Stem cells, regenerative orthopaedics and the knee

In the last 30 years orthopaedics has made great advances in the field of bioengineering, in which engineers have worked with surgeons to design a sophisticated array of artificial components, generally of metal and plastic, to replace worn out joints and even individual joint surfaces.

There have also been advances in the design of surgical procedures and instrumentation to allow improved ligament reconstruction, meniscal transplant and cartilage repair in an effort to prolong the life of a joint before replacing it. The phrase 'biologic knee replacement', coined by Dr Kevin Stone, seemed to encompass this latter group of procedures but the newer phrase of 'regenerative orthopaedics' takes the idea even further. Instead of transplanting mature donor tissue to a compromised recipient area, regenerative orthopaedics involves multiplying 'primitive' or unspecialised cells by a variety of methods and then transferring them to the damaged area to grow into mature tissue and restore or establish normal function.

KNEEguru readers will already be familiar with the concept of 'marrow stimulation' which is a simple form of regenerative orthopaedics. The early techniques of subchondral drilling developed in the 1950s by Dr Kenneth Pridie (ref 1) and the later microfracture technique of Dr Richard Steadman (ref 2) work by effectively breaching damaged joint cartilage with a blood clot into which can migrate unspecialised cells (stem cells or progenitor cells) from the underlying bone marrow. These cells then differentiate and attempt to fill the defect with adult cartilage tissue.

Marrow stimulation is considered part of the armamentarium of 'cartilage repair' techniques. One of the now familiar cartilage repair operations for the knee - ACI (autologous chondrocyte implantation) - is a another regenerative orthopaedic procedure for repairing small defects in the joint surface. It involves taking some of the patient's own adult cartilage cells (chondrocytes) and stimulating them to multiply in a culture in the laboratory before re-inserting them back into the knee behind a membrane of periosteum which is sewn over the defect. Ideas have moved on quite dramatically since the first ACI procedures, and now there are second generation and third generation procedures that have developed out of this original concept. These newer procedures involve different combinations of bioengineered scaffolds onto which cells are seeded, as well as different combinations of cells derived from the patient or from laboratory stem cell lines.

In the laboratory, meanwhile, the stem cells scientists are finding out what stem cells and progenitor cells are really all about - how and why certain cells retain the ability to reproduce themselves indefinitely, and how the cells know when to switch on or off their ability to differentiate into different cell and tissue types. They are learning how to replicate these processes in the laboratory, and are speculating about future applications in the clinical setting. However, this branch of medicine is still in its infancy and is being influenced by scientific, ethical, moral and political issues.

Human embryonic stem cells and the ethical dilemma

A human embryo starts off with only a single fertilised cell. This cell is 'totipotent' - it has the ability to divide and form a complete new embryo. After a few days the tiny embryo starts to take shape and individual cells lose their totipotent ability, although the inner cell mass retains the capacity to be 'pluripotent', capable of differentiating into all three of the tissue groups in the body. As the embryo matures to become a foetus and then an infant and finally an adult, most cells gradually change from pluripotent to multipotent (limited to one tissue group) and eventually towards being 'unipotent' (limited to a tissue type), but even in the adult a few specialist cells still retain some of their original ability to differentiate (ref4).

Pluripotent cells are popularly called stem cells, and the fact that the most potentially useful of these for regenerative medicine are harvested from human embryos (hESCs or human embryonic stem cells) is the source of a major ethical dilemma5. To gather and culture hESCs requires the laboratory creation and then subsequent destruction of a 5-6 day old human embryo cell mass. This embryo divides and within a few days a bubble-like structure has formed (the blastocyst), from the inner edge of which is suspended a tiny cell mass which can be removed and cultured in a laboratory to form a new pluripotent 'stem cell line'. The remainder of the embryo is discarded. The bubble-like blastocyst is clearly a mass of cells without a brain, but it still has the potential to form a complete human being, and this deliberate laboratory creation and destruction of a potential human being to harvest stem cells has been a hot issue since viable hESCs were first successfully cultured in 19986.

The human blastocyst from which the stem cells are taken, of course, may not have been created simply with this one purpose. As part of the in vitro fertilisation (IVF) process offered to infertile couples, the woman is chemically stimulated to produce a number of eggs. These are generally all sperm-fertilised in the laboratory into embryos and those 'excess' embryos that are not implanted into the woman's uterus to create a baby for the couple may be discarded. If permission has been granted by the couple they may alternatively be used for the extraction of stem cells.

