Surgical Manual

SURGICAL MANUAL Defect Assessment and Biopsy Procurement Cell Implantation Procedure Written by Tom Minas, M.D. Edited by Lars Peterson, M.D., Ph.D.

Surgical Manual AcknowledgmentThe surgical technique represented in this manual is acknowledged to its developers:Lars Peterson, M.D., Ph.D.; Mats Brittberg, M.D., Ph.D.; Anders Lindahl, M.D., Ph.D.;Anders Nilsson, M.D., Ph.D.; Olle Isaksson, M.D., Ph.D., and Claes Ohlsson, M.D., Ph.D. It istheir careful consideration, and analysis of experimental and clinical results which has con-tributed to the evolution of the surgical technique in this manual.Careful patient selection, correct indications, meticulous surgical technique and an appro-priate rehabilitation protocol are important for a satisfactory clinical result when usingautologous chondrocyte implantation.I am indebted to Dr. Peterson for his time and openness in teaching and sharing his tech-nique and thoughts on cartilage repair.Tom Minas, M.D. M.S. Manual Design and Medical Illustration by Amy Matson Walts

Surgical Manual for the Implantation of Cultured Autologous Chondrocytes Written by Edited byTom Minas, M.D. M.S. Lars Peterson, M.D., Ph.D.Assistant Professor of Orthopaedics Professor, Department of Orthopaedics Harvard Medical School University of Gothenburg Director Cartilage Repair Center Clinical Director Gothenburg Medical Center Brigham & Women’s Hospital Vastra Frolunda, Sweden Boston, Massachusetts64 Sidney St. N Cambridge N Massachusetts N 02139 N USA Carticel® is a registered trademark of Vericel Corporation in the United States.



Defect Assessment and Defect Assessment and Biopsy Procurement Biopsy Procurement

Radiographic Joint Assessment Radiographic Joint Assessment In order for a patient to be considered a candidate for the implantation of Carticel, the knee joint must be free from osteoarthritic conditions. Radiographic assessment may provide an indication of the presence of osteoarthritis. If this condition is appar- ent, the patient should not be consid- ered as a candidate for this technology. Standing X-rays may provide evidence to reach this conclusion. Radiographs of the patient’s legs should include a weight-bearing view at 45° of flexion, and a second view in full extension. If the X-ray interpretation is consistent with generalized osteoarthritis, implan- tation of autologous cultured chondro- cytes is not indicated.Figure 1.1: Subtle lateral joint Normal image.space narrowing. Figure 1.2: Arthroscopic appearance of entire lateral tibial plateau. Arthroscopic examination revealed generalized thinning, with near complete cartilage loss on the lateral tibial plateau.4

MRI AssessmentMRI AssessmentMRI scans may be useful to reveal meniscalpathology and/or detect bone bruising.However, the resolution and sequences ofroutine clinical scans may not be sensitiveenough to assess cartilage defects.Thispatient had a normal MRI scan, but anarthroscopic evaluation detected a largechondral defect. Figure 1.3: Medial Femoral Condyle (MFC) Scope arthroscopic assessment shows a lesion on the medial femoral condyle of 2cm x 1.5cm.Figure 1.4: Negative MRI MFCMRI reveals no cartilage abnormality. 5

Arthroscopic AssessmentArthroscopic AssessmentDetermine patient candidacy for treatment. Joint condition, concomitant pathologies, size, number and extent ofdefects are critical factors in this consideration.Defect AssessmentThe implantation of autologous cultured chondrocytes requires all damaged, loose, undermined and unhealthyfemoral articular cartilage surrounding the defect to be removed.The complete extent and size of the defect mustbe determined to assure that an appropriate cell volume is provided at the time of implantation. Defects must besurrounded by healthy, stable cartilage in order to maximize success. Figure 2.1 Figure 2.2 Carefully probe to assess quality of cartilage and rule out undermining.6

