Biol 501 – Hot Topics – Flashcards

Kobe did it, A-rod did it and even pope jean paul the second did it. It is a controversial new therapy that some are calling the end of pain. A treatment that is still untested in north America that uses the patients own blood to cure everything from bad knees to tennis elbow. Today my first topic will be on the treatment of osteo arthritis through autologous condition serums such as Platelet Rich Plasma therapies and Orthokine therapies

The Regenokine Program is a non-surgical treatment program used to treat Osteoarthritis, joint pain, low back pain and some muscle and tendon issues. The Regenokine Treatment Program uses your bodies own natural healing components to resolve pain, improve mobility, restore function and reduce inflammation in your joints. What you need to know about Regenokine: • Non-surgical treatment program • Uses your bodies own natural healing components • No restrictions on activity or movement during treatment • Reduces pain • Increases mobility • Helps to restore healthy joint function • Protects and preserves healthy cartilage • Results last for up to 2-4 years • Safe for use in all joints (all peripheral joints and spine) • Effective in 75% of people Some of the worlds best athletes are using it to stay in the game but what about the rest of us?

Osteo Arthritis is the most common cause of chronic joint pain affecting over 25 million Americans today, with its only primary symptom being pain. It commonly affects large weight bearing joints and those that undergo large amounts of mechanical stresses giving the body that wear and tear affect. 2 types of osteo arthritis exist. The first being type 1 coming from chronic wear and tear of life's activities and the second being type 2, which comes from acute injuries to the joints.

Its important to know that primary osteo arthritis has no single cause but is rather the end result of a variety of things which lead to the structural and functional degeneration of the joints It involves the progressive loss of cartilage within the joint. The cartilage tries to repair itself, the bone remodels, and the underlying subchondral bone hardens. This process has several phases. In addition to pain cause by friction due to the lack of articulating cartilage within the joint, adjustments within the joints can also occur, which cause the pinching of nerves. In an attempt to shift and adjust the area that is bearing the weight, bone remodels itself to form osteophytes or extensions of the bone, however the end effect often results in the pinching of nerves thus leading to more pain. this occurs in examples such as osteo-arthritis of the back and slipped or herniated discs which aggravate the nerves. One underlying cause of the degeneration however is from chronic inflammation of the affected joint.

Repetitive use of the worn joints over the years can irritate and inflame the cartilage, causing joint pain and swelling. Loss of the cartilage cushion causes friction between the bones, leading to pain and limitation of joint mobility. With aging, the water content of the cartilage increases, and the protein makeup of cartilage degenerates. Eventually, cartilage begins to degenerate by flaking or forming tiny crevasses. In advanced osteoarthritis, there is a total loss of the cartilage cushion between the bones of the joints. Chronic Inflammation of the joint as I stated earlier is generally the underlying symptom that presides with these 2 factors (that being ageing and repetitive use)

1: A normal or unaffected joint demonstrating the articular surfaces and the joint space. 2: The stationary phase of disease progression in osteoarthritis involves the formation of osteophytes or joint space narrowing. 3: Osteoarthritis progresses further with obliteration of the joint space. 4: The appearance of subchondral cysts (cysts in the bone underneath the cartilage) indicates the erosive phase of disease progression in osteoarthritis 5: The last phase in the disease progression involves bone repair and remodeling. Large osteophytes form. Goal of Osteoarthritis Treatment The overall goal of treatment is the appropriate treatment of pain to help people regain their mobility and joint function.

Platelet Rich Plasma (PRP) Therapy is a non surgical injection therapy using a concentrated mixture of the patients own blood. PRP treatments have the potential to relieve pain and accelerate the healing of damaged tissue and joint structure. The patient's blood is drawn and placed in a centrifuge causing the separation and sedimentation of the blood to separate out the platelets and other growth factors. The layer of platelet-rich plasma is then injected into the diseased portion of the tendon. In Regenokine however the layer rich in inflammatory cytokines is selected and injected into the diseased area.