Stem cells may also be collected from aborted foetuses and from the umbilical cord of a newborn baby, and in the adult progenitor cells can be identified and harvested from the blood and the bone marrow and other tissues. Cells from human umbilical cord and adult tissues do not, of course, carry the same ethical stigma as embryonic and foetal cells. Previously discarded after the birth of an infant, the umbilical cord is nowadays prized as an ethically-sound source of stem cells, and umbilical blood can be frozen and stored for later use in individuals with blood disorders like leukaemia. Parents may now choose to pay for the collection and storage of their own child's blood as a safeguard for the future.

hESCs, regulation and political involvement

With regard to hESCs, there are other issues of which any intended recipient needs to be aware, such as that of immune rejection. Basically stem cells from another human, albeit an embryo, are like transplant cells - the recipient's body may perceive them as foreign and there may be an immune rejection of the cells. Worse still there may be a rejection by the hESCs themselves of the body into which they are placed (graft versus host reaction). Another important issue issue is that of tumours called teratomas - if the medium in which the hESCs are nurtured is not properly adjusted prior to the cells being introduced into the recipient, they may not be sufficiently differentiated into the progenitor cells (precursor of the required tissue) and may start to generate within the recipient an embryo-like tumour called a teratoma.

Because of issues like these, four years after the first hESCs were first cultured, the White House announced (2001) that it was monitoring stem cell research with a view to keeping an eye on things7. President Bush, under pressure from pro-life movements, withdrew the majority of federal funding for hESC research, announcing that federal funding would be allowed for hESC research only on stem cell lines derived before 2001 but not for any new lines created subsequently nor for any research requiring destruction of new human embryos.

In 2003 a new organisation called the International Stem Cell Forum (ISCF) was conceived to bring together international consensus about the direction of human stem cell work and to discuss the many ethical issues. The Bush administration's restrictions had effectively choked American innovation, and only in early 2009 did President Obama order the lifting of this funding ban. He allowed a research lifeline of federal funding for research on hESCs from excess IVF embryos in storage since 2001 and any created after his announcement. Funding would still be banned, however, for the creation and destruction of human embryos specifically for their stem cells. The FDA in the same year approved the first human clinical trials using embryonic stem cells.

Adult stem cells - a different horizon

Because of this lost decade of federal funding in the USA in relation to embryo work, many research laboratories turned their attention to the unspecialised adult cells that occur normally in the adult tissues. Initially these cells proved difficult to identify, but before long they were found to exist in many tissues in the body. Not all these cells proved equally useful, but two cell types stood out above the others - haemopoietic stem cells (HSC) and mesenchymal stem cells (MSC). The former is already in routine clinical use for certain blood disorders (eg leukaemia). The latter can be isolated from bone marrow, fat, synovial membrane and periosteum8, and have the potential to differentiate into several musculoskeletal cell types, including bone, cartilage and tendon, and thus MSCs are highly relevant to orthopaedics9.

Using adult stem cells removes several of the issues that continue to haunt embryonic work. Firstly they generally originate from the patient him/herself (autologous) and not from someone else, so the issue of tissue rejection is less relevant. No human embryo is created or destroyed in acquiring the adult cells. Because the cells are more differentiated there is a much lower chance of teratoma formation. But the limited ability to differentiate means that the cells are restricted in their potential.

What research workers did determine is that the adult cell does not actually lose this genetic ability - but the process gets turned off as the cells mature10,11.

Then in 2006 a big breakthough occurred. Two Japanese scientists12 published a paper where they showed that such ordinary adult cells in mice could be 'reprogrammed' back to their primitive status by inserting four genes into the cells, triggering them to de-differentiate and behave very much like pluripotent hESCs. John Gurdon (a British scientist who initiated studies in this field 50 years ago) and Shinya Yamanaka of Japan in 2013 shared the Nobel Prize for this breakthrough. But it is still early days and a lot of problems have yet to be solved. Although it does not mean that routine use of such induced pluripotent stem cells (iPSCs) is just around the corner, it does mean that it is likely that in the not too distant future this may be the stem cell source of choice13.

