Cover Story

Promising cell therapy applications pose more questions than answers

With few — but encouraging — human clinical studies, researchers remain cautiously optimistic about the use of cell therapy in orthopedics.

“I foresee a bright future for the use of cell therapy in orthopedic applications. We are paving the way to ultimately develop systems in which cells can be used to induce and or accelerate tissue healing,” the director of The New England Musculoskeletal Institute and Chairman of the Department of Orthopaedic Surgery at the University of Connecticut, Augustus D. Mazzocca, MS, MD, told Orthopedics Today.

According to Mazzocca and George F. Muschler, MD, to use the term “stem cells” to define the cells that may be used in therapy is incorrect. The cells used in most therapies will not have the capacity to “self renew” or perpetuate themselves indefinitely, which is the defining feature of a true stem cell, Muschler, of the Lerner Research Institute’s Department of Biomechanical Engineering and orthopedic surgeon at Cleveland Clinic in Ohio, noted.

“The biological term, ‘stem cell,’ has fallen into a state of gross misuse in much of the recent literature,” Muschler said. “Virtually all of the cell populations that we currently target or transplant with our therapies should be called progenitor cells, not stem cells. The term stem cell is sexy, but it is just not accurate.”

George F. Muschler, MD, noted that several terms have been used to define purified cell populations that have been culture expanded in the laboratory, and the potential uses of these cell populations are only beginning to be tested in clinical trials.

George F. Muschler, MD, noted that several
terms have been used to define purified cell
populations that have been culture expanded
in the laboratory, and the potential uses of
these cell populations are only beginning to be
tested in clinical trials.

Image: The Cleveland Clinic

A progenitor cell is defined as a cell that can proliferate and generate progeny that may be able to differentiate into the desired tissue. The class of progenitors most important to orthopedic surgeons are those that are already present in native tissues, the connective tissue progenitors or CTPs. A CTP has the potential to be activated and then proliferate to give rise to progeny that can differentiate into one or more connective tissues such as bone, cartilage, muscle, fat or blood, according to Muschler. Endothelial progenitors, that proliferate to regenerate vascular endothelium during revascularization, are another key progenitor cell population that contribute to all tissue regeneration, including connective tissues.

Tissue CTPs are a heterogeneous class of cells that are found in multiple locations in vivo, he noted. CTP populations often demonstrate different properties, depending on the tissue source or site from which they are harvested. These sites include marrow, periosetum, endosteum and cartilage. They also include vascular pericytes found in fat, muscle and other tissues.

In contrast to the heterogeneous mix of cells in native tissues, several terms have been used to define purified cell populations that have been culture expanded in a laboratory. Mesenchymal stem cells (MSCs) are defined as a cell population that has been expanded in culture under conditions that create a population of cells that homogeneously express a defined set of cell surface markers and have the capacity to differentiate in culture into several connective tissue phenotypes. Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are another classes of culture expanded cells, which have more extensive capacity for proliferation and differentiation, according to Muschler. The potential uses of culture expanded cell populations is only beginning to be tested in clinical trials, he noted.

FDA regulations

Current FDA regulation stemming from The United States of America v. Regenerative Sciences LLC, Christopher J. Centeno, MD, John R. Schultz, MD, and Michelle R. Cheever prevent the removal, manipulation expansion and reimplantation of human cells without an FDA-approved clinical trial, according to Lawrence V. Gulotta, MD, of Hospital for Special Surgery in New York City and C. Thomas Vangsness Jr., MD, of the University of Southern California in Los Angeles. The FDA pursued these rules as part of their charge to ensure the safety and efficacy of new medical products, according to Muschler.

“Testing in clinical trials is limited by the high cost of executing clinical trials. Development and testing of cell therapy products is also limited by the lack of clearly defined consensus around the standards and quantitative metrics that should be used to define the nature of cells being used, and the methods and standards that should be used to screen cells for biological hazards, such as infection or mutations. This creates great uncertainty regarding the regulatory pathway that will be required now and in the future. Development is also challenged by uncertainty regarding the environment around reimbursement for these new therapies,” Muschler told Orthopedics Today.

Lawrence V. Gulotta

Lawrence
V. Gulotta

Vangsness cited work by Paul Lu, PhD, and colleagues in which they discovered that neural stem cells allowed paralyzed rats to walk again. However, Vangsness noted that Neuralstem (Rockvalle, Md.), a company developing neural stem cell treatment, has yet to get FDA approval.

The high cost of conducting the clinical trials needed to prove the safety and efficacy of stem cell products has also deterred many companies from investing in these projects, according to Vangsness, who noted that some stem cell companies have gone out of business trying to get their products to market.

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“The FDA wants good manufacturing practices with clinical trials showing statistical improvements of any treatment regimen before releasing these treatments to the public,” Vangsness told Orthopedics Today.

Promising early research

Sources who spoke withOrthopedics Today highlighted the early results and potential applications of cell therapy in orthopedic procedures and discussed the controversies among scientists in the field of stem cell research.

