Cartilage regeneration in osteoarthritis using msc cells
Cartilage regeneration in Osteoarthritis using MSC cells:
Cartilage provides the support and strength for the rest of the body. (1) The structure of cartilage is a matrix of collagen and elastic fibres,(2) which are set in a ground substance. Chrondocytes, the cartilage cells are embedded in this matrix and inhabit small cavities called lacunae. They are the only cells in the matrix, there are no blood vessels in cartilage therefore all nutrition absorption and waste removal must be occur by diffusion through the matrix. (1)
The matrix of the cartilage is made up of a ‘ternary compound that consists of cartilage proteoglycan monomer, link protein and hyaluronic acid,' (3) with many proteoglycan monomers and link proteins interacting with one hyaluronic acid, although the majority of the matrix is collagen type II. (9) ‘Type II, IX, XI collagen form fibrillar network, providing the structural framework for the matrix' (9) to form a homogenous mesh of fibers which are surrounded by the proteoglycan ternary complex. Proteoglycans contain a protein core with glycosaminoglycan chains, usually chondroitin sulphate and keratin sulphate. Smaller proteoglycans are used associated with the collagen fibrils. (9) There is no vascular system in cartilage due to chrondocytes producing antiangiogenesis factor, which depresses the formation of blood vessels.
There are three different types of cartilage, with unique properties and functions in the body. Hyaline cartilage is the most abundant type. It is normally covered by a perichondrium (1) except in the joints where it is coated in synovial fluids (9). The perichondrium allows cartilage to differ from surrounding tissue, it has to discrete layers; the outer region is more fibrous and dense. This region is used in ‘mechanical support, protection and attaching to other structures'. (1) The inner layer contains the cellular components and is vital in ‘growth and maintenance of cartilage' (1) This also provides the cartilage with nutrientsThe matrix of hyaline fibers are made with type II collagen and chondroitn sulphate (REFERENCE!!!), which allows the cartilage to be stiff and therefore gives the structure some support but provide some flexibility in order to reduce friction. (1) Hyaline cartilage is found in the ‘connection between the ribs and the sternum, nasal cartilages and respiratory tract.' Hyaline cartilage is also found covering the ends of bones in large joints such as the knee and elbow, here is it known as articular cartilage.
The other types of cartilage are elastic cartilage and fibrocartilage. Elastic also is used for support but it allows distortion to occur without the returning damage and it will then return to original structure. The flexibility is due to the presence of large amounts of elastic fibers, which are closely packaged between chondrocytes. (1). Elastic cartilage is found in the pinna and the cartilage in the larynx. Fibrocartilage contains a matrix largely of collagen fibres, which are tightly interlinked making the cartilage tough durable and able to resist compression.(1) These properties make it well adapted for its function, to prevent damage during bone-bone contact and shock absorption. It is therefore found between the vertebrae in the spin and pubic bones in the pelvis. (1)
The possibility of cartilage growth can occur in one of two ways interstitial or appositional. Interstitial growth occurs by the matrix of cartilage expanding between the dividing chondrocytes, causing growth of new matrix. They cause matrix to expand from within. (1) Appositional growth takes place as fibroblasts from the perichondrium inner, cellular layer differentiate into chondrocytes, which in turn produce new matrix. (1) With the expansion of the matrix, the fibroblasts are taken up into the matrix (1) and division of perichondrium cells replaces them. (1) Appositional growth takes place by increasing cartilage to the outer edge of the matrix. This method is used to repair minorly damaged cartilage. In more serious damage, the damaged cartilage is ‘replaced by a dense fibrous patch'.
ADD A SECTION IN HERE ON THE GROWTH OF CARTILAGE IN EMBRYOS, RELATE TO MSC STEM CELLS ETC.
Osteoarthritis is a chronic disease and according to NICE it is by far the most common form of arthritis and one of the leading causes of pain and disability worldwide (6). ‘Osteoarthritis was the sixth leading cause of years living with disability at a global level, accounting for 3% of the total global years of living with disability' (6, 7). The signs and symptoms of osteoarthritis are ‘pain during use of joint and relieved by rest, limited range of movement and stiffness, joint swelling bony enlargement, and malalignment,' (8,9) although many of these symptoms vary depending of the severity of condition, the number and location of affected joints. Any joint may be affected but the most common are hands, knees and hips, (6) but it can also commonly occur in the cervical and lumber spine. The condition is diagnosed by clinically assessing the patient and radiography.