At present there are two main strategies in current clinical use - cell therapy and tissue engineering -

Cell therapy - In autologous cell therapy, progenitor cells are taken from the patient, multiplied to useful numbers and then inserted back into the patient in suspension. They find their way to the damaged tissue by a process called 'homing' which is chemically mediated by the body, and when in place they can replace damaged ells, influence the ingrowth of blood vessels and help to moderate the inflammatory process. The concept seems simple but the details of the process is important, and institutions differ in the techniques used to isolate, multiply and re-insert the cells. For example, circulating blood has very few progenitor cells, but it is possible to stimulate the mobilisation of these from the bone marrow into the blood, allowing greater numbers for collection.
Tissue engineering - This is rather more technical. Cells are taken from the patient, multiplied, and then seeded onto a bioengineered scaffold before the whole construct is inserted into the damaged area. An example in the knee is the MACI procedure (matrix-induced autologous chondrocyte implantation), but there are now many related procedures differing in technical, chemical and bioengineering details. The drive appears to be to develop a procedure that involves only one surgical step, rather than two steps (one to harvest the cells and send them for culture and a second step to implant the construct). The two-step procedures can be very expensive of time and travel. An obvious approach is to use of stem cells lines already in existence in the laboratory (non-autologous or allogeneic) which means that the culture of cells and seeding of a scaffold can all be done without needing the patient to be there. Of interest is the clinical study by Dr Brian Cole's team at Rush University on the use of umbilical cord stem cells to repair isolated cartilage lesions in the knee14.

Finally, here is a very good video presentation by Marie Csete that offers further insights into this exciting field -

References

1. Pridie K (1959) A method of resurfacing osteoarthritic knee joints. J Bone Joint Surg Br 41-B(3):618-619.

2. Steadman JR, Rodkey WG, Singleton SB, Briggs KK (October 1997). "Microfracture technique for full-thickness chondral defects: Technique and clinical results". Oper Tech Orthop 7 (4): 300–304.

no longer online

4. The potential of stem cells in orthopaedic surgery Lee EH and Hui JHP. J Bone Joint Surg Br July 2006 vol. 88-B no. 7 841-851. https://www.bjj.boneandjoint.org.uk/content/88-B/7/841.full

5. The Embryonic Stem Cell Debate: A Brief Review. Jordan GR. https://www.qub.ac.uk/methics/JordanGR.pdf

6. Thomson JA et al., “Embryonic Stem Cell Lines Derived from Human Blastocysts,” Science 282, no. 5391 (1998): 1145-1147.

7. Embryo Stem Cell Research: Ten Years of Controversy. Robertson JA. journal of law, medicine & ethics. 2010. 191-203. https://www.utexas.edu/law/faculty/jrobertson/JLME-10-year-survery-Robertson-final.pdf

8. Periosteum as a source of mesenchymal stem cells: the effects of TGF-β3 on chondrogenesis. de Mara CS et al. Clinics (Sao Paulo). 2011 March; 66(3): 487–492.

9. Andreas Schmitt, Martijn van Griensven, Andreas B. Imhoff, and Stefan Buchmann, “Application of Stem Cells in orthopaedics,” Stem Cells International, vol. 2012, Article ID 394962, 11 pages, 2012. https://www.hindawi.com/journals/sci/2012/394962/cta/

10. Briggs R, King TJ 1952. Transplantation of living nuclei from blastula cells into enucleated frogs' eggs. Proc Natl Acad Sci 38: 455–463. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1063586/pdf/pnas01578-0075.pdf

11. Gurdon JB 1962. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J Embryol Exp Morphol 10: 622–640.

12. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Takahashi K and Yamanaka S. Cell 126, no. 4 (2006): 663-676. https://www.cell.com/action/showPdf?pii=S0092-8674%2806%2900976-7

13. The promise of induced pluripotent stem cells in research and therapy. Robinton DA and Daley GQ. Nature. 2012 January 18; 481(7381): 295–305. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3652331/

14. Rush University Medical Center (2013, January 24). Stem cell therapy to repair damaged knee cartilage. ScienceDaily. Retrieved June 4, 2013, from https://www.sciencedaily.com /releases/2013/01/130124163246.htm