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Orthobiology

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Ortho: to make straight or right. The use of biologic substances to prompt, stimulate or support a “healing event” within the body.The use of biologic substances to promote healing or reduce pain.The use of platelets and stem cells in treatment and management of musculoskeletal conditions

Transcripción

  1. 1. ORTHOBIOLOGY ;
  2. 2. ONCE UPON A TIME... Wednesday, 4 June 2 3000 BC : As perMahabharata,Adi Parva, chapter 114,shloka17, theKauravawerecreatedby splittingthe singleembryointo101partsandgrowing eachpartina separate kund or container. It is believedthatKaurva’s birthmentionedhereisfirst reportedmentionofwhatcould bepossibleuseof embryonic stem cell.
  3. 3. Orthobiologics: • Ortho: to make straight or right • The use of biologic substances to prompt, stimulate or support a “healing event” within the body • The use of biologic substances to promote healing or reduce pain • The use of platelets and stem cells in treatment and management of musculoskeletal conditions
  4. 4. ◦ Within orthopedics, focal chondral lesions, osteo- arthritis (OA), fracture healing, and soft tissue repair (tendon to tendon interface, tendon to bone interface, meniscus, muscle, and ligaments) are fertile fields for biological therapies. ◦ Biological and surgical treatments have been proposed to treat these pathologies. Biological treatments include ◦ platelet-rich plasma (PRP), ◦ bone marrow aspirate concentrate (BMAC), ◦ and cell-based therapies. ◦ Surgical interventions include marrow stimulation procedures and stem cells implanted in matrix
  5. 5. BMP’S IN ORTHOPEDICS ◦ Bone Morphogenic Proteins (BMPs) are a group of molecules which induce mesenchymal stem cells to differentiate into bone forming cell lines that form new bone. ◦ They are a group of noncollagenous glycoproteins that mostly belong to the transforming growth factor-beta (TGF-b) superfamily. ◦ BMPs have been widely used in the field of orthopaedics since the last two decades. ◦ BMPs are a group of osteoinductive proteins that are usually extracted from bone matrix which are responsible for skeletal regeneration and bone healing.
  6. 6. ◦ The primary functions of BMPs are to activate inactive mesenchymal stem cells and to differentiate them into osteoblast, chondroblasts and fibroblasts. ◦ Apart from the above functions, BMPs are also involved in other physiological and pathological processes such as inflammatory response, bone formation and resorption, growth signalling pathways, oncogenesis and immune response. ◦ The safety and efficacy of BMP as bone graft substitute are influenced by its purity, local effects, systemic effects, immunogenicity and biocompatibility.
  7. 7. ◦ Bone ossification occurs through both intramembranous ossification and endochondral ossification ◦ Both cellular events of intramembranous ossification and endochondral ossification involve MSCs which may be bone marrow derived or periosteum derived. ◦ These primitive MSCs are pluripotent progenitors that can differentiate into both osteoblasts and chondroblasts. ◦ This differentiation is regulated by molecules such as BMPs and Fibroblast growth factor (FGF).
  8. 8. Classification of BMPs • Till date more than 20 BMPs have been identified in vertebrates. • Based on structural homology, BMPs can be further classified into severa subgroups which includes BMP-2/- group, BMP-5/-6/-7/-8 group, BMP-9/ 10 group and BMP-12/-13/-14 group.
  9. 9. Functions of BMPs ◦ The actions and functions of BMPs depend on various factors which are: ◦ 1. Type of target cell. ◦ 2. Maturation phase of target cells. ◦ 3. Local concentration of BMPs. ◦ 4. Other biological signals.
  10. 10. Functions of BMPs ◦ BMPs are mitogens (growth factors) that stimulate the production and multiplication of morphogens (differentiation factors) that transform connective tissue cells into osteoprogenitor cells. ◦ They play a pivotal role in embryonic development through specification of positional information in the embryo. ◦ They regulate growth, differentiation, chemotaxis and apoptosis of cells such as epithelial, mesenchymal, haematopoietic and neuronal cells. ◦ Apart from inducing bone formation through intramembranous or endochondral ossification, they also induce osteoclasts leading to bone resorption.
  11. 11. Carriers of BMPs • BMP is a water soluble molecule which diffuses into the body fluid easily. • When administered alone, most of the protein molecule is lost rapidly due to diffusion and irrigation. • To negate these effects and have a prolonged localized effect at target site, BMPs are administered in a carrier. • Type I collagen is the most preferred carrier used for transport of BMPs.
  12. 12. Clinical Applications in Orthopaedics ◦ 1. BMP as an alternative to autograft for non-unions Long Bones – ◦ Currently, US Food and Drug Administration (FDA) approval of BMP use in long bones is limited to tibia non-unions as alternative to autograft. ◦ Pseudoarthrosis of tibia remains to be the most frequent site of long bone non-union due to its poor muscular coverage, high incidence of open injuries and its anatomical peculiarity. ◦ Friedlaender et al initially reported the efficacy of BMP-7 in the treatment of tibia non-unions. ◦ It was concluded that BMP-7 provided comparable results when compared with bone autograft, without donor site morbidity.
  13. 13. Scaphoid ◦ Scaphoid is very commonly affected with non-union due to its precarious vascular supply. ◦ Patients treated with autograft and BMP-7 healed successfully within 3 months ◦ However, all the evidences on scaphoid non-unions were poor. ◦ SPINE ◦ By far, spine is the most common site associated with BMP application. ◦ US FDA has approved the usage of rhBMP-2 in anterior lumbar interbody fusions. ◦ However recent studies have shown that BMP use has been extended to fusions performed with other approaches as well.
  14. 14. ◦ BMP as an alternative to autograft for fusion Foot and ankle – ◦ Few authors have used BMPs to improve the rates of union in ankle arthrodesis for patients with comorbidities affecting bony union. ◦ Initially use of BMP in complex foot and ankle arthrodesis seem to be a safe option considering its high failure rates. ◦ However, there is no randomized control trial available till date to give a suggestable conclusion.
  15. 15. Platelets ◦ Platelets develop from megakaryocytes in the marrow. ◦ Each megakaryocyte can produce 5000- 10000 platelets. Approximately 2/3rd of the platelets circulate in the blood and 1/3rd are stored in the spleen. ◦ An average healthy adult can produce 1011 platelets per day. ◦ The normal platelet count is (150– 400) × 103 per microliter of blood. ◦ The diameter of a mature platelet is 2-3 5μ. ◦ Platelets usually remain alive for 5-11days. ◦ Old platelets are destroyed by phagocytosis in the spleen and liver by Kupffer cells
  16. 16. + What is the response to platelets • Proliferation of Mesenchymal stem cells • Proliferation of fibroblasts • Production of Type 1 Collagen • Platelets itself release Growth factors
  17. 17. Healing pathway
  18. 18. PLATELET RICH PLASMA ◦ By definition, Volume of plasma fraction of autologous blood having a platelet concentration above baseline. ◦ To be labeled as PRP, a platelet count of 3-5 times of the baseline should be present in the platelet concentrate(many studies like Max et al mention the platelet count 1 million as standard valve for PRP). ◦ Platelets begin secreting these proteins within 10 minutes of activation. After initial release of growth factors, the platelets synthesize and secrete additional factors for the remaining days of their life span.