Mazzocca cited work by Philippe Hernigou, MD, PhD, and colleagues showing that concentrated marrow aspirated from anterior iliac crests led to union in 53 of 60 noninfected, atrophic tibia fractures.

According to Mazzocca, work that he and his colleagues published in Arthroscopy showed that aspirations of bone marrow taken from the proximal humerus and distal femur during surgery produced good concentrations of MSCs that could be used for soft tissue augmentation.

In addition, Vangsness and colleagues conducted an FDA-approved randomized controlled trial involving Chondrogen (Osiris Therapeutics Inc.; Columbia, Md.), which contains MSCs from human donors. The patients were randomized to receive an injection of hyaluronic acid, an injection of 50 million MSCs or an injection of 150 million MSCs into their knees 1 week after meniscectomy. The researchers could not prove meniscal regrowth in all patients, according to Vangsness. The study will be published within the coming months in the Journal of Bone & Joint Surgery, according to Vangsness.

“This is the first randomized clinical trial to be reported in the English literature with injections of cells into the human knee,” he said.

Restrictions by the FDA have contributed to the lack of clinical human studies involving culture expanded cells. To date, most feasibility studies have been done on animals, according to Vangsness and Gulotta. In her research on cell treatments in horses, Lisa A. Fortier, DVM, PhD,DACVS, of the College of Veterinary Medicine at Cornell University, and colleagues found haplotype or genetic code does determine whether the body accepts or rejects cells.

“We have a unique herd of horses here at Cornell [with which] we can do specific [genetic] matching and mismatching,” Fortier told Orthopedics Today.

The Cornell group is also studying whether bone marrow aspirate injected into the site of injury recruits stem cells to that area. Fortier and collaborators at The Rothman Institute in Philadelphia are also working on a clinical trial to examine if adipose-derived stem cells decrease inflammation and pain in humans with arthritis.

ES cells vs. MSCs

On the shoulders of such findings is the debate over whether ES cells or adult MSCs should be tapped for their healing potential, according to Mazzocca. ES cells offer more plasticity, meaning they can differentiate into any type of cell numbering in the millions as they continue to divide, compared to just thousands found from MSC sources, according to Vangsness.

MSCs are defined by the International Society of Cell Therapy as purified, homogeneous culture-expanded populations of cells that express the specific cell surface markers CD105, CD 90 and CD73, but not others such as CD34, CD14 or CD19, according to Muschler. MSCs can be derived from many adult human sources including aspirations of bone marrow extracted with a syringe, adipose tissue treated with the enzyme lipase to separate stem cells and peripheral blood spun down in a centrifuge, according to Mazzocca. MSCs can now be directed towards various lineages including bone, fat, cartilage, tendon, disc and muscle cells, he said.

“As yet, there is no convincing evidence that culture-expanded MSCs provide benefits beyond the use of native osteogenic connective tissue progenitors that can be harvested and processed to concentrate or enrich progenitor populations collected from native tissues in the operating room,” Muschler said.

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In blood, approximately one in 30,000 nucleated cells (the range is one in 10,000 to one in 100,000), Vangsness said. In adipose tissue, approximately one in 20,000 nucleated cells have an MSC, while embryonic stem cells multiply into a few trillion stem cells downstream, he said.

Muschler, pointed out, however, that there are few musculoskeletal settings requiring tissue regeneration where even large doses of culture expanded cells have outperformed modest processing of native CTP when compared head-to-head.

Induced pluripotent stem cells

Because of the potential of ES cells to divide into trillions more stem cells than MSCs, researchers had an interest in transforming adult tissue cells (non-stem cells) into ES cells in the lab, according to Vangsness. This concept was realized in 2006 when Shinya Yamanaka discovered mouse fibroblasts could be reprogrammed into immature cells or iPSCs, according to Gulotta. The National Institutes of Health (NIH) defines iPSCs as “adult cells that have been genetically reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells.” Scientists can inject viruses into MSCs to introduce genes that turn them into iPSCs.

C. Thomas Vangsness Jr.

C. Thomas
Vangsness Jr.

“Viruses are one way to introduce genes and there are concerns they could become cancerous, so researchers are searching for other alternatives,” Gulotta told Orthopedics Today.

According to the NIH, researchers are working on other methods besides viruses to deliver genes to adult stem cells to reprogram them into iPSCs.

Cell harvesting

MSCs can be found in many tissues of the body, but only certain areas are optimal for finding MSCs such as adipose tissue, bone marrow and peripheral blood, according to Mazzocca.

“The most important aspect of a connective tissue progenitor harvesting technique is the decision of where to harvest cells from,” Muschler said. “It has become increasingly clear that different tissues and tissue sites contain connective tissue progenitor populations that have different concentrations, prevalence and biological potential.”

For example, the periosteum is the richest sources of osteogenic and chondrogenic progenitor cells, while cells adherent to the surface of trabecular bone provide the second richest source of these cells, according to Muschler.