Arthroscopic Assessment Arthroscopic Assessment Figure 2.3 Hyperflexed;1 Create standard arthroscopic Posterior lesion evident. portals, medial and lateral. Figure 2.42 Hyperflex the defect knee to expose MFC @ 70-90o; entire condylar surface. Lesion 3cmL x 1cmW. Figures 2.3 and 2.4 Figure 2.53 Carefully inspect all articular Full-extension MFC cartilage, tibial plateaus, femoral and MTP. Carefully condyles, patella and trochlear groove. exam tibial surface to Figure 2.5 rule out kissing lesions.4 Arthroscopically assess the patella- femoral joint for abnormal facet tracking and joint surface contact.5 Using standard arthroscopic probe examine joint surface to determine cartilage resiliency.6 Carefully palpate fissures and tissue surrounding defect to determine integrity of surrounding cartilage. Figures 2.6 and 2.77 Joint assessment must rule out general- ized osteoarthritis. Inspect the oppos- ing surface of each defect. Full thick- ness cartilage defects on opposing articular surfaces, so called “bipolar lesions” are consistent with osteoarthritis and contraindicated for autologous cultured chondrocyte implantation. Figures 2.8 and 2.9 Figure 2.6 18 mos. MFC after drilling; Tissue delamination. Fibrocartilage repair. 7

Defect Assessment Figure 2.7 Defect Assessment MFC 1.5 x 2.0mm Contained lesion with 1 Isolate each individual defect. Assess loose flaps tissue surrounding defect. Figure 2.8 2 The borders of the defect must be Clearly not acceptable surrounded by healthy cartilage of reciprocal damage for normal thickness, measuring at least implantation of 2 to 3mm.This thickness is necessary autologous cultured to accommodate suture fixation of a chondrocytes periosteal patch to the defect for subsequent implantation of autologous cultured chondrocytes.Verify that the most posterior margin of the defect is visible and meets these requirements. 3 Using a graduated probe, measure the anterior-posterior and medial-lateral dimensions of each defect. Accurate measurement of each defect is critical to ensure adequate cell volume is pro- vided for implantation. Record defect measurements on the Patient Biopsy Transmittal Notice included in the Vericel Corporation Cartilage Biopsy Transport Kit. Figure 2.10 Figure 2.9 Generalized degeneration; Uncontained borders Figure 2.10 Use a 5mm probe tip to measure contained defect dimensions.8

Instrumentation List Instrumentation List for Biopsy ProcurementI Notchplasty GougeI Standard 6mm Ringed CurettesI Non-Toothed GraspersI Standard Arthroscopy EquipmentI Vericel Corporation Cartilage Biopsy Transport Kit 9

Biopsy ProcurementBiopsy Procurement Step 3: Medial Superior RidgeNotice: Before harvesting tissue, Step 4: Lateralensure that the Vericel Corporation Superior RidgeCartilage Biopsy Transport Kit is Step 5: Obtain 2 full-thicknesspresent.Verify that the biopsy biopsies measuring 5mm x 8mm.medium is not expired by inspectingthe expiration date noted on thebiopsy container. Also check thecontainer for leakage and themedium for any particulates,turbidity and color change.Cartilage tissue must be harvestedfrom a lesser load bearing,non-articulating surface.The superiormedial or lateral femoral articularcartilage margins are recommendedas tissue procurement sites.The tissue may be procured usingeither a ring curette or a curvednotchplasty gouge.The medial side is recommended asthe preferred biopsy site. It is easy toaccess and provides thick cartilage,which generally yields a superiorbiopsy. Rule out patellar overhang fromthe facets prior to biopsy, starting atthe cartilage bone junction. Harvestas close to the synovial junctionas possible.Careful assessment is required whenobtaining biopsies from the lateral sideof the femoral condyle. Again, rule outpatellar overhang prior to biopsy.Thecartilage is usually thinner, and ifharvested too far superiorly, it maybe fibrous tissue.To remove cartilage biopsy, graspcartilage at the site of attachment withnon-tooth graspers, and twist and pullto dislodge specimen. Obtain 2 full-thickness biopsies measuring 5mm x8mm. 8mm 5mm 200-300mg10

Biopsy ProcurementLesion Using Ring Curette Biopsy of healthy 1 Place leading edge of a 6mm ring cartilage curette at superior aspect of the medial or lateral articular margin of the femoral condyle 2 Apply pressure until the sharp edge penetrates the full-thickness of the cartilage down to subchondral bone. Using a twisting or “wiggle” motion, draw the curette down the margin to create a crescent-shaped specimen. 3 Leave the distal portion of the biopsy attached to enable retrieval from the joint using a non-tooth linear grasper. 4 The biopsy must be full-thickness and should include a small sample of bone. Superficial Tangential Articular Surface Zone (10-20%) Tide Mark Middle Zone (40-60%) Subchondral Bone Deep Zone (30%) Cancellous Bone Calcified CartilageStep 1:Medial superior ridgeFull-thickness specimens including small bone sample.Step 2: Engage back of curette. Step 3: Create a dowel of cartilage Step 4: Keep the distal stump with a side-to-side motion. intact. Remove with a non- tooth grasper. 11