  19. 19. ◦ In humans, the normal platelet count in whole blood ranges from approximately 150,000 to 350,000/μL , ◦ whereas platelet- rich plasma is often defined as at least 1,000,000 platelet/ μL suspended in plasma. ◦ In order to ensure that the platelets are suspended and do not form a clot, PRP must be made from anticoagulated blood. ◦ The logic behind PRP is that platelets are the first to arrive at the site of tissue injury, and thus have the potential to release growth factors that play a critical role in mediating healing
  20. 20. History ◦ The term PRP first used by KINGSLEY ◦ In 1970, MATRAR used fibrin glue in rats ◦ 1998 –PRP in regenerative medicine for bone healing ◦ 2001- 2nd generation platelet concentration identified
  21. 21. ◦ Physicians report that the demand for PRP has soared after pro golfer Tiger Woods received injections to accelerate healing after knee surgery.”
  22. 22. What is PRP ◦ Platelet Rich Plasma ◦ Utilizing growth factor (GF) content of platelets to aide in healing of musculoskeletal tissue ◦ Predominately tendons, ligaments and muscles ◦ High concentration of GF deposited locally in the area of an injury ◦ Anabolic effect enhances and supports healing
  23. 23. + What does it lead to?? • Chemotaxis • Directional movement in response to a chemical stimulus • Stems cells are attracted to the growth factors and migrate into the area • Cell proliferation • Significant increase in cellular reproduction activity • Possibly even a systemic effect • Performance enhancing?
  24. 24. PRP – MECHANISM OF ACTION 1st Mechanism: • Delivery of Growth Factors 2nd Mechanism : • “Up regulation” of local GF in tendons • TGF-Beta1 for 1st week post prp, • Increased IGF-1 for 4 weeks tenocytes post prp
  25. 25. 3rd mechanism: ◦ PRP Stimulates HA Release • PRGF significantly enhanced HA secretion compared with platelet- poor preparations 4th mechanism : ◦ Chemo-attractant for Stem Cells • Prompt Migration and Proliferation
  26. 26. Platelets ◦ Although platelets play a key role in haemostasis, they are central in mediating the anabolic effects of PRP by virtue of releasing growth factors stored in their alpha granules. ◦ During the initial phases of wound repair, activated platelets attract and foster cell migration into the wound by aggregating and forming a fibrin matrix. ◦ This matrix then serves as a tissuescaffold for sustained release of platelet growth factors and cytokines, which stimulate cell recruitment, differentiation, and communication
  27. 27. 2. Leukocytes ◦ Leukocytes are essential mediators of the inflammatory response, host defence against infectious agents, and wound healing. ◦ Neutrophils are involved in the inflammation phase of wound healing. ◦ Monocytes and macrophages facilitate tissue repair by debriding and phagocytising damaged tissue and debris. ◦ Similar to platelets, macrophages also secrete growth factors that are important in tissue repair and have been shown to contribute to subchondral bone regeneration.
  28. 28. 3. Red blood cells ◦ Red blood cells (RBC) content is typically reduced or absent in PRP because of the centrifugation process. ◦ Destructive process is thought to occur in human synoviocytes treated with RBC concentrates, leading to significantly greater cell death and cartilage degradation.
  29. 29. Classification of PRP Dohan et al in the year 2009 classified PRP based on presence of leukocytes and density of fibrin network
  30. 30. Mishra et al in the year 201 proposed a classification system which is based on platelet count, activation and leukocytes for clinical application in Sports medicine
  31. 31. + Advantages of having leucocytes?? Pure PRP has an advantage over traditional PRP in that it eliminates red blood cells (RBCs) and neutrophils. RBCs, have no therapeutic effect and more painful while injecting. Neutrophils, a type of white blood cell, have inflammatory components which may increase pain and inflammation post-treatment.
  32. 32. ◦ Equipment required for processing 40ml venous blood (figure1) 1. Centrifuge (Remi R-4C), 4000rpm with countdown timer fixed angle rotor, capacity 4x50 ml 2. First centrifugation (soft spin) : 2x20cc syringe, 6ml ACD-A (Acid citrate dextrose), 2xBD insulin syringe 3. Second centrifugation (hard spin): 20cc syringe, 3-way cannula, BD insulin syringe 4. PRP extraction: 10cc syringe, 3-way cannula.
  33. 33. STEP 1 ◦ Three ml ACD-A was taken in 20ml syringe and 17 ml blood drawn from the ante cubital vein. Blood (0.3 ml) was sent for initial platelet count. ◦ Nozzle of the syringe was blocked firmly with the cap of the insulin syringe. With a heavy scissors flanges on the either side of the syringe barrel were cut. ◦ The piston was cut leaving one inch projecting out of the barrel. ◦ The two syringes were placed in centrifugation tubes opposite to each other with the tip pointing upwards. ◦ This will help balance the centrifuge and minimize vibration. ◦ The first spin was set at 1500rpm for 15mins without brake (Soft spin).
  34. 34. STEP II ◦ After soft spin blood in the syringe was separated into three layers. ◦ The bottom layer comprises of RBC, the middle layer leucocytes and the top layer, plasma with platelets in suspension. ◦ The serum was drawn into the second 20cc syringe through the 3-way cannula. Care was taken to avoid aspiration of the buffy coat and RBC. ◦ The combined volume of plasma from both syringes ranged between 16-18ml. ◦ The syringe was again capped and modified to fit the centrifugation tube. ◦ The second spin was set at 3500rpm for 7minutes (Hard spin).
  35. 35. STEP III ◦ At the end of second spin the syringe contains Platelet Poor Plasma (PPP) on top and platelets along with scanty WBC at the bottom in the form of a pellet. ◦ The supernatant plasma is drawn and discarded leaving about four ml at the bottom which is reconstituted to form PRP fraction. This is sent for culture and counts.