The condition affects the both cartilage and bone and is ‘believed to be a result from an imbalance in erosive and reparative processes' (8) and involves the whole joint including the synovium and the artciular cartilage. (9) It is also thought to be due to inflammatory processes. (8) The inflammation occurs in the synovium, which is the non-articular part of the joint. Inflammation of the synovium can be known as synovitis (10) and it can lead to the overexpression of IL1, TNF alpha and beta. These are pro-inflammatory cytokines and are responsible for the ‘catabolic degenerative processes of articular cartilage.' (10) IL and TNF are also responsible for the production of Nitric Oxide, ‘by upregulating iNOS' and other inflammatory cytokines including IL6,17,18 LIF and chemokines. Increasing age raises the prevalence of osteoarthritis, (8) but other risk factors include being female, obesity and joint injury. There are two types of Osteoarthitis, primary osteoarthritis is seen to have no underlying cause, where as secondary is due to an underlying alterations, caused by a previous condition such as injury. (9)
The collagen fibrils in articular cartilage allow it to have non-linear properties to tension, allowing tension to be distributed from the tissue surface. This property decreases with age. This increased tension leads to the bind up ‘advanced glycation end products which will increase brittleness'. (9) Proteoglycans, especially chondroitin sulfate are associated with high numbers of negative charge. These molecules on an aggrecan molecule and HA to ensure the negative charge is fixed. The negative charges are needed for properties such as ‘large osmotic pressure and swelling, which can lead to ‘residual stresses that enhance support and distribution of applied loads. During Osteoarthritis, collagen, and the proteoglycans are broken down, causing a decrease in the tension and swelling and there is a reduction in the amount of articular cartilage and lesions may be formed. (9). During osteoarthritis, there is an increase in the amount of catabolic enzymes produced, causing the extracellular matrix of cartilage to be decreased. This is carried out by enzymes such as matrix metalloproteinase. These are up regulated by IL1 and TNF alpha in the chondrocytes of osteoarthritic patients and causes increased synthesis of MMP, causes the degradation of the extracellular matrix of cartilage (11) Normally repair is carried out by the appositional growth of the extracellular matrix. (8) During first stages of osteoarthritis, there is a greater amount of matrix degradation by proteases compared with chondrocyte activity to rebuild the scaffold. This results in a degradation of the cartilage with a loss of articular cartilage as the outcome. (8) This cycle will continue decreasing the amount of cartilage ever more, until late stage osteoarthritis where there will be ‘extreme or complete loss of cartilage' (8) and the narrowing of spaces between joints. Bony growths known as osteophytes may occur in the joints and bones at the joints may thicken, this is known as sclerosis. These late symptoms cause the clinical features of pain and less joint mobility. (8) In addition to this, there will be bone remodelling around the area where the Articular cartilage has been degraded. This can cause the bone density to become thicker, sclerosis, and the break down of bone, necrosis. (10) These changes in bone have been suggested to increase by osteoclasts of cytokines and cause the loss or damage of cartilage. There is also an effect on the MSCs in the bone marrow, from a leakage of synovial fluid into the medullar spaces. This causes osteophytes and cartilage nodules to be formed.