Biopsy Procurement Step 1 Using Notchplasty Gouge1 Place the leading edge of a notchplasty gouge approximately 10mm-15mm distal to the superior aspect of the medial or lateral articular margin of the femoral condyle.2 Apply pressure to ensure the sharp edge penetrates the full-thickness of the cartilage down to subchondral bone. Using a twisting or “wiggle” motion, push the notchplasty gouge proximally towards the superior aspect to create a crescent-shaped specimen.3 Leave the proximal portion of the biopsy attached to enable retrieval from the joint using a non-tooth linear grasper. Step 3: Angle the gouge on both sides, to ensure the superior proximal attachment is very thin. Step 2: When using the gouge, engage the cartilage and create a dowel with a side-to-side motion.12

Step 4: Entering from the medial portal, remove 5mm of Biopsy Procurementcartilage from the lateral half of the notch. Using Notchplasty Gouge at Intercondylar Notch 1 The intercondylar notch is an alternative biopsy site. 2 When harvesting from this location, flex the knee 30° to 40° and then fully extend while observing arthroscopical- ly to ensure that cartilage is not taken from a weight bearing area. 3 Start at the superior-lateral aspect (approximately 11:00 position on a left knee), and work down the lateral wall. 4 Place sharp edge of notchplasty gouge approximately 5mm lateral to the articular cartilage margin of the lateral notch wall and systematically begin to incise the outline of the biopsy to be taken prior to using the gouge to elevate the chondral biopsy. 5 Leave the distal portion of the biopsy attached. 6 Remove tissue with a non-tooth grasper.Step 6: Leaving the distal stump attached, reach in with anon-tooth grasper and remove the cartilage piece. 13

Biopsy Procurement Packaging Biopsy NOTE: The exterior of the container is NOT STERILE.1 Remove biopsy container from Cartilage Biopsy Transport Kit.2 Following sterile technique protocols, carefully place each biopsy sample into biopsy container containing transport media and seal with cap.3 Carefully read Cartilage Biopsy Transport Kit Instructions for Use provided with kit. Package in accordance with shipping and handling instructions.4 Complete all sections of Cartilage Biopsy Transmittal Notice included in Cartilage Biopsy Transport Kit and sign form.5 Include one copy of hospital’s / clinic’s Patient Face Sheet in Cartilage Biopsy Transport Kit.6 Immediately call Vericel Corporation Customer Care at 1-800-453-6948 (USA) to arrange for biopsy pick-up. 14

Cell Implantation Procedure Cell Implantation Procedure

Instrumentation List Instrumentation List for Cell Implantation Defect Preparation I Small Ringed Curettes 4mm or 6mm I Epinephrine I Neurosurgical Gauze Patties (Spinal) I Thrombin Spray or Gel I No.15 Surgical Blade Periosteal Patch Harvest and Fixation I Periosteal Elevator I 6mm, Curved or Blunt Tip Elevator I No.15 Surgical Blade I Ruler (sterile) I Ink Marker (sterile) I Non-toothed Tissue Forceps I 6.0 and 5.0 Coated Vicryl, Dyed Suture I P-I Cutting Needle I Optional Needles (PC-1, P-2) I Needle Driver I Sterile Mineral Oil I Dissecting Scissors Water Tight Testing I 1cc Syringe I Sterile Saline I 18 Gauge Blunt Tip Needle I IV Catheter, 18 gauge x 2” Fibrin Glue Application I Applicator Assembly 1cc I 26 Gauge x 3” Applicator Tip I Fibrin Glue CARTICEL Implantation I IV Catheter 18 Gauge x 2” I Alcohol Swab I Carticel Vial 16

