1. Standards in Europe for Orthopedic stem cells infusions Animal trials with stem cells in Orthopedics
Dr Dimitrios Tsoukas MD, MSc
Director MIOSMED CENTER ISAKOS APROVED TEACHING CENER

3. A challenge to the orthopaedic surgeon in the 21st century is the desire of the ageing patient to remain physically and mentally active and to continue to contribute to society.
Younger patients are participating in more physically demanding sporting activities, resulting in injuries that require biological solutions which can allow them to continue to be active throughout their lifetime.
Considerable benefits have resulted from the biomechanical solutions of the past 50 years, with better biomaterials and implants for joint replacements, more precise instrumentation and computer-aided navigation techniques.

4. A challenge to the orthopaedic surgeon in the 21st century is the desire of the ageing patient to remain physically and mentally active and to continue to contribute to society.
Younger patients are participating in more physically demanding sporting activities, resulting in injuries that require biological solutions which can allow them to continue to be active throughout their lifetime.
Considerable benefits have resulted from the biomechanical solutions of the past 50 years, with better biomaterials and implants for joint replacements, more precise instrumentation and computer-aided navigation techniques.

5. However, implants have a finite lifespan owing to loosening or other modes of failure and may require further surgery involving increased morbidity for the patient.
The future lies in regenerative medicine, with the potential to grow new tissues and organs to replace damaged or diseased ones by utilizing stem cells, which have the capacity to self-renew and differentiate into many different types of tissue.
Although this area of research holds infinite promise, it is also influenced by scientific, ethical, moral and political controversies.

6. What are stem cells
Stem cells must have a capacity for self-renewal giving rise to more stem cells, and the ability to differentiate into tissues of various lineages under appropriate conditions.
They may be pluripotent or multipotent, depending on type. Only the embryo is totipotent.
Embryonic stem cells (ESCs) are pluripotent, as they are capable of differentiating into many tissue types, whereas differentiation of adult stem cells is generally restricted to the tissue in which they reside, as with hepatocytes in the liver, and haemopoietic stem cells in blood.

7. However, under appropriate conditions some can differentiate into multilineages, becoming multipotent. This is true of mesenchymal stem cells (MSCs), which are found in bone marrow, skin, adipose tissue, and manyother tissues of mesenchymal origin.
These cells are capable of differentiating into bone, cartilage, tendon, ligament, fat and other tissues of mesenchymal origin.

9. STEM CELLS BASED THERAPIES
Stem cells based therapies encompassing collection, purification, manipulation, characterization delivery of cells for therapeutic purposes.
Presently, human embryonic stem cells (hESCs) are used in 13% of cell therapy procedures, fetal stem cells in 2%, umbilical cord stem cells in 10% and adult stem cells in majority (75%) of treatments.

11. Adult stem cells
Adult stem cells are lower in the hierarchy of stem cells and, although more limited in their ability to differentiate into many tissue types, present fewer ethical problems in their use. As many of their applications make use of autologous cells there are no problems with immunogenicity. However, the problem of control of differentiation still exists, as in the case of ESCs, but the propensity to form teratomas is much slighter.

12. Currently, there are two main types of adult stem cell with great clinical potential, haemopoietic stem cells (HSCs) and MSCs.
HSCs are already in use clinically for the treatment of leukaemia, thalassaemia and multiple myeloma. MSCs are found post-natally in the nonhaemopoietic bone marrow stroma, which comprises a heterogeneous population of cells including reticular cells, adipocytes, osteogenic cells, smooth muscle cells, endothelial cells and macrophages.
MSCs can also be derived from bone marrow, adipose tissue and skin. They are multipotent cells capable of differentiating into cartilage, bone, muscle, tendon, ligament and fat.

13. The ability of MSCs to differentiate into many types of musculoskeletal tissue has great potential in the repair and regeneration of bone and cartilage. This has prompted scientists to study the biology of these cells so that they can be appropriately converted to the desired tissues with the proper biological and mechanical properties.
With appropriate growth factors and scaffold technology, it is envisaged that in the near future it will be possible to repair large defects in bone and cartilage biologically.

14. Mesenchymal stem cells
MSCs are multipotent progenitor cells which are capable of differentiating into several connective tissue cell types, including osteocytes, chondrocytes, adipocytes, tenocytes and myocytes. They are characterised by cell surface markers and, under appropriate conditions, can be induced to differentiate into bone, cartilage, muscle and fat
MSCs from bone marrow share the following features:
1. No expression of HSC markers, such as CD34 and CD14
2. Expression of matrix receptors such as CD44, CD29 and CD71
3. Expression of MSC markers, SH2 (CD105) and SH3 (CD71)
4. Extracellular matrix components: collagen, proteoglycans and fibronectin
5. Growth factors and cytokine receptors for TGF-β group

16. Using appropriate induction solutions, MSCs have been shown to be capable of differentiating into bone, cartilage, muscle and adipose tissue.
Chondrogenic differentiation with genetic modulation has been attempted by transfecting stem cells with recombinant DNA constructs and coding for the expression of certain growth factors or proteins that promote chondrogenesis, such as SRY-box containing genes (SOX) 5-9, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β.