There are many different forms of preparing the platelet rich plasma however in the study I'm citing they ... Took a venous sample of peripheral blood from the patients arm and centrifuged it 2 times 1st time at 1800 rpm's to separate from erythrocytes 2nd time at 3500 rpm's to concentrate the platelet content (A) Withdrawal of the patient's peripheral blood (B) Centrifugation to obtain a concentration of platelets and cytokines. (C) The centrifuged product is stratified into 3 layers. The end products yields concentrations of platelets and leukocytes that are over 6 times above normal. Muskuloskeletal ultrasound is then used to properly identify the area of injury. then with the use of a local anesthetic the PRP is injected into the diseased area. The treatment is not a quick fix and is designed to promote long term healing of the injured tissue, regeneration of the cartilage collagen takes up to 4-6 months and may require multiple injections (with most cases needing 2-3 treatment sessions occurring at intervals of 4-6 weeks).

Platelets are typically involved as a homeostatic sealant or as a plug to stop bleeding. They can also however be used as a Scaffold for tissue regeneration, Growth factor concentration, and production, as well as Stem cell binding. In a peripheral blood smear you can see that platelets can be found scattered amongst the rest of the blood components. In platelet rich plasma however the platelets are seen to be much more concentrated.

now why is this important? once platelets become activated the lose their spherical shape and begin producing a variety of growth factors or cytokines which increase linearly with platelet concentration meaning the more platelets that are present the greater the number of growth factors as well. Growth Factors perform a variety of functions within the body one of which is Chemo Taxis, which is a directional movement in response to a chemical stimulus. Stem cells are attracted to these growth factors and then migrate into the area and proliferation or cell division then occurs

The growth factor binds to the stem cell membrane and stimulates cell division when the growth factor binds to a cell receptor is triggers intra-cellular changes causing a signalling cascade ending with a change in the function of the mesenchymal stem cell cell directing its growth into the desired cell type needed for the healing process and the regeneration of the tissue

PDGF or Platelet Derived Growth Factor which is chemoattractive for WBC's and Mesenchymal Stem Cells - 17ng/ml (5 fold increase over whole blood) attracting these cells allows neo-genesis of new healthy cells to replace the old and damaged ones. TGF-B or Transforming Growth Factor- beta, Promotes cell mitosis, or cell division and also Significantly increases type 1 collagen production - 42ng/ml (3.6 fold increase over whole blood) the increased profliferation caused by TGF accelerates the repair process as the newly replicated cells replace the old damaged cells. VEGF or Vascular Endothelial Growth Factor Stimulates angiogenesis or blood vessel formation - 955pg/ml (6fold increase over whole blood) the formation of blood vessels is important as cartilage generally has poor blood supply inhibitting much needed levels of healing factors from arriving thus preventing the healing process from occurring.

Regenokine is a patented method developed by molecular biologist Dr. Julio Reinecke and Dr. Peter Wehling exposure of blood to non-pyrogenic surfaces (e.g. medical-grade glass spheres) selectively elicite a vigorous, rapid increase in the concentration of anti-inflammatory cytokines, specifically IL-1Ra, resulting in higher concentrations of those factors in human blood serum. In Regenokine, According to this method, the process removes about 2 US fluid ounces (59 ml) of blood from a patient's arm and the venous blood is drawn into syringes containing pre-treated glass. Incubation of the blood reproducibly stimulates peripheral blood leukocytes to produce elevated amounts of endogenous anti-inflammatory cytokines (see Table 1). Table 1 also shows an amount of growth factor content in the ACS. There is little to no detectable induction of proinflammatory IL-1β or TNF-α. Significantly, when the blood is incubated with glass beads, extensive cell death does not occur, haemolysis is mild and not clinically relevant and the major serum protein composition is not affected. After incubation, the blood is centrifuged and the serum is extracted (see Figure 1). The resulting product, known as ACS, is then portioned and either stored frozen until use or immediately injected into the affected region of the patient. Injections are given once or twice weekly in a series of three to six (for spinal and muscle injuries) or six to eight (for joint diseases). Incubation at a slightly raised temperature, to help induce a feverish state thus increasing the concentration of anti interluekin factors by 100 fold. The liquid is then placed in a centrifuge until its constituent parts are separated. The middle yellowish layer is dense with agents that are believed to stop an arthritic agent known as interleukin-1, which causes degeneration of the joints and the breakdown of cartilage.That serum is injected into the patient's affected area. The procedure aims to reduce pain and discomfort in the joint. The treatment generally lasts five days, with six shots of the serum into the affected area. It is normal for a patient to receive annual injections to ease the joint discomfort. A two-year study of osteoarthritis of the knee, published in the medical journal Osteoarthritis and Cartilage, confirmed the safety and effectiveness of the therapy. Orthokine is less invasive than most, if not all, other forms of knee surgeries available.It focuses on treating the inflammation as opposed to mechanical problems in the joints

