Prof. Anthony Hollander had always believed that he would be able to save lives. He believed it so strongly, in fact, that he wrote to a television program as a child asking for a few tools to help him. As it was, he did not receive the necessary items, but he did go on to fulfill his prophecy: “making people or animals alive.” Prof. Hollander has been in the vanguard of research into bioengineered arthritis therapies for many years, and has established a world-renowned laboratory with an expert team of scientists and high-quality equipment. As a result, his group has been able to perfect stem cell culture protocols to provide the consistent starting material essential for all areas of their research. Furthermore, their knowledge and facilities were instrumental in the first-ever bioengineered tracheal graft.
Figure 1 - Prof. Anthony Hollander, ARC Professor of
Rheumatology & Tissue Engineering, Department of
Cellular & Molecular Medicine, University of Bristol, U.K.
(Figures 1–6 courtesy of Dr. Sally Dickinson, University
of Bristol.)With nearly 20 years’ experience in the field of cartilage biology and arthritis research, Prof. Hollander (see Figure 1) is presently the ARC Professor of Rheumatology & Tissue Engineering in the Department of Cellular & Molecular Medicine at the University of Bristol, U.K. He currently holds a number of significant research grants to look at various scientific and clinical aspects of tissue engineering and stem cell biology for cartilage repair. He has pioneered the development of new assays and methodological approaches for the measurement of repair tissue quality in very small biopsies of cartilage from patients with knee injuries.
Stem cells show therapeutic potential
Prof. Hollander and his colleagues spend much of their time working with stem cells as the basis for their tissue engineering research. Stem cells have shown the promise to revolutionize the treatment of many diseases, as noted by George Wolff in his book, The Biotech Investor’s Bible: “The damaged brains of Alzheimer’s disease patients may be restored. Severed spinal cords may be rejoined. Damaged organs may be rebuilt. Stem cells provide hope that this dream will become a reality.” Much has been written and discussed on embryonic stem cells, but for Prof. Hollander, the main focus has been on adult (somatic) stem cells. “Embryonic stem cells do have the potential to become every type of cell in the body, but they are very difficult to control fully—they form tumours relatively easily. Somatic stem cells do not possess the same breadth of differentiation capabilities as embryonic stem cells, but are more predictable and controllable.” Adult stem cells are found in a number of locations, such as the bone marrow, and can therefore be retrieved directly from patients for their own treatment. This removes the need for immunosuppression to prevent rejection, thereby greatly increasing the chance of grafting success.
Research provides foundation for tissue replacement
Figure 2 - Adherent human adult bone marrow stem cells in
culture.Bone marrow mesenchymal stem cells (BMSCs) harvested from the heads of femur bones are the major source of stem cells in Prof. Hollander’s laboratory.1 Dr. Sally Dickinson, a research associate in the group, explained, “Bone marrow mesenchymal stem cells are donated by patients undergoing hip replacement operations and are the perfect starting point for our research, as they are multipotent and can therefore form the major cell types involved in rheumatology applications.” The donated cells are suspended in a specialized stem cell culture medium formulated to promote the growth and differentiation of BMSCs (see Figure 2). To remove any bone remnants, the cells are washed several times in the medium and fat is then removed by gently centrifuging the cells in a Thermo Scientific Sorvall Legend RT-Plus centrifuge (Thermo Fisher Scientific, Asheville, NC) at 1500 rpm for 5 min and recovering the cell pellet. Once the cells are clean and free from bone or fat, they are seeded in 175-cm2 culture flasks at a density of 5–10 million cells. The cells are then placed in a Thermo Scientific Cytoperm 2 CO2 incubator (5% CO2, 37 ºC, 95% humidity), with media changes after four days and then every other day until adherent cells have reached 90% confluence.
Figure 3 - Tissue-engineered cartilage produced from
bone marrow stem cells (macroscopic appearance).Differentiation
Once successfully expanded, the BMSCs are further incubated in specially developed media to enable differentiation into either chondrogenic monolayers, osteogenic, or adipogenic cultures (see Figures 3 and 4). Alternatively, BMSCs can be added to polyglycolic acid (PGA) scaffolds and incubated for five weeks with regular media changes to create 3-D engineered cartilage (see Figure 5). The majority of research work conducted in Prof. Hollander’s laboratory focuses on chondrogenic cultures, either monolayer or 3-D, since these are the most important cell type for osteoarthritis applications.
Figure 4 - Bone marrow stem cells that have undergone
adipogenic differentiation (20× magnification).
Figure 5 - Bone marrow stem cells that have undergone
osteogenic differentiation (20× magnification).Analysis
Several analytical techniques are used to assess BMSC cultures and their differentiation, including histological staining and real-time PCR. However, the bulk of the analyses on the engineered cartilage are carried out using enzyme-linked immunosorbent assays (ELISAs) since they provide quantitative biochemical measurements for key molecules such as collagen types I and II. All ELISAs in Prof. Hollander’s laboratory are analyzed on a Thermo Scientific Multiskan microplate photometer.
