mesenchymal stem cells and blood-vessel-associated pericytes represent patient-specific stem cells that are excellent candidates for regenerative medicine. To that end being able to control the differentiation of these stem cells with drugs or small molecules is extremely desirable for eliciting targeted tissue and organ regeneration.

However, identifying these stem-cell-inducing molecules is time-consuming, expensive, and fraught with dead ends. Is there an easier way to control the behavior of stem cells in culture or in your own body?

Research from the City University of New York (CUNY) suggests that the answer to this question might be “yes.” According to Rein Ulijn from CUNY, “Simple small metabolites present in the body already can dictate cell behavior.”

In collaboration with Matthew Dalby from the University of Glasgow, Ulijn and his colleagues discovered that when they grew stem cells on a gel-like medium, the stiffness of which could be easily adjusted, they found molecules that could direct the differentiation of cultured stem cells. As an added bonus, they could direct the differentiation of cultured stem cells much more cheaply.

Ulijn and Dalby began their collaboration in 2011 after other laboratories had demonstrated that the stiffness of the medium could affect the differentiation of stem cells. “On a stiff gel you might get bone-like differentiation,” Ulijn explained. “On a softer gel differentiation into neurons is more likely.” They wanted to use such a system to identify small molecules that can control stem cell differentiation in culture. Such a finding could also “aid the discovery of natural metabolite-based drugs,” added Ulijn added. Such natural-based drugs could be used to, for example, reinforce bones in osteoporosis.

Dalby was interested in the role metabolites played in this stem cell differentiation. Unfortunately, these metabolites are present in fleetingly low concentrations. To complicate the picture, the different formulations of stiffer and floppier materials can mask subtle changes in metabolite concentration. Ulijn found a way around this problem by turning to the two-component peptide gels made by Biogelx (full disclosure: Ulijn serves as the chief scientific officer for Biogelx). Fine-tuning the concentration of the two different gel components changes the rigidity of the gel without changing any other components of the gel that might mask metabolite variation.

The researchers therefore studied concentration changes of hundreds of metabolites during stiffness-controlled stem cell differentiation of stem cells into bone or cartilage. Several metabolites that seemed to make a significant difference for stem cell differentiation were lysophosphatidic acid, which drove stem cells to form cartilage and cholesterol sulfate, which helped stem cells form bone. When Ulijn and his coworkers fed these metabolites to standard stem cell cultures, they differentiated into the desired cell type.

Helena Azevedo of Queen Mary University of London, said, “We will see, for sure, studies exploiting these metabolites for inducing controlled differentiation of stem cells.” She went on to called this study “highly innovative” and said that it might directly influence future stem cell differentiation experiments; particularly those that involve the formation of cartilage or bone.

Making Cartilage to Heal Broken Bones


Gage Crump and his colleagues at the University of Southern California have used the regeneration of zebrafish jawbone to demonstrate that the regeneration of damaged bones does not necessarily require a recapitulation of the same processes that occur during embryonic development. Even though this work used zebrafish as a model system, it may provide some of the underlying principles for treating difficult fractures.

Cartilage production is critical for healing full-thickness bone injuries. In order to understand how this bone-producing cartilage is generated, Crump and his coworkers turned to the genetically malleable and relatively more simple zebrafish system. Zebrafish are vertebrates, like humans, but these animals retain a remarkable capacity to regenerate many of their organs.

When human bones fracture, a small cartilage callus forms that is replaced by bone that bridges small, but not large, gaps in the bone.

In zebrafish, however, the cartilage callus continues to expand and fills even very large gaps in broken bones. This cartilage is replaced throughout the bone by bone. This allows zebrafish to heal even very large fractures.

These days, patients with severe bone fractures may have a surgeon insert metal pins and even plates to help set bone. In more severe cases, bone grafts are used to span gaps, and stem cell-based treatments have been tested in a few clinical trials as well.