Both chronic and extreme inflammation are associated with disruptions of anabolic signals initiating muscle growth. Chronic inflammation has been implicated as part of the cause of the muscle loss and cartilage degradation that occurs with aging By blocking the biological activity of IL-1, including inflammation and cartilage degradation associated with rheumatoid arthritis, by competitively inhibiting the binding of IL-1 to the Interleukin-1 type receptor. IL-1 is produced in response to inflammatory stimuli and mediates various physiologic responses. IL-1 additionally stimulates bone resorption and induces tissue damage like cartilage degradation as a result of loss of proteoglycans. In patients with rheumatoid arthritis the natural IL-1 receptor antagonist is not found in effective concentrations in synovium and synovial fluid to counteract the elevated IL-1 concentrations in these patients. IL-1RA is a member of the interleukin 1 cytokine family. IL1Ra is secreted by various types of cells including immune cells, and is a natural inhibitor of the pro-inflammatory effect of IL1.

German Orthokine Osteoarthritis Trial: The long-term results of a Randomized Controlled Trial (RCT) regarding the clinical efficacy and safety of ACS. Patients were randomised to receive a series of intra-articular (IA) injections of ACS, hyaluronan or saline over a period of three weeks. The study included 376 patients with pain rated no less than 50-100mm on a visual analogue scale (VAS). Efficacy was assessed by patient-administered outcome instruments and results were calculated in an intention-to-treat (ITT) analysis. Patients who participated in the trial were invited to return to the study centre after two years for follow-up by a blinded observer. To allow for pain severity to be accurately evaluated, analgesic and anti-inflammatory medications were discontinued prior to the start of treatment. In all three treatment groups, IA injections produced a significant reduction in VAS scores; however, responses to ACS were significantly stronger. The ACS group had the largest reduction in VAS. Weight-bearing pain in the ACS group decreased from a mean of 70.2 to 30.3mm. The mean decrease in pain for patients treated with HA and placebo was less than half that in the ACS group. The treatment difference between ACS and HA or placebo was over 20.0 mm. Two-year Results All three groups showed significant improvements in OA symptoms as measured by the VAS. After two years, the effects seen in the ACS group were still significantly stronger, with the greatest reduction of symptoms and lowest pain scores. The number of patients who needed further treatment after the initial injection series was lowest in the ACS group. Although patients who had received pharmacological or surgical treatment for their OA after the initial series of injections were not included in the assessment of symptoms, the number of patients who fell into this category was lowest in the ACS group.

Orthokine differs from platelet-rich plasma (PRP), as platelets are targeted instead of the interleukin antagonist. Also, PRP does not require the blood to be heated as Orthokine does. which increases the anti-inflammatory proteins by as much as 100 times. Additionally PRP does not require the use of glass beads or to be incubated, as this incubation period serves purely to produce elevated amounts of endogenous anti-inflammatory cytokines While Platelets are sought out to speed the healing process, Orthokine treatments are used to reduce the damages cause by chronic inflammation to the affected joint. The platelets produce a cocktail of Cytokines and Growth Factors, which trigger and accelerate the regeneration of chronic tissue problems, such as inflammation and degeneration Where as Regenokine and other Autologous condintioned serum therapies use anti inflammatory cytokines to inhibit inflammation as a whole allowing the body to heal itself on its own, and reduce pain to a fraction of what it was overall one is focused on joint regeneration and the other is focused on inflammation and pain control

Wrong method of biotechnological preparation Leading to undesired effects and side effects of the therapy in treatments that are prepared improperly Inflammation being a side affect from some of these potentially "botched" treatments Long term effects As a new treatment the long term effects of PRP are still yet unknown leading to questions of potential complications Vague terminology Thought to be a potential hindrance to differentiating between the various products and their respective protocols such as Speed of centrifugation Number of centrifugations Use of anticoagulant Presence of leukocytes in preparation Use of activator all of which alter the biological out comes and which may differ from your desired result going in to the treatment. overall both therapies are ones that are Strong on logic yet weak on any hard long term evidence