Harvest technique is also important, and may dramatically change the concentration of CTPs collected. The most effective method to harvest bone marrow is to extract 2 mL from the iliac crest bone marrow using small volume aspirates, changing the site of aspiration between each aspirate. Using this method, each aspirate will provide an average 20 to 40 million nucleated cells and 1,000 to 2,000 CTPs per mL for transplantation, according to Muschler.

To obtain stem cells from adipose tissue, a ‘lipase-like’ step is added to break fat cells free. However, this method is banned by the FDA, according to Vangsness.

“The Food and Drug Administration says using this ‘lipase’ is more than minimal manipulation,” Vangsness said.

Cell processing

Bone marrow is most commonly spun down in a centrifuge to concentrate stem cells, according to Gulotta. For example, iliac crest bone marrow is spun down in a centrifuge to extract marrow-derived stromal cells that could be used to treat fractures.

“Exploring and refining options for rapid, safe, effective and inexpensive intraoperative cell processing is one of our next great opportunities,” Muschler said. “In addition to processing using a centrifuge, CTPs can be concentrated and also enriched with respect to other nucleated cells in marrow based on their intrinsic biological tendency to attach to certain surfaces or implantable materials, without directly manipulating or modifying the cells themselves. We need to define the standards by which we measure the changes in concentration and prevalence that occur following various processing methods, and then competitively assess the change in clinical outcome that can be achieved in order to justify these new therapy approaches.”

Processing using magnetic fields is another option. Muschler’s lab recently reported that magnetically labeling cells that have hyaluronan on their surface and concentrating those cells using a magnetic field can increase the concentration and prevalence of CTPs from bone marrow, and that transplantation of this enriched population improved the quality of bone formed in a canine femoral bone defect.

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Muschler said, “Increasingly, we are finding ways to effectively harvest CTPs and also separate them from the vastly more numerous non-progenitor cells. This allows more connective tissue progenitors to be transplanted, with fewer competing cells, and in theory, will provide much better engraftment and performance.”

Stem cell differentiation

Once any cell is implanted, Mazzocca and Muschler noted the cells may need a certain chemical and mechanical agent to induce the differentiation that is desired. This may include the local or systemic delivery of drugs or growth factors. Gulotta and colleagues added helix-loop-helix transcription factor scleraxis to bone-marrow derived MSCs and found improved rotator cuff tendon healing in rats.

Another way to induce differentiation is to coculture stem cells, according to Gulotta. For example, adding end-stage cells, such as chondrocytes, in a petri dish with growing stem cells allows differentiation factors and cytokines released by the chondrocytes to induce differentiation in the stem cells.

Some molecules, such as SDF-1 or MCP-3, can increase the cell homing into an area when they are secreted or delivered locally, according to Muschler.

Tethering of a bioactive molecule to the surface of a biomaterial is a new method to deliver and control the location and effect of an active agent. Epidermal growth factors (EGFs) or bone morphogenetic proteins (BMPs) are among the molecules being explored, according to Muschler.

“[Tethering] increases the local concentration, controls the distribution of the agent and prolongs the time when it can be active by reducing diffusion and degradation,” Muschler said.

Genes may be added to stem cells to differentiate them into tendon, bone or other tissues such as BMP-13 to differentiate stem cells into tenocytes, Gulotta said.

“At this time, there are more questions than answers in terms of how to harvest stem cells, what are the best types of cells, how do you get enough of them, what do you do to them to convince them to form whatever tissue you need and what do you do postoperatively or post-treatment,” Gulotta said. – by Renee Blisard Buddle

References:
Beitzel K. Arthroscopy. 2013;doi:10.1016/j.arthro.2012.08.021.
Caralla T. Tissue Eng Part A. 2013;doi:10.1089.ten.tea.2011.0622.
Gurdon E. J Embryol Exp Morphol. 1962;10:622-640.
Gulotta L. Am J Sports Med. 2011;doi:10.1177/0363546510395485.
Hernigou P. J Bone Joint Surg Am. 2005;87(7):1430-1437.
Lu P. Cell. 2012. doi:10.1016/j.cell.2012.08.020.
http://stemcells.nih.gov/info/basics/pages/basics10.aspx.
Schnabel L. Vet J. 2013;doi:10.1016/j.tvjl.2013.04.018.
Yamanaka S. Cell. 2006;126(64):663-676.
For more information:
Lisa A. Fortier, DVM, PhD, DACVS, can be reached at Vet Box 32, College of Veterinary Medicine Cornell University, Ithaca, NY 14853; email: laf4@cornell.edu.
Lawrence V. Gulotta, MD, can be reached at Hospital for Special Surgery, 535 East 70th St., New York, NY; email: hickenbottomt@hss.edu.
Augustus D. Mazzocca, MS, MD, can be reached at The New England Musculoskeletal Institute, UConn Health Center, 263 Farmington Ave., Farmington, CT 06030; email: mazzocca@uchc.edu.
George F. Muschler, MD, can be reached at the Lerner Research Institute, Cleveland Clinic, Cleveland Clinic Main Campus, Mail Code A41, 9500 Euclid Ave., Cleveland, OH 44195; email: ambrol@ccf.org.
C. Thomas Vangsness Jr, MD, can be reached at the University of Southern California, 1520 San Pablo St., Suite 2000, Los Angeles, CA 90033; email: alison.trinidad@usc.edu.
Disclosures: Gulotta is a consultant for Biomet Inc. Mazzocca receives research support and is a consultant for Arthrex. Muschler receives fees as a consultant for the FDA and Smith & Nephew and receives grant funding from Harvest Technologies and Medtronic. Fortier and Vangsness have no relevant financial disclosures.