17. REGULATORY FRAMEWORK IN EU Legislation on cell therapy in Europe is based on 3 directives
Directive 2003/63/EC, which defines cell therapy products as clinical products and includes their specific requirements.
Directive 2001/20/EC, which emphasizes that Clinical Trials are mandatory for such cell therapy products and describes the special requirements for approval of such trials.
Directive 2004/23/EC, which establishes the standard quality, donation safety, harvesting, tests, processing, preservation, storag and distribution of human tissues and cells.

18. REGULATORY FRAMEWORK IN EU
The EU regulation (1394/2007) on Advanced Therapy Medicinal Products (ATMPs)- effective from December 2008-includes gene therapy medicinal products, somatic cell therapy products and tissue engineered products.
Cells fall under this regulation in case they have been subjected to substantial manipulation, resulting in a change of their biological characteristics, physiological functions or structural properties relevantfor the intended therapeutic application.
The Committee for Advanced Therapies (CAT) within European Medicines Agency (EMA) is responsible for preparing a draft opinion on the quality, safety and efficacy of ATMPs that follow the centralized marketing authorization (MA) procedure

19. US FDA CELL THERAPY REGULATION ‘Any procedure where human cells are manipulated for clinical use will be subject to federal manufacturing standards and oversight’
Human Cells,Tissues,Cellular/tissue-based products (HCT/Ps)
Lower risk HCT/Ps :Products361-Manufactured under good tissue practices-Regulated under PHSA 361-No pre-market clinical studies or approval prior to marketing.
Higher risk HCT/Ps : Products 351- Manufactured under good tissue practices and good manufacturing practices- Regulated under PHSA 351- Pre-clinical animal and clinical studies to prove safety and efficacy.

20. US FDA CELL THERAPY REGULATION 4 Criteria to Qualify as a low-risk biologic product
Minimal manipulation: manufacturing is limited to simple procedures
Advertised/labeled for homologous use only: Product must carry out the same biologic function as it normally would.
Non combination Product: Combining products increases complexity with exclusions involving simple electrolyte solutions and preservation agents.
Non systemic Effect or is Autologous: If the product may have a systemic effect,it must be autologous or from a close blood relative,in order to reduce the risk of an immune reaction.
The Center for Biologics Evaluation and research (CBER) is the division of US FDA that regulates stem cell based therapies based on the above criteria.

21. REGULATORY CHALLENGES
Safety testing is critical, including assays for potential microbial, fungal,endotoxin,mycoplasma and viral contamination.
In vitro functional assays designed to act as surrogate measures for clinical effectiveness.
Use of severely immuno-compromised small animals. For a variety of diseases, for example in orthopaedics, small animals are not capable of modeling the disease.
The clinical trials should be performed with the highest attention being paid to the safety and ethical issue involved.

22. Adult stem cells in orthopaedic research
Research on the therapeutic application of MSCs can be carried out by using MSCs as progenitor cells, injected directly into tissues to enhance the process of repair, or by using them as a vehicle for gene delivery.

24. CLINICAL CARTILAGE Pre-Clinical animal study. Publications.
Initial Pilot Clinical Trial.
Case Series. Published methodology .
Randomized controlled trial. Publications.
Case Series. Publications.

25. Articular cartilage
Articular cartilage is vulnerable to injury and has poor potential for repair. Damage can lead to arthritic changes many years after injury.
Procedures directed at the recruitment of stem cells from the marrow by penetration of the subchondral bone have been widely used to treat localised cartilage defects. The ‘microfracture’ technique is often used, but the fibrocartilage which results from these techniques has poor mechanical properties compared with normal cartilage.

28. Nevertheless, good short- to intermediate-term results from treating the lesions of osteochondritis dissecans or traumatic osteochondritis with these techniques have been described.
More recently, attempts to ‘regenerate’ normal articular cartilage have been introduced in clinical practice with autologous chondrocyte implantation (ACI).
Chondrocytes could be transplanted into articular cartilage defects, which improved healing compared with that in controls. Chondrocytes, stem cells, periosteum containing stem cells, chondrocyte precursors or any combinaton of these can be used.