  36. 36. RESULTS
  37. 37. PRF ◦ Under strict aseptic conditions, ten to twenty millilitres of patient's blood was taken and centrifuged at 3000 rpm for 15 min. ◦ PRFM was separated from red corpuscles at the base using a sterile forceps and scissor, preserving a small RBC layer measuring around one mm in length, which was transferred onto a sterile gauze
  38. 38. ◦ Middle membrane so obtained was compressed between two gauze pieces gently and applied on a healthy wound followed by application of a secondary non-absorbable dressing. ◦ The secondary dressing of the patient and the dried PRFM was removed from the wound bed after a minimum of five days. ◦ The same procedure was repeated every week for six weeks.
  39. 39. + Where can it be used??  Everything and anything!  Internet marketing for host of ailments  Snake oil of today “Cures Rheumatism, Sports injuries, Grows hair, Gets rid of wrinkles, etc., etc.”
  40. 40. +  Meniscus repair  Tendonitis (office based injection)  Arthritis  Ligament surgery  Tendon repairs  Acute injury – Debatable!
  41. 41. +  Epicondylitis, Achillodynia  Runners / Jumpers Knee  Plantar fascitis  Osteoarthritis (Grad I-III)  Muscle- and Ligament Lesions - Ultrasound-Guided Injections  Peri – Post operatively - Achilles Tendon Repair - HTO, Microfracturing - Meniscal Suture, RC Repair etc
  42. 42.  Analgesic?  Potential primary analgesic effect  Some human studies state decreased post-op pain levels  Stimulation of thrombin receptors (ie, PAR-1) shown to increase pain threshold in laboratory animals through opioid pathways and haemostasis  Antimicrobial  Against Staphylococcus aureus (Sutter 2012)
  43. 43. Arthritis What happens in an arthritic joint?
  44. 44. • Cartilage wears out • chondrocytes unhealthy/poor quality matrix - breaksdown • Poor lubrication - hyaluronic acid/lubricin (SZP -Proteoglycan 4) • increased friction - wears out Inflammation - pain
  45. 45. Arthritic Joint - The “BAD” Molecules • MMP’s - matrix metalloproteinases - collagenases, gelatinases, stromelysins - degrade collagen, proteoglycans, elastin, etc. • IL-1, TNF - interleukins - inflammatory - enhance MMP’s • ADAMTS - disintegrins - prevent platelet function • CATABOLIC - BREAKDOWN
  46. 46. What About “GOOD” Molecules? • TGF - tissue growth factor • TIMP-1&2 - tissue inhibitor of metalloproteinases • IRAP - interleukin receptor antagonist protein • A2M - alpha 2 macroglobulin (useless by itself • - so drug companies barking up the wrong tree) • Block the BAD molecules - • ANTI-CATABOLIC (ANABOLIC)
  47. 47. PRP (Platelet Rich Plasma) Reduces “BAD” Molecules Increases “GOOD” Molecules
  48. 48. What About “Traditional” Treatments? Are there problems? NSAIDS Corticosteroid Surgery (Physical Therapy ALWAYS GOOD!)
  49. 49. NSAIDS • Commonly taken • Multitude of side-effect Very significant problems • ulcers, bleeding, heart attack, stroke, kidney and liver failure • Inhibit chondrocytes and stem cells
  50. 50. Corticosteroids Injections • Commonly performed • Temporarily reduce inflammation • Inhibit healing • Cause damage to cartilage and soft tissue structures
  51. 51. PRP Intra-articular Pathways • Decreases BAD molecules - MMP, IL - reduces CATABOLIC PROCESSES • Increases GOOD molecules - TGF, TIMP, IRAP, A2M • REBALANCES the micro- environment of the joint - back to favoring the normal healing status
  52. 52. • Produced by synovial cells • Important for lubrication/viscosity of joint fluid • Hyaluronic Acid - Hyaluronan • Viscosupplementation -Synvisc, Orthovisc - from rooster combs • HA alone not enough to protect the joint PRP Stimulates Hyaluronic Acid Production
  53. 53. + Tennis elbow • Common Disorder • Repetitive activities – Most common cause – May occur from injury (acutely) • PRP Application Technique – Inject 2-3 cc of PRP into the ECRB--Peppering technique – Average Dose 3.3 million platelets per patient
  54. 54. Lateral Epicondylitis - PRP • PRP provides excellent and superior results • Results are longer lasting • Particularly better than corticosteroid injection Am J Sports Med 2006, 2014, J Clin Diag Res 2015, J Hand Microsurg 2015
  55. 55. Compared to Surgery: PRP - showed 83% improvement Surgery - showed 46% improvement PRP improved 2X as much as surgery SO WHY ARE WE STILL DOING SURGERY FOR THIS PROBLEM? J Orthopaedics March 2016
  56. 56. Planar Fasciitis PRP • more effective and durable than corticosteroid • 3X (300%) the functionality and improvement of pain • Actually heal structures - rather than mask Sx (Foot Ankle Int, April 2014)
  57. 57. Shoulder Impingement • Rotator cuff tendons weaken, develop tears, degenerative changes • Poor blood supply Unable to heal itself • Gets stuck in early phase of repair Chronic changes, inflammation, pain Poor healing environment
  58. 58. Shoulder Impingement NSAIDS – minimal benefit, inhibit healing Corticosteroids - if used - no longer see healing response - Br J Sports Med 2014 kill rotator cuff cells -Bone Joint Res 2014
  59. 59. Shoulder Impingement - PRP • PRP more effective than corticosteroid • in pain relief (30% better) • in ROM improvement (3X more improvement) • 16X less likely to undergo surgery (3/100 vs 48/100) O’Donnell, et al. AAOS 2013
  60. 60. + TendoAchilles
  61. 61. + Post Injection Therapy • Rest (sling/crutches) for a few days to a week • NO NSAID’S • Slow stretching program • No high loading activities until • No rest pain • Minimal tenderness
  62. 62. + COMPLICATIONS  No adverse complications have been reported  Symptoms may be worsened for few days because of induced inflammation  Infection  Persisting symptoms
  63. 63. + Limitations • Efficacy • Lack of clinical data • Number of studies being done • Indications are being developed • Uniformity of prep • PRP classification • Insurance recognition
  64. 64. Stem Cell • An undifferentiated progenitor/primitive cell • A: capable of renewing itself through cell division, sometimes after long periods of inactivity • B: Under certain physiologic or experimental conditions, it can be induced to become a tissue- or organ-specific cell with special functions
  65. 65. STEM CELL RESEARCH TIMELINE
  66. 66. STEM CELL CHARACTERISTICS Wednesday, 4 June 14 77 Undifferentiated cells with the capacity for unlimited or prolonged self-renewal that can give rise to differentiated cells. Self renewing for long periods. Unipotent /pluripotent /multipotent e.g. Intestinal stem cells produce one or more of the 4 classes of mature differentiated cells (absorptive, goblet, Paneth and enteroendocrine cells)
  67. 67. Sources of stem cells ◦ Adult Tissue: Hematopoeitic SC, Mesenchymal SC, Neural, muscle derived, Pancreatic, Hepatic, Epithelial, Dental. ◦ Fetal tissue ◦ Cord blood ◦ Embryos
  68. 68. BMAC ◦ In adult skeleton there are two types of bone marrow which are red marrow and yellow marrow. ◦ Yellow marrow is filled with adipose tissue and is inactive by function. Red marrow possesses hematopoietic cells and mesenchymal stem cells. ◦ Mesenchymal stem cells (MSC) have the capability to differentiate into cell lines like cartilage, bone, tendon, muscle and nerves. ◦ Benefits of BMAc are achieved either by proliferation and differentiation into variety of cell lineages or by elution of growth factors and cytokines to hasten the process of healing in tissues with attenuated healing potential.