Factors and RecommendationsPredisposing FactorsTo create a favorable environment for healing, predisposing factors must be addressed prior to cellimplantation.These conditions include: Meniscal Pathology: Unstable meniscus tears should be repaired or resected. If the patient has had a total meniscectomy, absent meniscus should be reconstructed. Cruciate Instability: Instability of the knee may adversely affect the success of the procedure and should be corrected. The anterior and posterior cruciate ligaments should be free of laxity as well as stable and intact. It is recommended that cruciate deficiencies be corrected. Malalignment: Abnormal weight-distribution within the joint may adversely affect the success of the procedure and should be corrected.The tibial/femoral joint should be properly aligned.When treating trochlear defects, abnormal patellar mechanics should be assessed and corrected.Tourniquet UseRecommended options for use: 1) Use tourniquet throughout the entire procedure. 2) Lower tourniquet after periosteum procurement. 3) Do not use a tourniquet.Hemostasis of the defect bone bed is a critical component to the success of the surgical procedure and mustbe achieved prior to cell implantation.The effectiveness of hemostatic agents applied to the defect bed can be assessed by lowering the tourniquetimmediately after harvesting the periosteum. Complete hemostasis must be achieved prior to periosteal fixationand cell implantation.HemostasisBleeding through the subchondral plate must be controlled. Epinephrine is recommended as a first-line hemostaticagent as follows: 1) Mix a vial of 1-1000 epinephrine with 20cc of sterile saline. 2) Soak neuropatties in epinephrine solution, then pack defect with soaked patties. 3) If a second agent is needed to achieve hemostasis,Thrombin, either in gel form or spray, is recommended. Apply Thrombin only at the point of penetration or source of bleeding. 17

Defect PreparationDefect Preparation Step 1: Use an open ring curette to excise damaged cartilage from the defect.ArthrotomyTo treat an isolated defect on the medialor lateral femoral condyle, a mini-arthrotomymay be performed.When treating multipledefects or defects that are more difficultto access, a traditional medial arthrotomyis recommended.Use a sharp blade to circumscribe the defectback to healthy cartilage. Eliminate all clefts,fissures, and cartilage that is undermined ordetached from subchondral bone.To further assess the cartilage excision,focus on: discoloration, irregular surfaceareas, absence of normal resiliency, thinningcartilage, and gritty texture.Healthy cartilage of normal thickness andresiliency must surround the defect. If thedefect is close to the intercondylar notch andno cartilage rim is available, the periosteumcan be sutured to the synovial lining.Use an open curette to excise all damaged Good stable vertical borders.or unhealthy cartilage from the perimeter of Step 5: The bone plate must be intact. Removethe defect. Remove as little healthy cartilage fibrous covering.as possible.The base of the defect must be carefully andcompletely debrided by removing all fibroustissue and cartilage remnants. Use a curetteto scrape defect base to expose calcifiedcartilage layer.Take care not to penetratesubchondral bone.Key points:Criteria for Optimal Defect Bed: Cartilage surroundingthe defect is healthy with sharp, vertical walls surroundingthe entire defect.This will aid suturing the periosteum, andthe containment of implanted cells.The subchondral bone plate is completely intact, and free offibrous tissue, and bleeding.Criteria for Optimal Periosteum:Oval or circular shaped defects simplify suturing periosteum,and help to secure a watertight seal.Periosteum must remain moist throughout the procedure.The incision exposing the cartilage defect must allow Defect contained by healthy cartilage.adequate room for securing the periosteal patch.18

Periosteum ProcurementStep 1: Measure the defect at its widest point.Add 2mm to both the vertical and horizontalmeasurement.Step 3: Insertion of pes Periosteum Procurementanserinus. 1 Use a disposable ruler to accurately measure the defect at its widest (medial to lateral, and superior to inferior) points. A sterile paper template may be used to aid in marking difficult defect shapes. 2 Add 2mm to both vertical and horizon- tal measurements to compensate for shrinkage of the periosteum after removal from the bone surface. Keep the periosteum moist to decrease shrinkage. 3 The tibia provides optimal periosteal tissue. Obtain this tissue from the proximal medial anterior cortex of the tibia with a separate incision approxi- mately 2.5cm below the pes anserinus.Incision located 2.5 cm belowthe pes anserinus. 19