There are many treatment options used in Osteoarthritis, but all have the same objectives, according to Walker and Whittlesea these are ‘reducing pain, increase mobility, reduce disability, and minimize disease progression. (8) This can be achieved using pharmacological and non pharmacological methods. Non pharmacological methods include educating patients on the condition about methods of protecting the joints involved. These methods include exercise therapy, weight loss, physiotherapy, muscle strengthening exercise, treating the joints with heat/cold. (10) The non-pharmacological methods stated are used to ‘improve muscle strength and regain some of the movement of the affected joints'. (8) The pain suffered in osteoarthritis can be helped using transcutaneous electrical nerve stimulation (TENS) or acupuncture. (10) Other treatments which may help the patient which are not directly related to the Osteoarthritis are to treat depression and anxiety which is common in sufferers of the condition. This can help to reduce the pain and debilitating effects of the condition, although most sufferers are likely to need social support. (8)
The pain in osteoarthritis is caused by damaging the bone and cartilage in the affected joints. If there is no inflammation, then mild analgesic can be enough to reduce the treatment. Standard treatment is 4g of paracetamol daily (6). This treatment is more successful if Paracetamol is given on a regular basis instead of when required. Commonly there is an inflammation component associated with Osteoarthritis. if this is the case then NSAIDs such as ibuprofen or a COX2 inhibitor should be used, instead of a simple analgesic. Although NSAIDs are have a greater effect of the symptoms of OA, they have many more side effects, including upper gastrointestinal problems and renal toxicity (8) and a PPI should be given in high risk patients for gastroprotection. The other option for systemic pain relief is to use Opioids. These are usually used as a last resort if failure or contraindications to all other treatment, due to the adverse side effects, ‘risks of dependence, addiction and hyperanalgesia. (11) If opioids are used they must be given in small doses, especially if treating the elderly. It is likely that opioids will be excellent analgesics but they only have ‘moderate effects of the physical function of the joint',(11) although Fentanyl was shown by Langford et al, to both decrease pain experienced and improve joint function.(15) Fentanyl cannot be given to opioid niave patients and still has many of the same side effects as other opioids including nausea, dizziness, somnolence. (11) Topical NSAIDs can also be used for localized therapy or if it not possible to give systemic analgesic therapy. Alternative topical therapies can include ‘topical capsaicin or simple rubefacients.' (8) Other short term pain relievers include intra-articular injections of glutacorticoid, although this has the added side effect profile of short acting efficacy and differing metabolic effects. This restricts its use to only one injection every three or four months, but can potentially be excellent treatment for patients experiencing inflammation including a build up of fluid in the joints.
Longer term treatment for Osteoarthritis, although relieving pain, has other objectives including decreasing cartilage breakdown. (10) Glucosamine and chondroitin are both naturally occurring substances, which may help to slow down the articular cartilage erosion, although there has not been conclusive evidence. Glucosamine is an aminomonosaccharide (11) which acts as precursor to glycoaminoglycan (GAG) causing the stimulation of GAG production through chondrocytes, and also collagen synthesis. This will cause the frequency of joint space narrowing to decrease (10). Glucosamine can also form proteoglycans and hyaluronic acid. Chondroitin, normally found as Chondroitin sulphate is a major material of the cartilage extracellular matrix. It also works by ‘decreasing the activities of the catabolic enzymes in osteoarthritic cartilage and to stimulate the synthesis of GAG and collagens. Although according to the Glucosamine/Chrondrotin Arthritis Trial, there is no conclusive evidence that pain will be significantly treated after 24 weeks of treatment with glucosamine, chondroitin only or a combination of both, there has been other evidence that these naturally occurring agents have been shown to have some efficacy in treating Osteoarthritis. (11, 12) Due to the lack of severe side effects, with normal side effects being ‘nausea, abdominal pain, indigestion, diarrhoea(13), and potential action, glucosamine and chrondroitin can be given to osteoarthritic patients with a mild to moderate presentation.
Other longer acting medications include hyaluronic acid and Disease Modulating Osteoarthritis Agents (DMOADS). Hyaluronic acid is a polysaccharide located in the extracellular tissue, and its role is as a lubricant. Hyaluronic acid helps to restore the viscosity of the cartilage, providing an analgesic effect by increasing lubrication in the joints. (10) It is injected through an intra-articular injection and although it is most expensive, compared to steroids it has a longer time of action. (11) It has significant improves the symptoms of pain and joint movement, (15-16), although it is ‘not indicated for patients with severe osteoarthritis or patients with an articular misalignment.' Disease Modulating Osteoarthritis Agents are not used for the treatment of pain in the condition, but instead they are used to prevent further joint damage which will inevitably occur during the progression of the disease. This treatment will only work in joints where cartilage is already being broken down by proteases. The disease modifying groups of choice are tetracyclines. They have certain anti-inflammatory effects and also work as inhibitors of collagenases, the enzyme used to break down collagen, gelatinase, breaking down gelatin, the concentrations of both are increased in Osteoarthritis.(11) In the future, it is likely that there will be many more DMOADS, targeting a variety of ‘catabolic enzymes and cytokine-activated signalling cascades. (10)
The last option at the moment for patients with osteoarthritis is surgery. Surgery is only thought of as an option when all non invasive procedures have failed. The main surgical options available to osteoarthritic patients are total joint replacement, joint lavages and debridements, although both lavages and debridement are not clinically proven to be any more effective at relieving both pain or improving movement then pharmacological methods for treating these symptoms. (11) Arthoplasty/joint replacement is known to help relieve and improve symptoms for patients who are end stage before surgery and once surgery is deemed the best course of action, it should occur as soon as the decision is made in order to preserve as much of the joint as possible, for increased function. (11) Complications with surgery include rejection and infection around the joint replacement.