  69. 69. Components of BMAC ◦ In normal bone marrow aspirate (BMA),common components present are erythrocytes(22-28%), neutrophils (32-37%),lymphocytes(13%), eosinophils (2.2%), blast cells(1.4%), monocytes (1.3), basophils(0.1%) and megakaryocytes in variable percentages. ◦ Apart from the above components, BMA contains mesenchymal stem cells and various growth factors. ◦ An adult bone marrow has about 0.001% to 0.01% of mesenchymal stem cells in 7-30 cells per million nucleated cells. ◦ Growth factors like PDGF, TGF-â, BMP2and7 and IL-1RA are also present in the BMA in variable concentrations
  70. 70. BONE MARROW CONCENTRATE Wednesday, 4 June 14 84 Enhances levels of Bone Marrow-BM Mesenchymal Stem MS) Cells BM-MS cells decrease with age Concentrating mimics the cell load and healing potential of young patients 4-5x progenitor cell increase above baseline
  71. 71. ◦ Main aim of bone marrow concentration is to increase the cell count of mesenchymal stem cells. ◦ The concentrations of MSC in bone marrow alone is relatively low, hence the aspirate is concentrated by centrifugation in order to increase the percentage of MSCs. ◦ Cell count in bone marrow aspirate is 612±134 per cm3 compared to bone marrow aspirate concentrate is 2579±1121 per cm3 which is a fivefold increase in the number of mono nuclear cells
  72. 72. Sources of BMAC ◦ Commonest source of bone marrow aspiration is from iliac crest, tibia or calcaneum. ◦ Among all of the sources,iliac crest yields highest concentration of mesenchymal stem cells. ◦ Graft is harvested bysector rule in iliac crest, which roughly divides the entire iliac crest into six zones. ◦ Of these six zones the posteriorcrest is preferred site as it contains higher concentration of MSCs (higher by 1.6 times compared to anterior iliac crest).
  73. 73. ◦ The patient can be positioned either supine or prone. In the supine position, BMAC is harvested from the anterior superior iliac spine or the iliac crest. In the prone position, BMAC is harvested from the posterior superior iliac crest region. ◦ Monitored anesthesia (conscious sedation), local anesthesia, or general anesthesia can be used for this BMAC harvest procedure, we prefer positioning the patient in the prone position with light sedation monitored by an anesthesiologist for access to the posterior superior iliac crest. ◦ After palpation of the bony landmarks, the procedural site is sterilely prepared and widely draped to ensure an adequate field.
  74. 74. • The BMAC aspiration kit is opened and then the battery-powered BMAC aspiration device is sterilely drape • A bone marrow aspiration kit (MarrowStim; Biomet Biologics, Warsaw, IN) is used for bone marrow aspiration • First the bony landmarks of the posterior iliac crest and sacroiliac joint are palpated. The skin is then injected down to and including the periosteum with 1% lidocaine without epinephrine.
  75. 75. • Then, the bone marrow aspiration trochar and needle are percutaneously inserted through the skin and subcutaneous tissues until it reaches the posterior iliac crest. • Then, manual pressure is used to position the bone marrow aspiration trochar against the dense cortical bone, attempting to center it over the middle of the posterior crest cortical walls. • The trajectory of the needle should be parallel to the iliac crest, or perpendicular to the ASIS or PSIS, depending on the harvest site used
  76. 76. • A battery-powered power instrument is then used to drill the trochar and needle into the medullary cavity of the posterior iliac crest • After the trochar is inserted into the posterior iliac crest but prior to aspiration, 1 mL of heparin (1,000 U/mL) should be preloaded into the syringe. • Preloading the needles avoids clot formation and coagulation, which can diminish the ultimate yield from the aspiration. • Approximately 60 mL of bone marrow is aspirated, which requires the use of two 30-mL syringes.
  77. 77. Processing of the Bone Marrow Aspirate ◦ The bone marrow aspirate (BMA) sample must be processed after it is harvested. BMA is filtered through a 200-μm-mesh filter into 50-mL conical tubes. ◦ then, 1 to 1.5 mL of the filtered BMA is pipetted into a 2-mL microcentrifuge tube for hemanalysis and the sample complete blood count with differential is automatically recorded. ◦ Subsequently, 60 to 90 mL of BMA is transferred into 2 × 50-mL conical tubes, and initially centrifuged at 2,400 rpm for 10 minutes.
  78. 78. ◦ After completion of this process, the buffy coat layer and platelet-poor plasma layer are extracted from the conical tube and discarded. ◦ The red blood cell layers are combined into 1 × 50-mL conical tube for second centrifugation (3,400 rpm for 6 minutes). ◦ Finally, the BMAC/white cell pellet is resuspended in platelet poor plasma, hemanalysis is performed and complete blood count with differential is recorded (including monocyte count) to assess the final product to inject
  79. 79. ◦ After density gradient centrifugation to remove red blood cells, granulocytes, immature myeloid precursors, and platelets, progenitor cells account for a small population within the bone marrow (0.001% to 0.01%). ◦ However, a high concentration of growth factors, including platelet- derived growth factor, transforming growth factor-β, and bone morphogenetic proteins 2 and 7, which are reported to have anabolic and anti-inflammatory effects are present in BMAC. ◦ Of note, it has been reported that BMAC has a considerable concentration of interleukin-1 receptor antagonist (IL-1RA). ◦ This molecule inhibits IL-1 catabolism and therefore may be responsible for the beneficial symptomatic pain relief with this biologic approach.