Periosteum Procurement Step 1: Start with the edges around the periphery. Identifying the Cambium Layer It is important to identify the cambium Step 2: Angle the periosteal surface when applying the periosteum to elevator. Do not damage the the defect area.To distinguish the cambium subchondral bone. side of the periosteum from the outer fibrous side, use a sterile ink pen to mark Step 3: Elevate periosteum. the center of the tissue to be harvested, Flap may roll or curl up; gently prior to removing from the bone. straighten after removal. With a sterile ink pen, use the recorded measurements and mark the widest dimensions of the corresponding defect and then grossly outline the shape of the patch with the ink pen. To allow for complete exposure of tissue, use a scissor dissection and a damp sponge to carefully remove all tissue layers superfi- cial to the periosteum. Use a number 15 surgical blade to cut sharply around the marked outline down to the bone.1 Use a small periosteal elevator to create an edge around the borders.Work distal to proximal. If the tissue is laminated, it may be in the distal portion of the pes.2 Care must be taken not to damage the delicate cambium layer of the perios- teum when removing tissue from the bone.3 Angle the elevator and carefully push the periosteum off the bone, holding elevator as parallel as possible to the bone surface. Roll tissue up and away from the bone to avoid damage. Avoid gouging the underlying bone when removing the tissue. Key points: Keep periosteum and surface cartilage moist at all times. Place periosteum on damp sponge. A sterile paper template may aid periosteal patch sizing. When treating multiple defects, mark each periosteal patch specifically for its intended location. If the periosteum rips, it can be sutured; if it is too small, a second piece of tissue must be harvested and sutured to the original piece. Suture the two pieces of periosteum together, end to end. Do not overlap the tissues. Fibrin glue may be applied to create a watertight seal. 20

Periosteum Fixation Step 2 Periosteum FixationStep 4:Start by anchoring Tourniquetdown the corners. o When using a tourniquet, let tourniquet down and closely inspect the base of each defect to confirm that hemostasis has been achieved. o If hemostasis has not been achieved, apply throm- bin in pinpoint fashion at the source of bleeding. Suture fixation 1 Determine proper orientation of the periosteum and cover defect with cambium layer down (facing towards the defect). Identify cambium layer by: o Ink marked periosteum. o Cambium layer will appear glossy. o When inspected closely, the outer fibrous surface of the periosteum will reveal visible fatty tissue. 2 The periosteum must fit into, and not overlap, the edges of the defect. 3 Trim away excess tissue which overlaps cartilage surface prior to securing it to the cartilage. 4 Anchor periosteum at 4 corners of the defect. 5 A 6.0 Vicryl suture on a P-1 cutting needle is recommended to suture the periosteum to the defect rim. A variety of needle sizes and configurations may be needed depending on cartilage quality and/or defect location. 6 Lubricated sutures are necessary for passage through cartilage. Prior to use, coat or soak sutures and needle in sterile glycerin or mineral oil. 7 Angle needle towards periosteum surface approximately 2mm from its edge and pass through tissue into the cartilage wall following curvature of needle. Needle must enter cartilage wall approximately 2mm below the condylar surface and have a 3mm bite. 8 Tie-off sutures and place knots on the side of the periosteum ensuring knots are not prominent to the condylar surface. 9 Continue to place interrupted sutures in a diametrically opposed pattern (z-pattern).This will evenly distribute the tension of the periosteum and provide a secure, watertight fit.Step 7: Angle the needle toward the surface to obtain acartilage bite.Tie knot on side of periosteum, not on thecartilage rim. 21

Periosteum Fixation (continued) 3mm superior opening 3 to 4mm10 Space sutures approximately 3-4mm apart to achieve a watertight seal.11 Leave an opening large enough to accommodate an 18 gauge catheter at the most superior point of the defect.This opening will be used to test the periosteal integrity by injecting saline under the perios- teum to test for leakage prior to cell implantation. Key Points: Cut well-lubricated sutures to 8 inches. Follow the path and curvature of needle during placement. Do not force the path of the needle; this may damage the cartilage or periosteum. Periosteum and suture knots must be flush and not prominent to the surface of the defect; to assess knot placement, run finger over the periphery of the defect. If knots are prominent, push them off the cartilage rim toward the center of the periosteum. To test suture knot tightness, use forceps and lightly pull the sutures away from the cartilage. If the knots move or appear loose, they must be tightened. If no cartilage rim is available, suture the periosteum to the synovial membrane. Placement of sutures needs to be closer together to ensure watertightness. Smaller needles (P-2) are recommended when suturing into synovium.With very soft cartilage, longer needles (PC-1) will provide a longer bite. A vascular needle driver may be helpful when suturing the periosteum to a deep posterior defect.22