There are problems with all of the pharmacological/non pharmacological and surgical methods currently used to treat Osteoarthritis, whether these are problems with efficacy, side effects or not being appropriate for severe cases. All the pharmacological interventions do not manage to cure the disease, they only manage treat the symptoms and postpone the degeneration of the joint.
Recently the push has been towards repairs the cartilages with either a patch or a graft. (19) These can be either Osteochondral autograft or allograft and are commonly known as cartilage replacement techniques. Osteochondral autograft works on medium sized defects often involving the bone. In this process a ‘multiple cyclinder of cartilage and subchondral bone are taken for the non weight bearing area of the joint' and are then press fitted into the defect. (11) This process is known mosaicplasty. The donor cartilage is ‘self secured to the subschondral bone without any additional fixing procedures or devices.' Advantages A number of cylinders have been used in order to fix larger pores. of this process is that the cartilage used to treat the problems will be autogenetic so therefore will avoid the host immune system (11) and also there will be immediate treatment through ‘harvesting a patients own cartilage, although it does have the disadvantage that the amount of cartilage is limited without having affecting the weight bearing regions of the joint.
Allograft is different as it is used to repair much larger defects. The transplantation during allograft is from a different member of the same species. This can be both an advantage and a disadvantage. The advantages include the ability for a very close match as the donor cartilage can be taken from the same location in the donor as the recipient requires the transplant and the donor site will not contract disease. This disadvantage of this type of donation is that osteochondral tissue must be freshly transplanted into the donor as soon as possible, as it is possible for the ‘chondrocytes to expire during the process' although due to the improvement of preservation, it is now possible for the tissue to be transplanted up to three weeks later. The other problem with this type of replacement technique is the small but possible risk of disease transmission. (11,19)
The gold standard treatment from patients with Osteoarthritis is Autologous Chondrocyte implantation. This principle is based on transplanting cells with the properties to make cartilage into the defect. This technique has three main steps to it, ‘cartilage collection, isolation and in vitro expansion of chondrocytes in monolayer culture and implantation of the cultured chrondocutes in the lesion under a periosteal flap.' (10) This process is used clinically around the world, and has been since the approval by the FDA in the states in 1997. Clinical trials have shown that there is encouraging improvement in the clinical manifestations of patients with Osteoarthritis. (10)
Tissue engineering can be defined as using biological, chemical and engineering principles in order to ‘repair, restore and regenerate of native tissue by using scaffolds, cells and growth factors.'(25) They can be used alone or in combination. (26) If just cells are used then the correct cells are extracted from the patient, cultured in an in vitro conditions and then replanted back into the body, in the location of the defect requiring cartilage regeneration. (25) ‘Using a 3D porous material, a scaffold, can stimulate the growth of new tissue', whilst using biological factors including small molecules and growth factors can have a similar result. When constructing all three, it is common practise to for cells, often stem cells to be encased in a biodegradable scaffold and is often subject to certain environmental and chemical factors.
The principle of articular cartilage regeneration is to develop the extracellular matrix to repair cartilage by depositing chondocytes into the defect of the cartilage in order for them to produce the ECM. This currently carried out by implanting chondrocytes which are ‘seeded onto a porous absorbable scaffold' which provides the chrondocytes with support during both the culturing process and the early post-implantation stage, (19) although it is likely in the future that growth factors and stem cells will be introduced. There are three crucial areas of tissue engineering, which are the cells, the scaffolds and the culture conditions.