Clinical collaborations lead to patient therapies
As one of the foremost scientists in this rheumatology field and the development of chondrogenic cultures from BMSCs, Prof. Hollander works closely with clinical teams to develop patient-specific cartilage autografts. These are generated by extracting cartilage cells (rather than BMSCs) from the patient and culturing them to provide autologous chondrocytes, which are then seeded into a 3-D biodegradable material (derived from the total esterification of hyaluronan with benzyl alcohol and constructed into a nonwoven configuration). These engineered grafts are then placed at the site of cartilage injury, often without the need to glue or suture them in place. Furthermore, the procedure does not necessitate open surgery since a mini-arthrotomy is usually sufficient. Once in place, the graft quickly integrates with the patient’s existing tissues, providing good collagen composition and integration with the underlying bone. The autograft technique provides several distinct advantages, namely less stressful surgical procedures and, perhaps more importantly, the lack of any immune response. This is a major advance over allografts, which require the use of powerful immune-suppressing drugs for extended periods post-transplant.
First-ever bioengineered tracheal graft
Last summer, Prof. Hollander received a request for help from a friend and colleague, Prof. Martin Birchall, a surgical professor at the University of Bristol. A patient of Dr. Birchall’s had suffered serious damage to her trachea as a result of contracting tuberculosis (TB). The patient, Claudia Castillo, was a young mother whose only chance of survival at that point in time was to have one lung removed, which would have seriously affected her quality of life and her ability to look after her children. After much discussion, Prof. Hollander and his team very quickly set to work adapting their existing osteoarthritis-based protocols to enable Prof. Birchall to grow a large population of chondrocytes derived from Ms. Castillo’s BMSCs. A section of human trachea was donated for use as a scaffold on which the new tissue could be grown. The trachea was stripped of the donor’s cells, leaving a trunk of nonimmunogenic connective tissue, onto which the chondrocytes were seeded.2 This seeding process used a novel bioreactor developed at the Politechnico di Milano, Italy, which provided the right environment for the cells to form the cartilaginous part of the trachea within four days of seeding.
The graft was then lined with epithelial cells and transplanted into Ms. Castillo, who responded very quickly to the new airway section, without any sign of rejection (no antibodies to the graft were found). Subsequent biopsies have shown that the new section is fully integrated with the existing airway and is fully supplied with blood vessels. She is now able to live life as if she had not been struck down with TB, a result that would never have been possible if her lung had been removed.
Discussion
Stem cell-based therapies have promised huge changes in the treatments of many diseases and disorders, but much research is still required to ensure safety and consistency before they can be applied more extensively. Prof. Hollander and his colleagues at the Department of Cellular & Molecular Medicine at the University of Bristol have been investigating the fundamental principles governing the differentiation of bone marrow stem cells into chondrocytes—the source of cartilage. Through this research they aim to further improve the processes used to generate chondrocyte-based autografts, which have already started to prove their value in the treatment of cartilage damage. Throughout their pioneering research, Prof. Hollander’s team have come to rely on the dependability and functionality of a broad array of standard and advanced laboratory equipment specifically designed to provide the highest quality and reliability in the cell biology laboratory. Central among these are a number of Thermo Scientific instruments and tools, from Finnpipette manual pipets through HERAsafe KS12 biosaftey cabinets and Revco ultralow-temperature freezers to Cytoperm CO2 incubators and a Multiskan microplate reader (see Figure 6).
Figure 6 - Dr. Sally Dickinson and Mr. Henry Jia with some of the
Thermo Scientific equipment, including one of three HERAsafe KS12
biological safety cabinets, two Cytoperm 2 gased incubators, and two
HERAcell CO2 incubators.As a result of their dedicated work, Prof. Hollander’s team was able to take part in the amazing feat of the first-ever bioengineered tracheal graft, in some way fulfilling Prof. Hollander’s own childhood prophecy. Their work has enabled Claudio Castillo to regain an amazing quality of life following a life-threatening condition, while increasing the drive among researchers and clinicians to more expansive use of stem cell-based therapies.
Reference
- Kafienah, W.; Mistry, S.; Perry, M.J.; Politopoulou, G.; Hollander, A.P. Pharmacological regulation of adult stem cells: chondrogenesis can be induced using a synthetic inhibitor of the retinoic acid receptor. Stem Cells2007, 25, 2460–68.
- Macchiarini, P.; Jungebluth, P.; Go, T.; Asnaghi, M.A.; Rees, L.E.; Cogan, T.A.; Dodson, A.; Martorell, J.; Bellini, S.; Parnigotto, P.P.; Dickinson, S.C.; Hollander, A.P.; Mantero, S.; Conconi, M.T.; Birchall, M.A. Clinical transplantation of a tissue-engineered airway. Lancet 2008, 372, 2023–30.
Mr. Meech is Commercial Product Manager, Thermo Fisher Scientific, Basingstoke RG21 6YH, U.K.; tel.: +44 0 870 609 9203; e-mail: [email protected].