POINTCOUNTER

What is the best method to obtain cells for cell therapy and why?

POINT

Adult-derived cells safer, more readily available

Despite the obvious potential of embryonic stem cells, I will take the position of favoring adult-derived cells. I base this opinion on considerations related to biologic potential, safety and availability. The current standard sources of mesenchymal stem cells for orthopedic applications include bone marrow and adipose tissue. The use of cell-sorting techniques in the future will allow isolation of the small number of true stem cells (estimated as 0.01% of bone marrow cells). These cells have the ability to differentiate into several mesenchymal tissues, including bone, cartilage, adipose, and dense fibrous connective tissue resembling tendon and ligament. It is becoming increasingly evident that numerous tissues in the adult contain a niche of cells that are multipotent. For example, such cells have been identified in tendon, ligaments, bone, muscle, cartilage and the meniscus. These cells are usually located in proximity to vessel walls. Our challenge is to identify how to stimulate these cells to participate in tissue repair and regeneration.

Scott A. Rodeo

Scott A. Rodeo

The importance of the cells’ local environment is also a critical factor. First, cells from the local environment appear to have superior repair capacity compared to distant cells. For example, stem cells derived from synovium likely have better a ability to differentiate into a chondrogenic phenotype than bone marrow-derived cells. Second, the biologic activity of transplanted cells is highly dependent upon the environment into which the cells are transplanted. The biological signals present in the local environment may be as important as the specific cell type used. Thus, even a pluripotent cell, such as an embryonic stem cell, will not realize its optimal potential in a nonsupportive environment, such as that seen in an elderly patient.

It should be noted that embryonic cells are pluripotent while adult-derived cells are multipotent, indicating greater plasticity for embryonic stem cells. However, such pluripotency also comes with a higher risk for tumorigenicity. There are also ethical issues related to the use of embryonic stem cells. Finally, another factor to be considered in favor of autologous (adult-derived) cells is the potential for an immune response against allogeneic cells, which has been demonstrated.

Scott A. Rodeo, MD, is an Orthopedics Today Editorial Board member and is co-chief of the Sports Medicine and Shoulder Service at Hospital for Special Surgery in New York City.
Disclosure: Rodeo receives research support from the American Orthopedic Society for Sports Medicine, is a consultant for Smith & Nephew and owns stock in Cayenne Medical.

COUNTER

Use of cell therapy is complex, and shared decision-making is necessary

We have entered an era of evidence-based medicine and shared decision-making. Physicians must thoroughly consider the efficacy of treatments, educate their patients and enable them to make appropriate decisions regarding care. While stem cell therapy offers great promise, most patients do not appreciate the limited evidence of efficacy in human trials or the time it will take to establish proven treatments.

Regis J. O’Keefe

Regis J. O’Keefe

Stem cell therapy has the potential to profoundly alter the impact of musculoskeletal disease. Preclinical animal studies show that stem cell treatments can delay or prevent the onset of degenerative diseases of the skeleton, joints and muscle tissues. Stem cells have been shown to stimulate tissue regeneration following injury or disease in animal models of cardiac, endocrine and neurological diseases as well. Development of these treatments is essential for our patients and to maintain orthopedics at the leading edge of discovery and innovative treatment.

Education and shared decision-making are particularly important as new therapies emerge. During the years, we have had some sobering missteps with the initiation of new therapies. The enormous promise of gene therapy has been tempered by the severe complications noted in some patients. We are re-examining the use of bone morphogenetic proteins in spine fusion, and our recent experience with metal-on-metal hip implants has been discouraging despite its initial promise.

Numerous Internet sites promise amazing outcomes and potential cures from stem cell therapy. The use of stem cells in clinical medicine is complex – and patients suffering from orthopedic disease are vulnerable. The work by Drs. Mazzocca, Muschler and others to determine the clinical efficacy of stem cell approaches are essential. Additional work must be done so that we can provide patients with solid information based on multiple evidence-based clinical studies. Until then, we need to inform patients that these therapies have promise, and keep abreast of this innovative therapy as it emerges.

Regis J. O’Keefe, MD, PhD, is chairperson of the Department of Orthopaedics at the University of Rochester Medical Center in Rochester, NY.
Disclosure: O’Keefe has no relevant financial disclosures.