The cells important in the production of cartilage are chondrocytes, and are able to produce the ‘extracellular matrix of type II collagen and glycoaminoglycans' During an autologous transplant, there will not be enough chondrocytes available for the demand of cartilage required to fix the defect. The chrondocytes can be execrated from various sources including articular, nasal and costal. (10, 31), although there have been recent studies to suggest that chondrocytes from the nasal source are the appropriate for use in tissue engineering. The other major problem with using chondrocytes for engineer cartilage is dedifferentiation; this is due to a phenotypic instability and causes a' decrease in expression of type II collagen and an increase expression of type I'. Also there will be a change in numbers of round cells to fusiform shape cells. (10) To solve this problem, in vitro chondrocytes are harvested in appropriate biomaterials, before seeding, (19This problem can be solved using tissue engineering.
One way to achieve this engineering in cartilage is to extract chondrocytes, allow them to expand in vitro and transplant these enlarged cells into the location of the defect. This can be done either with or without a biomaterial scaffold. (27,25) This can also be achieved by the use of neocartilage, which is ‘generated from chrondocytes, obtained from juvenile cadaveric donors.' (28) These cells can then by scaled up from a single donor are able to treat multiple recipients are able to be avoid the T lymphocytes.
Up until recently, it was thought that were only two types of stem cells, embryonic stem cells and non embryonic stem cells, also known as somatic or adult stem cells.
If there is a defiency of chondrocytic cells, it is possible for ‘bone marrow derived mesenchymal stem cells' (MSC) can be induced to form the chondrogenic cells, if the stem cells are placed in the correct media and the specific growth factor ‘TGF Beta' is given to the cells. (19) If the cells are subjected to different media and growth factor, the same cells can form osteogenic cells, responsible for the production of bone. Although MSCs undergo better chrondogenesis under certain conditions, it is also possible for chondrocytes to be developed from MSCs in the ‘periosteum, perichondrium, adipose tissue, placenta and fetal tissues. (19) Location of the cells is not the only factor affecting the ability of cells to produce cartilage; older chrondocytes have less ability to produce cartilage. Chondrocytic cell behaviour will also be affected by the incubation conditions and the ‘physical, chemical and biological factors applied to the cell.' (19) It is likely that the variables will affect the ability of the cell to survive, produce correct cartilage tissue and present ‘proper chondrogenic phenotype'. (19) The ideal conditions for MSC cells to form chondrocytes is a medium containing ‘1.5 x 10 4mg ml-1 ascorbic acid, 1ng ml -1 human recombinant TFG-B. (30) MORE DETAIL HERE
Cells require a specific habitat in order to regenerate, in the process of tissue engineering, this is a biomaterial scaffold. The scaffold allows the chondrocytic cells to ‘survive, multiply and produce extracellular matrix.' Scaffolds are also used to transfer the chondrocytes to the correct location. (19) The scaffold should be degradable, as over a long period of time, the cellular components will replace the scaffold and remain in place. These scaffolds, usually made from biomaterials need to be compatible with the ‘native tissue around the recipient site.' (19)
The biological scaffolds should be porous, in order for the cartilage extracellular matrix to allow the cells to migrate, a supply of nutrients to enter and when dealing with bone the formation of blood vessels. (25) Scaffolds may have issues attaching themselves to the area of defect, in order to overcome this problem by either attaching cell adhesion proteins or adding ‘hydroxyl, carboxyl, or amine groups'. Cell adhesion proteins can be used to specifically attach the scaffold to a certain type of cell; where as the smaller molecules are more appropriate for the attachment of biological molecules including proteins. Cell differentiation and metabolism may increased by using growth factors, present in the scaffold.
Natural products can be used as a scaffold to deliver the cells. Many of these products are hydrogels. Hydrogels can be injected in the liquid form and they will mix with the chrondogenic cells. (19). Hydrogels have the advantage that after injection into the recipient site, they will gelate and set into any size and shape of defect in cartilage. (19). Fibrin can be used to ‘adhere other engineered cartilage to the site of the defect', a scaffold in its own right, or as a growth factor. There is limited use of fibrin due to the possibility of ‘provoking an immune and inflammatory response' and the lack of ability for host cells to migrate, and the lack of beneficial mechanical properties. The problems with immunogenicity will occur for all non native compounds, therefore for the best biocompatibility, native substances found in joints should be used. Collagen would be an excellent biological scaffold, but unfortunately has pitfalls due to the fact it is only produced in living creatures, causing it to be expensive and the transmission of disease borne in collagen. There may still be an immunogenic response as well if animal collagen is used in humans, leading to the disturbance or destruction of implanted cells. (19) Other examples of natural products include chitosan, and hyaluronan. Chitosan a polysaccharide which produces ‘a minimum inflammatory response. It can be prepared as a injectable liquid which at body temperature will set to a gel. (21, 19)Hyaluronon is found in the native joint and helps to make up both synovial fluid and the extracellular matrix.