About six million people in the U.S. suffer bone breaks each year, and even though most of these patients recover fully, about 300,000 are slow to heal and some may not heal at all. Complications include post-traumatic arthritis, growth abnormalities, delayed union and misaligned union.

Hundreds of professional football players have invested in stem cell treatments to treat injuries, even though the evidence for the efficacy of such treatments is, sometimes, sparse. One report even tells of an NFL linebacker who paid $6,000 for a 1-milliliter vial of donated placenta tissue containing stem cells to be injected into his injured knee.

The bone surface contains thin lining called the “periosteum” that contains a stem cell population that helps maintain bone mass throughout one’s life. In Gage’s laboratory, his team identified a gene called Indian Hedgehog a (IHHa), which is responsible for inducing these periosteal stem cells to switch from bone production to cartilage production. Mutant zebrafish strains that lack the IHHa gene are unable to make cartilage in response to bone injury and heal poorly from bone fractures.

Periosteum

Crump said that an “exciting finding from our work is that, somewhat counterintuitively, cartilage is critical for healing full thickness bone injuries. By understanding how this bone-producing cartilage is generated in the simpler zebrafish model, we hope to find ways to create more of this unique cartilage tissue in patients to better heal their bones.”

According to this paper, which was published in the journal Development, 2016; dev.131292 DOI: 10.1242/dev.121292; instead of the more traditional approach of using bone cells or bone-like materials to heal broken bones, stimulating endogenous bone-based stem cells that make this special kind of fracture-healing cartilage might be a more effective strategy.

A Common Osteoporosis Drug Protects Bone Marrow Stem Cells from DNA Damage


A commonly used treatment for osteoporosis can protect stem cells in bone from the ravages of aging, according to a new study from the University of Sheffield.

Ilaria Bellantuono and her colleagues have discovered that zoledronate can extend the lifespan of bone marrow mesenchymal stem cells by reducing the degree of DNA damage experienced by these stem cells.

As stem cells age, they accumulate DNA damage, and this seems to be one of the most important mechanisms of aging. DNA damage can cause stem cells to lose their capacity to maintain tissues and repair them when those tissues are damaged. This new research from Bellantuono’s laboratory shows that zoledronate can protect mesenchymal stem cells from DNA damage, which enhances their survival and maintains their function.

According the Professor Bellantuono, “The drug enhances the repair of the damage in DNA occurring with age in stem cells in the bone. It is also likely to work in other stem cells too.”

She continued: “This drug has been shown to delay mortality in patients affected by osteoporosis but until now we didn’t know why. These findings provide an explanation as to why it may help people to live longer.

“Now we want to understand whether the drug can be used to delay or revert the aging in stem cells in older people and improve the maintenance of tissues such as the heart, the muscle and immune cells, keeping them healthier for longer.

“We want to understand whether it improves the ability of stem cells to repair those tissues after injury, such as when older patients with cancer undergo radiotherapy.”

Almost half of elderly patients over 75 years of age have three or more diseases at the same time, such as osteoporosis, diabetes, cardiovascular disease, infections, and muscle weakness. However, work like this suggests that drugs like zoledronate could be used to treat, prevent or perhaps even delay the onset of such diseases.

Dr Bellantuono added: “We are hopeful that this research will pave the way for a better cure for cancer patients and keeping older people healthier for longer by reducing the risk of developing multiple age-related diseases.”

How Stem Cell Therapy Protects Bone In Lupus


Systemic Lupus Erythematosis, otherwise known as lupus, is an autoimmune disease cause your own immune system attacking various cells and tissues in your body. Lupus patients can suffer from fatigue, joint pain and selling and show a marked increased risk or osteoporosis.

Clinical trials have established that infusions of mesenchymal stem cells (MSCs) can significantly improve the condition of lupus patients, but exactly why these cells help these patients is not completely clear. Certainly suppression of inflammation is probably part of the mechanism by which these cells help lupus patients, but how do these cells improve the bone health of lupus patients?