29. Bone
Trauma and some pathological conditions may lead to extensive loss of bone which requires transplantation of bone tissue or bone substitutes to restore structural integrity.
The use of autologous and allogenic bone grafts is associated with donor site morbidity and the possible transmission of infection. Several researchers have described the purification and expansion of bone marrow cells from mice, rats, rabbits, dogs and humans, and the ability of these cell populations to form bone when implanted ectopically with hydroxyapatite or an appropriate carrier
Methods have also been developed for the expansion of bone marrow osteoprogenitors, which indicates the possibility of using autologous human stromal progenitors in the regeneration of large bone defects

30. Lieberman et al, used rat bone marrow-derived cells transduced with BMP-2 to heal critically-sized femoral defects and observed more robust formation of bone than in the control group. BMP regulates chemotaxis, mitosis and differentiation, and is fundamental in initiating fracture repair.
Other growth factors, such as TGF-β and IGF may stimulate fracture repair and minimise the rate of nonunion.
In order for BMP to induce bone formation effectively, its dose must be of sufficient concentration for a sustained period. However, these proteins have short biological half-lives and must be maintained at therapeutic concentrations at the fracture site to be effective. Gene therapy has been used to deliver therapeutic doses of protein for sustained periods.

32. Successful tissue engineering of bone requires osteoproduction, osteoinduction, osteoconduction and mechanical stimulation. Osteoproduction is the ability of the cell to secrete bone material.
Bruder et al and Bruder, Fink and Caplan obtained healing of critically sized bone defects with purified MSCs derived from bone marrow.
Quarto et al, used autologous MSCs to treat segmental bone defects in a limited group of patients for whom a traditional therapeutic alternative was very difficult or had already failed.
Recently, muscle-derived stem cells have received much attention as a source of osteoproductive cells. Osteoinduction refers to the growth factors that attract osteogenic cells to the site of the defect. In recent years the isolation of factors such as TGF-β3 and its analogues, bone morphogenetic proteins (BMPs), BMP-2 and BMP-7, has led to their use clinically to enhance and accelerate the repair of bone, and also to replace it.

33. Tendons and ligaments
Injuries to tendons and ligaments heal by forming inferiorquality tissue. Autografts, allografts and resorbable biomaterials have been used to repair defects in tendons and ligaments.
The problems associated with biological grafts include donor site morbidity, scarcity, and tissue rejection.

34. A number of studies on the use of MSCs to improve the repair of tendon defects have been carried out in animals.
Young et al showed that when MSCs were seeded on a defect of 1 cm in the tendo Achillis of a rabbit, the repair had a significantly larger cross-sectional area and the collagen fibres appeared better aligned than those in matched controls.
In another study, MSCs seeded with collagen composites were implanted into full-thickness central defects created in the patellar tendons of the animals providing the cells.
The healing of these autologous defects was compared with the natural repair of identical lesions on the contralateral side.

35. The repairs containing the MSC collagen composites showed significantly higher maximum stresses and moduli at 12 and 26 weeks than those which healed naturally, although greater concentrations of MSCs produced no additional significant histological or biomechanical improvement.
However, in these studies, as collagen gel could not be used alone as a control it would not contract without cells.
Ouyang et al, used knitted polylactide-coglycolide (PLGA) as a scaffold loaded with MSCs in the repair and regeneration of the tendo Achillis of rabbits. Their results suggest that this construct has the potential to regenerate and repair such defects and to restore structure and function effectively.

36. Meniscus
Tears in the avascular inner third of the meniscus have limited or no potential for repair as the reparative process cannot occur without the presence of bleeding. The standard biological healing process produces limited results. Meniscectomy has been shown to have a strong association with the subsequent development of osteoarthritis.
Recently, cell-based therapy for meniscal injuries has been attempted. It has been demonstrated that isolated chondrocytes seeded on to meniscal matrices were able to bond separate pieces together.
Histological and biomechanical analyses showed that the strength of the adhesion increased over time by the formation of a newly synthesised cartilaginous matrix.

37. Dutton et al, assessed the capability of autologous seeded BMSCs to repair an avascular meniscal lesion in the pig. They showed that a meniscal lesion involving the inner, avascular, one-third of the meniscus benefitted from the bonding capabilities of the transplant (next figure). These results represent a promising beginning for the potential of cellbased therapy to repair a tear in the avascular inner third of the meniscus rather than proceeding to surgical resection.

40. ADIPOSE TISSUE STEM CELLS The stromal fraction that is harvested from adipose tissue is a mixture of regenerative cells

42. WHAT IS THE EVIDENCE?

43. Stem cell therapy holds great promise for the repair and regeneration of musculoskeletal tissues. In order to realize the full therapeutic potential of stem cells, a number of challenges need to be overcome.
One of the most important prerequisites is to be able to generate adequate numbers of the correct phenotypes of cells under animal-free culture. This will require expertise in characterization, differentiation and expansion of these cells.

44. The regenerated tissue has to have the appropriate three-dimensional structure, with the production of extracellular matrix, and be structurally and biomechanically compliant with the demands of native tissue.
Bioengineers will play an important role in this endeavour. Ultimately the tissue must be fully integrated andimmunologically compatible, with the host tissue. Here the input from developmental biologists, molecular biologists and immunologists will be crucial.
It is important to continue research to understand the basic biology of stem cells and their potential for clinical and therapeutic applications