Although they may cause more immunogenic responses, there is also many advantages to synthetic polymer scaffolds. These include reliable production sources, variation and flexibility in the design and manufacture stages. Commonly used synthetic scaffolds include poly-alpha-hydroxy esters, with polylactic acid and polyglycolic acid being the most popular. These have clinically approved in the US and are routinely used in surgery. (19) These polymers can make better scaffolds then natural products as they have better mechanical properties including strength. They are more resilient to stress caused by motion in the joint and is it much simpler to attach them to the recipient site. (19. 23) The degradation rate of the polymer is an important balance, the scaffold must stay intact long enough to act as a scaffold, but must degrade before it hinders in the regeneration of tissue. The content of the biomaterial and the structural architecture will affect the cells which are seeded on the scaffold. Due to the density of chondocytes and collagen in different locations of the joints, with chondocytes near the join between cartilage and bone and collagen nearer the articular surface. It is beneficial for growth of seeded chondocytes if the scaffold mimics the natural environment.
Another potential for the regeneration of cartilage is the use of osteochondral repair. This method has the advantage of preventing the growing cartilage from detaching whilst growth is occurring and providing the chondral phase with a rigid support and allows it to ‘secure to the recipient site by press fit. (19) This is known as hydrid scaffold and contains a biphasic scaffold, containing a compartment for cartilage regeneration and a separate compartment for bone and allows bone and cartilage to be generated separately. (25)
During the engineering of cartilage, the chrondocytes or MSC stem cells must be able to produce new extracellular matrix in order to help treat Osteoarthritis. Optimum conditions for the chondocytes will incease the cellular presentation, and will be affected by factors such as ‘friction caused by surface motion, compressive stress, oxygen tension, hydrostatic force and dynamic mechanical stimulation. In order to get the ideal environmental conditions for optimum conditions, a bioreactor should be used. TALK ABOUT BIOREACTORS. (19)
If MSC cells are used instead of autogenous chondrocytes, more factors are required; in order to induce the cells become chondogenic. This process is done with the introduction into the media of cytokines and growth factors, including TGF-beta, which works by affecting cells ‘through transmembrane receptor complexs and intracellular pathways'.(19) Other growth factors which are enhance the production of cartilage include ‘bone morphogenetic protein-2, and insulin-like growth factor. (34) TALK ABOUT OTHER GROWTH FACTORS. Not only proteins can work as growth factors to induce chondrogenesis, ‘prostaglandin E2, thyroxin, 1.25dihydroxy vitamin D can all work in this way. (24) These compounds have the advantage of proteins of longer half life and therefore can work for several weeks.
Articular cartilage transplants can a distinct advantage over types of organ transplantation. It is an immune privileged structure, and this means it can be successfully be transplanted without eliciting an immune response and therefore be rejected. Immune responses are caused T lymphocytes can normally distinguish between self and non self. This is caused by the recipients T cells have either direct contact with the cell surface molecules or be presenting an antigen on the recipients APC, antigen presenting cells. (27) This immune response has been stimulated by the ‘T Cell receptor ligation and the engagement of CD28. This reaction is co-stimulated by the molecules of b7-1(CD80) and B7-2 (CD86) (28,29) Cartilage does not have this problem and therefore can be transplanted without having to prescribe anti-rejection therapy, which would occur with other transplants; this means that the allograft has increased survival. It does not require anti rejection therapy, due to the lack of APC cells and the inability of chondrocytes to produce an immune response. (28) The survival of allograft is further improved by the lack of vascular system to the cartilage, will limit the access of the cartilage to leukocytes.
Although, still in its infancy, there is research being carried out in the uses of gene therapy into the treatment of osteoarthritis. The area of interest in osteoarthritis is using cells for the production in-vitro of proteins required for therapy. (32,10) The therapy would aim to ‘stimulate the expression of genes involved in the tissue regeneration process' and the targets for such therapies would include the TGF superfamily, IGF-1, Sox family, PGF-3 and SMADs. (33,34,10)