With few — but encouraging — human clinical studies, researchers remain cautiously optimistic about the use of cell therapy in orthopedics.

“I foresee a bright future for the use of cell therapy in orthopedic applications. We are paving the way to ultimately develop systems in which cells can be used to induce and or accelerate tissue healing,” the director of The New England Musculoskeletal Institute and Chairman of the Department of Orthopaedic Surgery at the University of Connecticut, Augustus D. Mazzocca, MS, MD, told Orthopedics Today.

According to Mazzocca and George F. Muschler, MD, to use the term “stem cells” to define the cells that may be used in therapy is incorrect. The cells used in most therapies will not have the capacity to “self renew” or perpetuate themselves indefinitely, which is the defining feature of a true stem cell, Muschler, of the Lerner Research Institute’s Department of Biomechanical Engineering and orthopedic surgeon at Cleveland Clinic in Ohio, noted.

“The biological term, ‘stem cell,’ has fallen into a state of gross misuse in much of the recent literature,” Muschler said. “Virtually all of the cell populations that we currently target or transplant with our therapies should be called progenitor cells, not stem cells. The term stem cell is sexy, but it is just not accurate.”

George F. Muschler, MD, noted that several terms have been used to define purified cell populations that have been culture expanded in the laboratory, and the potential uses of these cell populations are only beginning to be tested in clinical trials.

George F. Muschler, MD, noted that several
terms have been used to define purified cell
populations that have been culture expanded
in the laboratory, and the potential uses of
these cell populations are only beginning to be
tested in clinical trials.

Image: The Cleveland Clinic

A progenitor cell is defined as a cell that can proliferate and generate progeny that may be able to differentiate into the desired tissue. The class of progenitors most important to orthopedic surgeons are those that are already present in native tissues, the connective tissue progenitors or CTPs. A CTP has the potential to be activated and then proliferate to give rise to progeny that can differentiate into one or more connective tissues such as bone, cartilage, muscle, fat or blood, according to Muschler. Endothelial progenitors, that proliferate to regenerate vascular endothelium during revascularization, are another key progenitor cell population that contribute to all tissue regeneration, including connective tissues.

Tissue CTPs are a heterogeneous class of cells that are found in multiple locations in vivo, he noted. CTP populations often demonstrate different properties, depending on the tissue source or site from which they are harvested. These sites include marrow, periosetum, endosteum and cartilage. They also include vascular pericytes found in fat, muscle and other tissues.

In contrast to the heterogeneous mix of cells in native tissues, several terms have been used to define purified cell populations that have been culture expanded in a laboratory. Mesenchymal stem cells (MSCs) are defined as a cell population that has been expanded in culture under conditions that create a population of cells that homogeneously express a defined set of cell surface markers and have the capacity to differentiate in culture into several connective tissue phenotypes. Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are another classes of culture expanded cells, which have more extensive capacity for proliferation and differentiation, according to Muschler. The potential uses of culture expanded cell populations is only beginning to be tested in clinical trials, he noted.

FDA regulations

Current FDA regulation stemming from The United States of America v. Regenerative Sciences LLC, Christopher J. Centeno, MD, John R. Schultz, MD, and Michelle R. Cheever prevent the removal, manipulation expansion and reimplantation of human cells without an FDA-approved clinical trial, according to Lawrence V. Gulotta, MD, of Hospital for Special Surgery in New York City and C. Thomas Vangsness Jr., MD, of the University of Southern California in Los Angeles. The FDA pursued these rules as part of their charge to ensure the safety and efficacy of new medical products, according to Muschler.

“Testing in clinical trials is limited by the high cost of executing clinical trials. Development and testing of cell therapy products is also limited by the lack of clearly defined consensus around the standards and quantitative metrics that should be used to define the nature of cells being used, and the methods and standards that should be used to screen cells for biological hazards, such as infection or mutations. This creates great uncertainty regarding the regulatory pathway that will be required now and in the future. Development is also challenged by uncertainty regarding the environment around reimbursement for these new therapies,” Muschler told Orthopedics Today.

Lawrence V. Gulotta

Lawrence
V. Gulotta

Vangsness cited work by Paul Lu, PhD, and colleagues in which they discovered that neural stem cells allowed paralyzed rats to walk again. However, Vangsness noted that Neuralstem (Rockvalle, Md.), a company developing neural stem cell treatment, has yet to get FDA approval.

The high cost of conducting the clinical trials needed to prove the safety and efficacy of stem cell products has also deterred many companies from investing in these projects, according to Vangsness, who noted that some stem cell companies have gone out of business trying to get their products to market.

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“The FDA wants good manufacturing practices with clinical trials showing statistical improvements of any treatment regimen before releasing these treatments to the public,” Vangsness told Orthopedics Today.

Promising early research

Sources who spoke withOrthopedics Today highlighted the early results and potential applications of cell therapy in orthopedic procedures and discussed the controversies among scientists in the field of stem cell research.