Songtao Shi and his team at the University of Pennsylvania have used an animal model of lupus to investigate this very question. In their hands, transplanted MSCs improve the function of bone marrow stem cells by providing a source of the FAS protein. FAS stimulates bone marrow stem cell function by means of a multi-step, epigenetic mechanism.

This work by Shi and his colleagues has implications for other cell-based treatment strategies for not only lupus, but other diseases as well.

“When we used transplanted stem cells for these diseases, we didn’t know exactly what they were doing, but saw that they were effective,” said Shi. “Now we’ve seen in a model of lupus that bone-forming mesenchymal stem cell function was rescued by a mechanism that was totally unexpected.”

In earlier work, Shi and his group showed that mesenchymal stem cell infusions can be used to treat various autoimmune diseases in particular animals models. While these were certainly highly desirable results, no one could fully understand why these cells worked as well as they did. Shi began to suspect that some sort of epigenetic mechanism was at work since the infused MSCs seemed to permanently recalibrate the gene expression patterns in cells.

In order to test this possibility, Shi and others found that lupus mice had a malfunctioning FAS protein that prevented their bone marrow MSCs from releasing pro-bone molecules that are integral for bone maintenance and deposition.

A deficiency for the FAS protein prevents bone marrow stem cells from releasing a microRNA called miR-29b.  The failure to release miR-29b causes its concentrations to increase inside the cells.  miR-29b can down-regulate an enzyme called DNA methyltransferase 1 (Dnmt1), and the buildup of miR-29b inhibits Dnmt1, which causes decreased methylation of the Notch1 promoter and activation of Notch signaling.  Methylation of the promoters of genes tends to shut down gene expression, and the lack of methylation of the Notch promoter increases Notch gene expression, activating Notch signaling.  Unfortunately, increased Notch signaling impaired the differentiation of bone marrow stem cells into bone-making cells.  Transplantation of MSCs brings FAS protein to the bone marrow stem cells by means of exosomes secreted by the MSCs.  The FAS protein in the MSC-provided exosomes reduce intracellular levels of miR-29b, which leads to higher levels of Dnmt1.  Dnmt1 methylates the Notch1 promoter, thus shutting down the expression of the Notch gene, and restoring bone-specific differentiation.

Shi and others are presently investigating if this FAS-dependent process is also at work in other autoimmune diseases.  If so, then stem cell treatments might convey similar bone-specific benefits.

Faster Bone Regeneration With a Little Wnt


Nick Evans and his colleagues at the University of Southampton, UK have discovered that transient stimulation of the Wnt signaling pathway in bone marrow stem cells expands them and enhances their bone-making ability. This finding has led to an intense search for drugs that can stimulate the Wnt pathway in order to stimulate bone formation in wounded patients.

The Wnt pathway is a highly conserved pathway found in sponges, starfish, sharks, and people. Wnt signaling controls pattern formation during development, and the growth of stem cells during healing.

When it comes to healing, bone fractures represent a sizeable societal problem, particularly among the aged. While most fractures heal on their own, approximately 10 percent of all fractures take over six months to heal or never heal at all. In the worse cases, fracture patients can require several surgeries or might need amputation in desperate cases.

According the Evans, he and his research group are screening a wide range of chemicals to determine if they stimulate Wnt signaling. If such chemicals prove safe to use in laboratory animals, then they might become clinical tools to help stimulate bone formation and healing in patients with recalcitrant fractures.

Research from Evans’ group has shown that transient stimulation of the Wnt signaling pathway in isolated bone marrow cells increases the number of bone-making progenitor cells. However, if the Wnt pathway is activated for too long a time period, this regenerative effect is lost or even reversed. Hence the need to develop treatments that deliver small molecules that stimulate Wnt signaling in bone marrow cells for a specified period of time and in a targeted fashion.

Evans and his group have used nanoparticles loaded with Wnt proteins to do exactly that. The feasibility of this technology and its effectiveness requires further work, but the promise is there and the idea is more than a little intriguing.