  80. 80. BMAC USES Wednesday, 4 June 14 96
  81. 81. BMAC IN NON UNIONS Wednesday, 4 June 14 97
  82. 82. BMAC IN SPINAL FUSION Wednesday, 4 June 14 98
  83. 83. BMAC IN AVN Wednesday, 4 June 14 99
  84. 84. BMAC FOR CARTILAGE REPAIR Wednesday, 4 June 14 100

Descripción

Ortho: to make straight or right. The use of biologic substances to prompt, stimulate or support a “healing event” within the body.The use of biologic substances to promote healing or reduce pain.The use of platelets and stem cells in treatment and management of musculoskeletal conditions

Transcripción

  1. 1. ORTHOBIOLOGY ;
  2. 2. ONCE UPON A TIME... Wednesday, 4 June 2 3000 BC : As perMahabharata,Adi Parva, chapter 114,shloka17, theKauravawerecreatedby splittingthe singleembryointo101partsandgrowing eachpartina separate kund or container. It is believedthatKaurva’s birthmentionedhereisfirst reportedmentionofwhatcould bepossibleuseof embryonic stem cell.
  3. 3. Orthobiologics: • Ortho: to make straight or right • The use of biologic substances to prompt, stimulate or support a “healing event” within the body • The use of biologic substances to promote healing or reduce pain • The use of platelets and stem cells in treatment and management of musculoskeletal conditions
  4. 4. ◦ Within orthopedics, focal chondral lesions, osteo- arthritis (OA), fracture healing, and soft tissue repair (tendon to tendon interface, tendon to bone interface, meniscus, muscle, and ligaments) are fertile fields for biological therapies. ◦ Biological and surgical treatments have been proposed to treat these pathologies. Biological treatments include ◦ platelet-rich plasma (PRP), ◦ bone marrow aspirate concentrate (BMAC), ◦ and cell-based therapies. ◦ Surgical interventions include marrow stimulation procedures and stem cells implanted in matrix
  5. 5. BMP’S IN ORTHOPEDICS ◦ Bone Morphogenic Proteins (BMPs) are a group of molecules which induce mesenchymal stem cells to differentiate into bone forming cell lines that form new bone. ◦ They are a group of noncollagenous glycoproteins that mostly belong to the transforming growth factor-beta (TGF-b) superfamily. ◦ BMPs have been widely used in the field of orthopaedics since the last two decades. ◦ BMPs are a group of osteoinductive proteins that are usually extracted from bone matrix which are responsible for skeletal regeneration and bone healing.
  6. 6. ◦ The primary functions of BMPs are to activate inactive mesenchymal stem cells and to differentiate them into osteoblast, chondroblasts and fibroblasts. ◦ Apart from the above functions, BMPs are also involved in other physiological and pathological processes such as inflammatory response, bone formation and resorption, growth signalling pathways, oncogenesis and immune response. ◦ The safety and efficacy of BMP as bone graft substitute are influenced by its purity, local effects, systemic effects, immunogenicity and biocompatibility.
  7. 7. ◦ Bone ossification occurs through both intramembranous ossification and endochondral ossification ◦ Both cellular events of intramembranous ossification and endochondral ossification involve MSCs which may be bone marrow derived or periosteum derived. ◦ These primitive MSCs are pluripotent progenitors that can differentiate into both osteoblasts and chondroblasts. ◦ This differentiation is regulated by molecules such as BMPs and Fibroblast growth factor (FGF).
  8. 8. Classification of BMPs • Till date more than 20 BMPs have been identified in vertebrates. • Based on structural homology, BMPs can be further classified into severa subgroups which includes BMP-2/- group, BMP-5/-6/-7/-8 group, BMP-9/ 10 group and BMP-12/-13/-14 group.
  9. 9. Functions of BMPs ◦ The actions and functions of BMPs depend on various factors which are: ◦ 1. Type of target cell. ◦ 2. Maturation phase of target cells. ◦ 3. Local concentration of BMPs. ◦ 4. Other biological signals.
  10. 10. Functions of BMPs ◦ BMPs are mitogens (growth factors) that stimulate the production and multiplication of morphogens (differentiation factors) that transform connective tissue cells into osteoprogenitor cells. ◦ They play a pivotal role in embryonic development through specification of positional information in the embryo. ◦ They regulate growth, differentiation, chemotaxis and apoptosis of cells such as epithelial, mesenchymal, haematopoietic and neuronal cells. ◦ Apart from inducing bone formation through intramembranous or endochondral ossification, they also induce osteoclasts leading to bone resorption.
  11. 11. Carriers of BMPs • BMP is a water soluble molecule which diffuses into the body fluid easily. • When administered alone, most of the protein molecule is lost rapidly due to diffusion and irrigation. • To negate these effects and have a prolonged localized effect at target site, BMPs are administered in a carrier. • Type I collagen is the most preferred carrier used for transport of BMPs.
  12. 12. Clinical Applications in Orthopaedics ◦ 1. BMP as an alternative to autograft for non-unions Long Bones – ◦ Currently, US Food and Drug Administration (FDA) approval of BMP use in long bones is limited to tibia non-unions as alternative to autograft. ◦ Pseudoarthrosis of tibia remains to be the most frequent site of long bone non-union due to its poor muscular coverage, high incidence of open injuries and its anatomical peculiarity. ◦ Friedlaender et al initially reported the efficacy of BMP-7 in the treatment of tibia non-unions. ◦ It was concluded that BMP-7 provided comparable results when compared with bone autograft, without donor site morbidity.
  13. 13. Scaphoid ◦ Scaphoid is very commonly affected with non-union due to its precarious vascular supply. ◦ Patients treated with autograft and BMP-7 healed successfully within 3 months ◦ However, all the evidences on scaphoid non-unions were poor. ◦ SPINE ◦ By far, spine is the most common site associated with BMP application. ◦ US FDA has approved the usage of rhBMP-2 in anterior lumbar interbody fusions. ◦ However recent studies have shown that BMP use has been extended to fusions performed with other approaches as well.
  14. 14. ◦ BMP as an alternative to autograft for fusion Foot and ankle – ◦ Few authors have used BMPs to improve the rates of union in ankle arthrodesis for patients with comorbidities affecting bony union. ◦ Initially use of BMP in complex foot and ankle arthrodesis seem to be a safe option considering its high failure rates. ◦ However, there is no randomized control trial available till date to give a suggestable conclusion.
  15. 15. Platelets ◦ Platelets develop from megakaryocytes in the marrow. ◦ Each megakaryocyte can produce 5000- 10000 platelets. Approximately 2/3rd of the platelets circulate in the blood and 1/3rd are stored in the spleen. ◦ An average healthy adult can produce 1011 platelets per day. ◦ The normal platelet count is (150– 400) × 103 per microliter of blood. ◦ The diameter of a mature platelet is 2-3 5μ. ◦ Platelets usually remain alive for 5-11days. ◦ Old platelets are destroyed by phagocytosis in the spleen and liver by Kupffer cells
  16. 16. + What is the response to platelets • Proliferation of Mesenchymal stem cells • Proliferation of fibroblasts • Production of Type 1 Collagen • Platelets itself release Growth factors
  17. 17. Healing pathway
  18. 18. PLATELET RICH PLASMA ◦ By definition, Volume of plasma fraction of autologous blood having a platelet concentration above baseline. ◦ To be labeled as PRP, a platelet count of 3-5 times of the baseline should be present in the platelet concentrate(many studies like Max et al mention the platelet count 1 million as standard valve for PRP). ◦ Platelets begin secreting these proteins within 10 minutes of activation. After initial release of growth factors, the platelets synthesize and secrete additional factors for the remaining days of their life span.