Mazzocca cited work by Philippe Hernigou, MD, PhD, and colleagues showing that concentrated marrow aspirated from anterior iliac crests led to union in 53 of 60 noninfected, atrophic tibia fractures.

According to Mazzocca, work that he and his colleagues published in Arthroscopy showed that aspirations of bone marrow taken from the proximal humerus and distal femur during surgery produced good concentrations of MSCs that could be used for soft tissue augmentation.

In addition, Vangsness and colleagues conducted an FDA-approved randomized controlled trial involving Chondrogen (Osiris Therapeutics Inc.; Columbia, Md.), which contains MSCs from human donors. The patients were randomized to receive an injection of hyaluronic acid, an injection of 50 million MSCs or an injection of 150 million MSCs into their knees 1 week after meniscectomy. The researchers could not prove meniscal regrowth in all patients, according to Vangsness. The study will be published within the coming months in the Journal of Bone & Joint Surgery, according to Vangsness.

“This is the first randomized clinical trial to be reported in the English literature with injections of cells into the human knee,” he said.

Restrictions by the FDA have contributed to the lack of clinical human studies involving culture expanded cells. To date, most feasibility studies have been done on animals, according to Vangsness and Gulotta. In her research on cell treatments in horses, Lisa A. Fortier, DVM, PhD,DACVS, of the College of Veterinary Medicine at Cornell University, and colleagues found haplotype or genetic code does determine whether the body accepts or rejects cells.

“We have a unique herd of horses here at Cornell [with which] we can do specific [genetic] matching and mismatching,” Fortier told Orthopedics Today.

The Cornell group is also studying whether bone marrow aspirate injected into the site of injury recruits stem cells to that area. Fortier and collaborators at The Rothman Institute in Philadelphia are also working on a clinical trial to examine if adipose-derived stem cells decrease inflammation and pain in humans with arthritis.

ES cells vs. MSCs

On the shoulders of such findings is the debate over whether ES cells or adult MSCs should be tapped for their healing potential, according to Mazzocca. ES cells offer more plasticity, meaning they can differentiate into any type of cell numbering in the millions as they continue to divide, compared to just thousands found from MSC sources, according to Vangsness.

MSCs are defined by the International Society of Cell Therapy as purified, homogeneous culture-expanded populations of cells that express the specific cell surface markers CD105, CD 90 and CD73, but not others such as CD34, CD14 or CD19, according to Muschler. MSCs can be derived from many adult human sources including aspirations of bone marrow extracted with a syringe, adipose tissue treated with the enzyme lipase to separate stem cells and peripheral blood spun down in a centrifuge, according to Mazzocca. MSCs can now be directed towards various lineages including bone, fat, cartilage, tendon, disc and muscle cells, he said.

“As yet, there is no convincing evidence that culture-expanded MSCs provide benefits beyond the use of native osteogenic connective tissue progenitors that can be harvested and processed to concentrate or enrich progenitor populations collected from native tissues in the operating room,” Muschler said.

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In blood, approximately one in 30,000 nucleated cells (the range is one in 10,000 to one in 100,000), Vangsness said. In adipose tissue, approximately one in 20,000 nucleated cells have an MSC, while embryonic stem cells multiply into a few trillion stem cells downstream, he said.

Muschler, pointed out, however, that there are few musculoskeletal settings requiring tissue regeneration where even large doses of culture expanded cells have outperformed modest processing of native CTP when compared head-to-head.

Induced pluripotent stem cells

Because of the potential of ES cells to divide into trillions more stem cells than MSCs, researchers had an interest in transforming adult tissue cells (non-stem cells) into ES cells in the lab, according to Vangsness. This concept was realized in 2006 when Shinya Yamanaka discovered mouse fibroblasts could be reprogrammed into immature cells or iPSCs, according to Gulotta. The National Institutes of Health (NIH) defines iPSCs as “adult cells that have been genetically reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells.” Scientists can inject viruses into MSCs to introduce genes that turn them into iPSCs.

C. Thomas Vangsness Jr.

C. Thomas
Vangsness Jr.

“Viruses are one way to introduce genes and there are concerns they could become cancerous, so researchers are searching for other alternatives,” Gulotta told Orthopedics Today.

According to the NIH, researchers are working on other methods besides viruses to deliver genes to adult stem cells to reprogram them into iPSCs.

Cell harvesting

MSCs can be found in many tissues of the body, but only certain areas are optimal for finding MSCs such as adipose tissue, bone marrow and peripheral blood, according to Mazzocca.

“The most important aspect of a connective tissue progenitor harvesting technique is the decision of where to harvest cells from,” Muschler said. “It has become increasingly clear that different tissues and tissue sites contain connective tissue progenitor populations that have different concentrations, prevalence and biological potential.”

For example, the periosteum is the richest sources of osteogenic and chondrogenic progenitor cells, while cells adherent to the surface of trabecular bone provide the second richest source of these cells, according to Muschler.