Safe due to its autologous nature Minimally invasive 3 step treatments shown to have effectiveness up to 4 years Minimal recovery time Applicable to several other injuries and or conditions PRP and regenokin are autologous blood products with high concentrations of blood components sought to help with osteo arthritis

Biological scaffold materials composed of extracellular matrix (ECM) have been shown to facilitate the constructive remodeling of many different tissues part of animal tissue that usually provides structural support to the cells and includes the interstitial matrix and basement membrane

Extracellular matrix is a structure which is commonly retrieved from the lining of the small intestine (SIS), or bladder (UBM) of a pig. This lining, once processed to remove all of the pig cells, leaves only a complex weave of collagen which is common to all humans and animals. Proteoglycans - help to trap and store growth factors within the ECM Heparan sulfate - Stimulates angiogenesis, blood coagulation Chondroitin sulfate - contribute to the tensile strength of cartilage, tendons, and ligaments Keratan sulfate Hyaluronic acid - ability to resist compression by providing counteracting turgor force and Regulates cell behavior healing processes, inflammation and tumor development

Collagen - give structural support to resident cells Elastin - give elasticity to tissues Fibronectin - connect cells with collagen fibers in the ECM, allowing cells to move through the ECM help at the site of tissue injury by binding to platelets during blood clotting and facilitating cell movement to the affected area during wound healing Laminin - web-like structures that resist tensile forces in the basal lamina and also assist in cell adhesion

ECM scaffolds consist of the structural and functional molecules secreted by the resident cells of each tissue. Therefore, the specific composition and distribution of the ECM constituents will vary depending on the tissue source. By dry weight, SIS (small intestinal submucosa)-ECM scaffold is composed of greater than 90% collagen glycosaminoglycans (GAGs), including heparin, heparan sulfate, chondroitin sulfate and hyaluronic acid. The amount of GAGs remaining in a tissue after decellularization depends greatly on the method of decellularization adhesion molecules such as fibronectin and laminin

ECM exists in all tissues and organs but can be harvested for use as a therapeutic scaffold from relatively few sources. Porcine derived ECM scaffolds that have not been modified, except for the decellurization process and terminal sterilization How it is applied to the target site: After sterilization and testing, theECM becomes an exceptionally strong but a very pliable, thin sheet of collagen that can be stitched onto the human heart and tissues to stimulate healing and re-growth. It acts as a sort of scaffolding onto which the patient's own cells grow to cover the collagen matrix. The result of this regenerative healing is a permanent repair with the patient's own tissue.

ECM elicits a host response that promotes cell infiltration and rapid scaffold degradation, deposition of host derived neomatrix, and eventually constructive tissue remodeling with a minimum of scar tissue. Organize cells into tissues by endogenous recruitment and using the cells provided in the composition Co-ordinate the function of the recruited cells and also allow for cell migration within the matrix Allow and provide normal metabolism of the cells once the vascular supply bringing nutrients is established Establish signal transduction pathways for growth differentiation, proliferation, and gene expression One thing we know from matrix biology is that a cell cannot survive if it is not attached to a healthy local matrix. so with out a doubt, no matter what type of cell or what organ it is, if the donor cell does not have a healthy matrix to adhere to it simply wont survive

read off slide shows a histological slide of an esophogeal scaffold part way through its intergration process with the body that had been transplanted 1 - There is an intact but not entirely normal appearing squamous epithelium, 2 - a lack of normal complement of submucosal glands, 3 - partially organized bundles of skeletal muscle and tissue organization that resembles the normal laminar arrangement of tissue types found in the esophagus. Of note, there is a lack of inflammatory cells or scar tissue, and there is no histologic evidence of the originally implanted scaffold.

Been Shown to have Many other biomedical applications Tissue regeneration Hair loss reversal Surgical patch Arterial replacement Treatment of Sclera

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ECM is a network of non living tissue that provides structure and support to the cell as well as providing a pathway for communication. Additionally ECM is very valuable in the healing process. The ECM represents nature's scaffold for tissue development and tissue repair