  19. 19. ◦ In humans, the normal platelet count in whole blood ranges from approximately 150,000 to 350,000/μL , ◦ whereas platelet- rich plasma is often defined as at least 1,000,000 platelet/ μL suspended in plasma. ◦ In order to ensure that the platelets are suspended and do not form a clot, PRP must be made from anticoagulated blood. ◦ The logic behind PRP is that platelets are the first to arrive at the site of tissue injury, and thus have the potential to release growth factors that play a critical role in mediating healing
  20. 20. History ◦ The term PRP first used by KINGSLEY ◦ In 1970, MATRAR used fibrin glue in rats ◦ 1998 –PRP in regenerative medicine for bone healing ◦ 2001- 2nd generation platelet concentration identified
  21. 21. ◦ Physicians report that the demand for PRP has soared after pro golfer Tiger Woods received injections to accelerate healing after knee surgery.”
  22. 22. What is PRP ◦ Platelet Rich Plasma ◦ Utilizing growth factor (GF) content of platelets to aide in healing of musculoskeletal tissue ◦ Predominately tendons, ligaments and muscles ◦ High concentration of GF deposited locally in the area of an injury ◦ Anabolic effect enhances and supports healing
  23. 23. + What does it lead to?? • Chemotaxis • Directional movement in response to a chemical stimulus • Stems cells are attracted to the growth factors and migrate into the area • Cell proliferation • Significant increase in cellular reproduction activity • Possibly even a systemic effect • Performance enhancing?
  24. 24. PRP – MECHANISM OF ACTION 1st Mechanism: • Delivery of Growth Factors 2nd Mechanism : • “Up regulation” of local GF in tendons • TGF-Beta1 for 1st week post prp, • Increased IGF-1 for 4 weeks tenocytes post prp
  25. 25. 3rd mechanism: ◦ PRP Stimulates HA Release • PRGF significantly enhanced HA secretion compared with platelet- poor preparations 4th mechanism : ◦ Chemo-attractant for Stem Cells • Prompt Migration and Proliferation
  26. 26. Platelets ◦ Although platelets play a key role in haemostasis, they are central in mediating the anabolic effects of PRP by virtue of releasing growth factors stored in their alpha granules. ◦ During the initial phases of wound repair, activated platelets attract and foster cell migration into the wound by aggregating and forming a fibrin matrix. ◦ This matrix then serves as a tissuescaffold for sustained release of platelet growth factors and cytokines, which stimulate cell recruitment, differentiation, and communication
  27. 27. 2. Leukocytes ◦ Leukocytes are essential mediators of the inflammatory response, host defence against infectious agents, and wound healing. ◦ Neutrophils are involved in the inflammation phase of wound healing. ◦ Monocytes and macrophages facilitate tissue repair by debriding and phagocytising damaged tissue and debris. ◦ Similar to platelets, macrophages also secrete growth factors that are important in tissue repair and have been shown to contribute to subchondral bone regeneration.
  28. 28. 3. Red blood cells ◦ Red blood cells (RBC) content is typically reduced or absent in PRP because of the centrifugation process. ◦ Destructive process is thought to occur in human synoviocytes treated with RBC concentrates, leading to significantly greater cell death and cartilage degradation.
  29. 29. Classification of PRP Dohan et al in the year 2009 classified PRP based on presence of leukocytes and density of fibrin network
  30. 30. Mishra et al in the year 201 proposed a classification system which is based on platelet count, activation and leukocytes for clinical application in Sports medicine
  31. 31. + Advantages of having leucocytes?? Pure PRP has an advantage over traditional PRP in that it eliminates red blood cells (RBCs) and neutrophils. RBCs, have no therapeutic effect and more painful while injecting. Neutrophils, a type of white blood cell, have inflammatory components which may increase pain and inflammation post-treatment.
  32. 32. ◦ Equipment required for processing 40ml venous blood (figure1) 1. Centrifuge (Remi R-4C), 4000rpm with countdown timer fixed angle rotor, capacity 4x50 ml 2. First centrifugation (soft spin) : 2x20cc syringe, 6ml ACD-A (Acid citrate dextrose), 2xBD insulin syringe 3. Second centrifugation (hard spin): 20cc syringe, 3-way cannula, BD insulin syringe 4. PRP extraction: 10cc syringe, 3-way cannula.
  33. 33. STEP 1 ◦ Three ml ACD-A was taken in 20ml syringe and 17 ml blood drawn from the ante cubital vein. Blood (0.3 ml) was sent for initial platelet count. ◦ Nozzle of the syringe was blocked firmly with the cap of the insulin syringe. With a heavy scissors flanges on the either side of the syringe barrel were cut. ◦ The piston was cut leaving one inch projecting out of the barrel. ◦ The two syringes were placed in centrifugation tubes opposite to each other with the tip pointing upwards. ◦ This will help balance the centrifuge and minimize vibration. ◦ The first spin was set at 1500rpm for 15mins without brake (Soft spin).
  34. 34. STEP II ◦ After soft spin blood in the syringe was separated into three layers. ◦ The bottom layer comprises of RBC, the middle layer leucocytes and the top layer, plasma with platelets in suspension. ◦ The serum was drawn into the second 20cc syringe through the 3-way cannula. Care was taken to avoid aspiration of the buffy coat and RBC. ◦ The combined volume of plasma from both syringes ranged between 16-18ml. ◦ The syringe was again capped and modified to fit the centrifugation tube. ◦ The second spin was set at 3500rpm for 7minutes (Hard spin).
  35. 35. STEP III ◦ At the end of second spin the syringe contains Platelet Poor Plasma (PPP) on top and platelets along with scanty WBC at the bottom in the form of a pellet. ◦ The supernatant plasma is drawn and discarded leaving about four ml at the bottom which is reconstituted to form PRP fraction. This is sent for culture and counts.