Harvest technique is also important, and may dramatically change the concentration of CTPs collected. The most effective method to harvest bone marrow is to extract 2 mL from the iliac crest bone marrow using small volume aspirates, changing the site of aspiration between each aspirate. Using this method, each aspirate will provide an average 20 to 40 million nucleated cells and 1,000 to 2,000 CTPs per mL for transplantation, according to Muschler.

To obtain stem cells from adipose tissue, a ‘lipase-like’ step is added to break fat cells free. However, this method is banned by the FDA, according to Vangsness.

“The Food and Drug Administration says using this ‘lipase’ is more than minimal manipulation,” Vangsness said.

Cell processing

Bone marrow is most commonly spun down in a centrifuge to concentrate stem cells, according to Gulotta. For example, iliac crest bone marrow is spun down in a centrifuge to extract marrow-derived stromal cells that could be used to treat fractures.

“Exploring and refining options for rapid, safe, effective and inexpensive intraoperative cell processing is one of our next great opportunities,” Muschler said. “In addition to processing using a centrifuge, CTPs can be concentrated and also enriched with respect to other nucleated cells in marrow based on their intrinsic biological tendency to attach to certain surfaces or implantable materials, without directly manipulating or modifying the cells themselves. We need to define the standards by which we measure the changes in concentration and prevalence that occur following various processing methods, and then competitively assess the change in clinical outcome that can be achieved in order to justify these new therapy approaches.”

Processing using magnetic fields is another option. Muschler’s lab recently reported that magnetically labeling cells that have hyaluronan on their surface and concentrating those cells using a magnetic field can increase the concentration and prevalence of CTPs from bone marrow, and that transplantation of this enriched population improved the quality of bone formed in a canine femoral bone defect.

PAGE BREAK

Muschler said, “Increasingly, we are finding ways to effectively harvest CTPs and also separate them from the vastly more numerous non-progenitor cells. This allows more connective tissue progenitors to be transplanted, with fewer competing cells, and in theory, will provide much better engraftment and performance.”

Stem cell differentiation

Once any cell is implanted, Mazzocca and Muschler noted the cells may need a certain chemical and mechanical agent to induce the differentiation that is desired. This may include the local or systemic delivery of drugs or growth factors. Gulotta and colleagues added helix-loop-helix transcription factor scleraxis to bone-marrow derived MSCs and found improved rotator cuff tendon healing in rats.

Another way to induce differentiation is to coculture stem cells, according to Gulotta. For example, adding end-stage cells, such as chondrocytes, in a petri dish with growing stem cells allows differentiation factors and cytokines released by the chondrocytes to induce differentiation in the stem cells.

Some molecules, such as SDF-1 or MCP-3, can increase the cell homing into an area when they are secreted or delivered locally, according to Muschler.

Tethering of a bioactive molecule to the surface of a biomaterial is a new method to deliver and control the location and effect of an active agent. Epidermal growth factors (EGFs) or bone morphogenetic proteins (BMPs) are among the molecules being explored, according to Muschler.

“[Tethering] increases the local concentration, controls the distribution of the agent and prolongs the time when it can be active by reducing diffusion and degradation,” Muschler said.

Genes may be added to stem cells to differentiate them into tendon, bone or other tissues such as BMP-13 to differentiate stem cells into tenocytes, Gulotta said.

“At this time, there are more questions than answers in terms of how to harvest stem cells, what are the best types of cells, how do you get enough of them, what do you do to them to convince them to form whatever tissue you need and what do you do postoperatively or post-treatment,” Gulotta said. – by Renee Blisard Buddle

References:
Beitzel K. Arthroscopy. 2013;doi:10.1016/j.arthro.2012.08.021.
Caralla T. Tissue Eng Part A. 2013;doi:10.1089.ten.tea.2011.0622.
Gurdon E. J Embryol Exp Morphol. 1962;10:622-640.
Gulotta L. Am J Sports Med. 2011;doi:10.1177/0363546510395485.
Hernigou P. J Bone Joint Surg Am. 2005;87(7):1430-1437.
Lu P. Cell. 2012. doi:10.1016/j.cell.2012.08.020.
http://stemcells.nih.gov/info/basics/pages/basics10.aspx.
Schnabel L. Vet J. 2013;doi:10.1016/j.tvjl.2013.04.018.
Yamanaka S. Cell. 2006;126(64):663-676.
For more information:
Lisa A. Fortier, DVM, PhD, DACVS, can be reached at Vet Box 32, College of Veterinary Medicine Cornell University, Ithaca, NY 14853; email: laf4@cornell.edu.
Lawrence V. Gulotta, MD, can be reached at Hospital for Special Surgery, 535 East 70th St., New York, NY; email: hickenbottomt@hss.edu.
Augustus D. Mazzocca, MS, MD, can be reached at The New England Musculoskeletal Institute, UConn Health Center, 263 Farmington Ave., Farmington, CT 06030; email: mazzocca@uchc.edu.
George F. Muschler, MD, can be reached at the Lerner Research Institute, Cleveland Clinic, Cleveland Clinic Main Campus, Mail Code A41, 9500 Euclid Ave., Cleveland, OH 44195; email: ambrol@ccf.org.
C. Thomas Vangsness Jr, MD, can be reached at the University of Southern California, 1520 San Pablo St., Suite 2000, Los Angeles, CA 90033; email: alison.trinidad@usc.edu.
Disclosures: Gulotta is a consultant for Biomet Inc. Mazzocca receives research support and is a consultant for Arthrex. Muschler receives fees as a consultant for the FDA and Smith & Nephew and receives grant funding from Harvest Technologies and Medtronic. Fortier and Vangsness have no relevant financial disclosures.

POINTCOUNTER

What is the best method to obtain cells for cell therapy and why?

POINT

Adult-derived cells safer, more readily available

Despite the obvious potential of embryonic stem cells, I will take the position of favoring adult-derived cells. I base this opinion on considerations related to biologic potential, safety and availability. The current standard sources of mesenchymal stem cells for orthopedic applications include bone marrow and adipose tissue. The use of cell-sorting techniques in the future will allow isolation of the small number of true stem cells (estimated as 0.01% of bone marrow cells). These cells have the ability to differentiate into several mesenchymal tissues, including bone, cartilage, adipose, and dense fibrous connective tissue resembling tendon and ligament. It is becoming increasingly evident that numerous tissues in the adult contain a niche of cells that are multipotent. For example, such cells have been identified in tendon, ligaments, bone, muscle, cartilage and the meniscus. These cells are usually located in proximity to vessel walls. Our challenge is to identify how to stimulate these cells to participate in tissue repair and regeneration.

Scott A. Rodeo

Scott A. Rodeo

The importance of the cells’ local environment is also a critical factor. First, cells from the local environment appear to have superior repair capacity compared to distant cells. For example, stem cells derived from synovium likely have better a ability to differentiate into a chondrogenic phenotype than bone marrow-derived cells. Second, the biologic activity of transplanted cells is highly dependent upon the environment into which the cells are transplanted. The biological signals present in the local environment may be as important as the specific cell type used. Thus, even a pluripotent cell, such as an embryonic stem cell, will not realize its optimal potential in a nonsupportive environment, such as that seen in an elderly patient.

It should be noted that embryonic cells are pluripotent while adult-derived cells are multipotent, indicating greater plasticity for embryonic stem cells. However, such pluripotency also comes with a higher risk for tumorigenicity. There are also ethical issues related to the use of embryonic stem cells. Finally, another factor to be considered in favor of autologous (adult-derived) cells is the potential for an immune response against allogeneic cells, which has been demonstrated.

Scott A. Rodeo, MD, is an Orthopedics Today Editorial Board member and is co-chief of the Sports Medicine and Shoulder Service at Hospital for Special Surgery in New York City.
Disclosure: Rodeo receives research support from the American Orthopedic Society for Sports Medicine, is a consultant for Smith & Nephew and owns stock in Cayenne Medical.

COUNTER

Use of cell therapy is complex, and shared decision-making is necessary

We have entered an era of evidence-based medicine and shared decision-making. Physicians must thoroughly consider the efficacy of treatments, educate their patients and enable them to make appropriate decisions regarding care. While stem cell therapy offers great promise, most patients do not appreciate the limited evidence of efficacy in human trials or the time it will take to establish proven treatments.

Regis J. O’Keefe

Regis J. O’Keefe

Stem cell therapy has the potential to profoundly alter the impact of musculoskeletal disease. Preclinical animal studies show that stem cell treatments can delay or prevent the onset of degenerative diseases of the skeleton, joints and muscle tissues. Stem cells have been shown to stimulate tissue regeneration following injury or disease in animal models of cardiac, endocrine and neurological diseases as well. Development of these treatments is essential for our patients and to maintain orthopedics at the leading edge of discovery and innovative treatment.

Education and shared decision-making are particularly important as new therapies emerge. During the years, we have had some sobering missteps with the initiation of new therapies. The enormous promise of gene therapy has been tempered by the severe complications noted in some patients. We are re-examining the use of bone morphogenetic proteins in spine fusion, and our recent experience with metal-on-metal hip implants has been discouraging despite its initial promise.

Numerous Internet sites promise amazing outcomes and potential cures from stem cell therapy. The use of stem cells in clinical medicine is complex – and patients suffering from orthopedic disease are vulnerable. The work by Drs. Mazzocca, Muschler and others to determine the clinical efficacy of stem cell approaches are essential. Additional work must be done so that we can provide patients with solid information based on multiple evidence-based clinical studies. Until then, we need to inform patients that these therapies have promise, and keep abreast of this innovative therapy as it emerges.

Regis J. O’Keefe, MD, PhD, is chairperson of the Department of Orthopaedics at the University of Rochester Medical Center in Rochester, NY.
Disclosure: O’Keefe has no relevant financial disclosures.