  36. 36. RESULTS
  37. 37. PRF ◦ Under strict aseptic conditions, ten to twenty millilitres of patient's blood was taken and centrifuged at 3000 rpm for 15 min. ◦ PRFM was separated from red corpuscles at the base using a sterile forceps and scissor, preserving a small RBC layer measuring around one mm in length, which was transferred onto a sterile gauze
  38. 38. ◦ Middle membrane so obtained was compressed between two gauze pieces gently and applied on a healthy wound followed by application of a secondary non-absorbable dressing. ◦ The secondary dressing of the patient and the dried PRFM was removed from the wound bed after a minimum of five days. ◦ The same procedure was repeated every week for six weeks.
  39. 39. + Where can it be used??  Everything and anything!  Internet marketing for host of ailments  Snake oil of today “Cures Rheumatism, Sports injuries, Grows hair, Gets rid of wrinkles, etc., etc.”
  40. 40. +  Meniscus repair  Tendonitis (office based injection)  Arthritis  Ligament surgery  Tendon repairs  Acute injury – Debatable!
  41. 41. +  Epicondylitis, Achillodynia  Runners / Jumpers Knee  Plantar fascitis  Osteoarthritis (Grad I-III)  Muscle- and Ligament Lesions - Ultrasound-Guided Injections  Peri – Post operatively - Achilles Tendon Repair - HTO, Microfracturing - Meniscal Suture, RC Repair etc
  42. 42.  Analgesic?  Potential primary analgesic effect  Some human studies state decreased post-op pain levels  Stimulation of thrombin receptors (ie, PAR-1) shown to increase pain threshold in laboratory animals through opioid pathways and haemostasis  Antimicrobial  Against Staphylococcus aureus (Sutter 2012)
  43. 43. Arthritis What happens in an arthritic joint?
  44. 44. • Cartilage wears out • chondrocytes unhealthy/poor quality matrix - breaksdown • Poor lubrication - hyaluronic acid/lubricin (SZP -Proteoglycan 4) • increased friction - wears out Inflammation - pain
  45. 45. Arthritic Joint - The “BAD” Molecules • MMP’s - matrix metalloproteinases - collagenases, gelatinases, stromelysins - degrade collagen, proteoglycans, elastin, etc. • IL-1, TNF - interleukins - inflammatory - enhance MMP’s • ADAMTS - disintegrins - prevent platelet function • CATABOLIC - BREAKDOWN
  46. 46. What About “GOOD” Molecules? • TGF - tissue growth factor • TIMP-1&2 - tissue inhibitor of metalloproteinases • IRAP - interleukin receptor antagonist protein • A2M - alpha 2 macroglobulin (useless by itself • - so drug companies barking up the wrong tree) • Block the BAD molecules - • ANTI-CATABOLIC (ANABOLIC)
  47. 47. PRP (Platelet Rich Plasma) Reduces “BAD” Molecules Increases “GOOD” Molecules
  48. 48. What About “Traditional” Treatments? Are there problems? NSAIDS Corticosteroid Surgery (Physical Therapy ALWAYS GOOD!)
  49. 49. NSAIDS • Commonly taken • Multitude of side-effect Very significant problems • ulcers, bleeding, heart attack, stroke, kidney and liver failure • Inhibit chondrocytes and stem cells
  50. 50. Corticosteroids Injections • Commonly performed • Temporarily reduce inflammation • Inhibit healing • Cause damage to cartilage and soft tissue structures
  51. 51. PRP Intra-articular Pathways • Decreases BAD molecules - MMP, IL - reduces CATABOLIC PROCESSES • Increases GOOD molecules - TGF, TIMP, IRAP, A2M • REBALANCES the micro- environment of the joint - back to favoring the normal healing status
  52. 52. • Produced by synovial cells • Important for lubrication/viscosity of joint fluid • Hyaluronic Acid - Hyaluronan • Viscosupplementation -Synvisc, Orthovisc - from rooster combs • HA alone not enough to protect the joint PRP Stimulates Hyaluronic Acid Production
  53. 53. + Tennis elbow • Common Disorder • Repetitive activities – Most common cause – May occur from injury (acutely) • PRP Application Technique – Inject 2-3 cc of PRP into the ECRB--Peppering technique – Average Dose 3.3 million platelets per patient
  54. 54. Lateral Epicondylitis - PRP • PRP provides excellent and superior results • Results are longer lasting • Particularly better than corticosteroid injection Am J Sports Med 2006, 2014, J Clin Diag Res 2015, J Hand Microsurg 2015
  55. 55. Compared to Surgery: PRP - showed 83% improvement Surgery - showed 46% improvement PRP improved 2X as much as surgery SO WHY ARE WE STILL DOING SURGERY FOR THIS PROBLEM? J Orthopaedics March 2016
  56. 56. Planar Fasciitis PRP • more effective and durable than corticosteroid • 3X (300%) the functionality and improvement of pain • Actually heal structures - rather than mask Sx (Foot Ankle Int, April 2014)
  57. 57. Shoulder Impingement • Rotator cuff tendons weaken, develop tears, degenerative changes • Poor blood supply Unable to heal itself • Gets stuck in early phase of repair Chronic changes, inflammation, pain Poor healing environment
  58. 58. Shoulder Impingement NSAIDS – minimal benefit, inhibit healing Corticosteroids - if used - no longer see healing response - Br J Sports Med 2014 kill rotator cuff cells -Bone Joint Res 2014
  59. 59. Shoulder Impingement - PRP • PRP more effective than corticosteroid • in pain relief (30% better) • in ROM improvement (3X more improvement) • 16X less likely to undergo surgery (3/100 vs 48/100) O’Donnell, et al. AAOS 2013
  60. 60. + TendoAchilles
  61. 61. + Post Injection Therapy • Rest (sling/crutches) for a few days to a week • NO NSAID’S • Slow stretching program • No high loading activities until • No rest pain • Minimal tenderness
  62. 62. + COMPLICATIONS  No adverse complications have been reported  Symptoms may be worsened for few days because of induced inflammation  Infection  Persisting symptoms
  63. 63. + Limitations • Efficacy • Lack of clinical data • Number of studies being done • Indications are being developed • Uniformity of prep • PRP classification • Insurance recognition
  64. 64. Stem Cell • An undifferentiated progenitor/primitive cell • A: capable of renewing itself through cell division, sometimes after long periods of inactivity • B: Under certain physiologic or experimental conditions, it can be induced to become a tissue- or organ-specific cell with special functions
  65. 65. STEM CELL RESEARCH TIMELINE
  66. 66. STEM CELL CHARACTERISTICS Wednesday, 4 June 14 77 Undifferentiated cells with the capacity for unlimited or prolonged self-renewal that can give rise to differentiated cells. Self renewing for long periods. Unipotent /pluripotent /multipotent e.g. Intestinal stem cells produce one or more of the 4 classes of mature differentiated cells (absorptive, goblet, Paneth and enteroendocrine cells)
  67. 67. Sources of stem cells ◦ Adult Tissue: Hematopoeitic SC, Mesenchymal SC, Neural, muscle derived, Pancreatic, Hepatic, Epithelial, Dental. ◦ Fetal tissue ◦ Cord blood ◦ Embryos
  68. 68. BMAC ◦ In adult skeleton there are two types of bone marrow which are red marrow and yellow marrow. ◦ Yellow marrow is filled with adipose tissue and is inactive by function. Red marrow possesses hematopoietic cells and mesenchymal stem cells. ◦ Mesenchymal stem cells (MSC) have the capability to differentiate into cell lines like cartilage, bone, tendon, muscle and nerves. ◦ Benefits of BMAc are achieved either by proliferation and differentiation into variety of cell lineages or by elution of growth factors and cytokines to hasten the process of healing in tissues with attenuated healing potential.
  69. 69. Components of BMAC ◦ In normal bone marrow aspirate (BMA),common components present are erythrocytes(22-28%), neutrophils (32-37%),lymphocytes(13%), eosinophils (2.2%), blast cells(1.4%), monocytes (1.3), basophils(0.1%) and megakaryocytes in variable percentages. ◦ Apart from the above components, BMA contains mesenchymal stem cells and various growth factors. ◦ An adult bone marrow has about 0.001% to 0.01% of mesenchymal stem cells in 7-30 cells per million nucleated cells. ◦ Growth factors like PDGF, TGF-â, BMP2and7 and IL-1RA are also present in the BMA in variable concentrations
  70. 70. BONE MARROW CONCENTRATE Wednesday, 4 June 14 84 Enhances levels of Bone Marrow-BM Mesenchymal Stem MS) Cells BM-MS cells decrease with age Concentrating mimics the cell load and healing potential of young patients 4-5x progenitor cell increase above baseline
  71. 71. ◦ Main aim of bone marrow concentration is to increase the cell count of mesenchymal stem cells. ◦ The concentrations of MSC in bone marrow alone is relatively low, hence the aspirate is concentrated by centrifugation in order to increase the percentage of MSCs. ◦ Cell count in bone marrow aspirate is 612±134 per cm3 compared to bone marrow aspirate concentrate is 2579±1121 per cm3 which is a fivefold increase in the number of mono nuclear cells
  72. 72. Sources of BMAC ◦ Commonest source of bone marrow aspiration is from iliac crest, tibia or calcaneum. ◦ Among all of the sources,iliac crest yields highest concentration of mesenchymal stem cells. ◦ Graft is harvested bysector rule in iliac crest, which roughly divides the entire iliac crest into six zones. ◦ Of these six zones the posteriorcrest is preferred site as it contains higher concentration of MSCs (higher by 1.6 times compared to anterior iliac crest).
  73. 73. ◦ The patient can be positioned either supine or prone. In the supine position, BMAC is harvested from the anterior superior iliac spine or the iliac crest. In the prone position, BMAC is harvested from the posterior superior iliac crest region. ◦ Monitored anesthesia (conscious sedation), local anesthesia, or general anesthesia can be used for this BMAC harvest procedure, we prefer positioning the patient in the prone position with light sedation monitored by an anesthesiologist for access to the posterior superior iliac crest. ◦ After palpation of the bony landmarks, the procedural site is sterilely prepared and widely draped to ensure an adequate field.
  74. 74. • The BMAC aspiration kit is opened and then the battery-powered BMAC aspiration device is sterilely drape • A bone marrow aspiration kit (MarrowStim; Biomet Biologics, Warsaw, IN) is used for bone marrow aspiration • First the bony landmarks of the posterior iliac crest and sacroiliac joint are palpated. The skin is then injected down to and including the periosteum with 1% lidocaine without epinephrine.
  75. 75. • Then, the bone marrow aspiration trochar and needle are percutaneously inserted through the skin and subcutaneous tissues until it reaches the posterior iliac crest. • Then, manual pressure is used to position the bone marrow aspiration trochar against the dense cortical bone, attempting to center it over the middle of the posterior crest cortical walls. • The trajectory of the needle should be parallel to the iliac crest, or perpendicular to the ASIS or PSIS, depending on the harvest site used
  76. 76. • A battery-powered power instrument is then used to drill the trochar and needle into the medullary cavity of the posterior iliac crest • After the trochar is inserted into the posterior iliac crest but prior to aspiration, 1 mL of heparin (1,000 U/mL) should be preloaded into the syringe. • Preloading the needles avoids clot formation and coagulation, which can diminish the ultimate yield from the aspiration. • Approximately 60 mL of bone marrow is aspirated, which requires the use of two 30-mL syringes.
  77. 77. Processing of the Bone Marrow Aspirate ◦ The bone marrow aspirate (BMA) sample must be processed after it is harvested. BMA is filtered through a 200-μm-mesh filter into 50-mL conical tubes. ◦ then, 1 to 1.5 mL of the filtered BMA is pipetted into a 2-mL microcentrifuge tube for hemanalysis and the sample complete blood count with differential is automatically recorded. ◦ Subsequently, 60 to 90 mL of BMA is transferred into 2 × 50-mL conical tubes, and initially centrifuged at 2,400 rpm for 10 minutes.
  78. 78. ◦ After completion of this process, the buffy coat layer and platelet-poor plasma layer are extracted from the conical tube and discarded. ◦ The red blood cell layers are combined into 1 × 50-mL conical tube for second centrifugation (3,400 rpm for 6 minutes). ◦ Finally, the BMAC/white cell pellet is resuspended in platelet poor plasma, hemanalysis is performed and complete blood count with differential is recorded (including monocyte count) to assess the final product to inject
  79. 79. ◦ After density gradient centrifugation to remove red blood cells, granulocytes, immature myeloid precursors, and platelets, progenitor cells account for a small population within the bone marrow (0.001% to 0.01%). ◦ However, a high concentration of growth factors, including platelet- derived growth factor, transforming growth factor-β, and bone morphogenetic proteins 2 and 7, which are reported to have anabolic and anti-inflammatory effects are present in BMAC. ◦ Of note, it has been reported that BMAC has a considerable concentration of interleukin-1 receptor antagonist (IL-1RA). ◦ This molecule inhibits IL-1 catabolism and therefore may be responsible for the beneficial symptomatic pain relief with this biologic approach.
  80. 80. BMAC USES Wednesday, 4 June 14 96
  81. 81. BMAC IN NON UNIONS Wednesday, 4 June 14 97
  82. 82. BMAC IN SPINAL FUSION Wednesday, 4 June 14 98
  83. 83. BMAC IN AVN Wednesday, 4 June 14 99
  84. 84. BMAC FOR CARTILAGE REPAIR Wednesday, 4 June 14 100

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