Watertight TestingStep 1: Periosteum WatertightEnsure that the superior opening Integrity Testingaccommodates an 18 gaugecatheter. A watertight seal serves two functions: prevents implanted chondrocytes from leaking out of the defect; and seals the defect from interior hemoarthrosis. a secure periosteum has a flush and taut appearance over the defect. 1 To test periosteum’s watertight integrity, use an 18 gauge angiocatheter tip or blunt needle to inject sterile saline into the defect through the superior opening. 2 Inspect for leakage around the perimeter of the defect at the periosteum and cartilage border. 3 If leakage is apparent, additional sutures will be necessary. 4 Once a watertight seal is achieved, aspirate all remaining saline from under periosteum. A dry defect bed enhances chondrocyte adherence and prevents dilution of cells.Step 2:Carefully check for leakageat the inferior apex of thedefect. 23

Autologous Fibrin Glue Application If you choose to use autologous fibrin glue, the following instructions are provided for the administration.* Autologous Fibrin Glue Apply fibrin glue one drop at a time around Fibrin glue consists of autologous the defect periphery. cryoprecipitate and a mixture of Thrombin and calcium chloride.To Additional fibrin glue is prepare, please consult Spotnitz needed when suturing WD, Mintz PD, Avery N, Bithell TC, to soft tissue. Kaul S, Nolan SP, “Fibrin glue from stored human plasma” The Key Points: American Surgeon, 1987, Vol. 53, pages 460-462 regarding its Y-needle may clog. Ensure additional units are readily available. preparation. Fibrin glue will dry in approximately 10 to 15 seconds. Application of Fibrin Glue1 Fibrin glue may be applied to the periphery of the defect at the periosteum-cartilage margin.2 When using autologous blood, the amount of fibrinogen will vary from patient to patient.The fluid consistency, and time to congeal and set will vary.3 Use a double-barreled syringe. Bring a single drop of fibrin glue to the tip of the Y-needle.4 When fibrin glue starts to congeal, place a drop on a plastic surface and note set time.This will indicate set time for glue solution.5 Proceed to defect periphery and again bring a single drop to the tip of the Y-needle and hold for several seconds. Apply drops as glue begins to congeal, one at a time, along entire suture line.6 If periosteum is of poor quality or contains tears, apply fibrin glue to the surface. Do not allow to leak under periosteum. Once fibrin glue application is complete and glue is dry, use syringe to inject approximately .4cc of air under patch. This will reveal if the periosteum is adhered to the defect bed, and if there is adequate space beneath the perios- teum for cell implantation. *In the databases evaluated for the approval of Carticel, over 90% of patients had fibrin glue applied during implantation of autologous cultured chondrocytes to prevent cell leakage.24

Cell Aspiration Cell Aspiration Vericel Exterior of the Carticel vial containing the cultured cells is NOT sterile. Follow strict sterileat start cell pellet aspirate resuspended technique protocols. medium cells Match the patient name and ID number on the certificate of analysis to the patient name on your patient chart and the patient ID number on the shipping box, transport cylinder and vial. See the figure to the left identifying the location of the patient ID number on each label. When treating a defect which requires multiple vials of cells, aspirate and inject one vial at a time. 1 Remove red plastic lid from vial.Wipe the vial surface and lid with alcohol. 2 Inspect vial contents for particulates, discoloration or turbidity.The cellular product appears as a yellowish clump in the bottom of the vial. Do not administer if contents appear turbid prior to cell suspension. 3 While holding vial in a vertical position, inset metal needle of the intraspinal catheter into the vial.The needle must be positioned just above the fluid level. Slowly remove the inner needle from the catheter, leaving the flexible tip behind.Attach tuberculin syringe to catheter. 4 Lower the catheter tip into the medium and position just above cell pellet.Aspirate all the medium from the vial leaving only the cell pellet behind. Slowly expel medium back into the vial.This action will break cell pellet and resuspend the cells in the medium. 5 Lower the catheter tip to the base of vial and aspirate all contents into syringe, leaving the vial empty. Slowly inject contents into the vial again.This will assure complete suspension of the cells. Repeat the steps as needed to ensure all cells are resuspended. Cell resuspension is complete when cell particles are no longer apparent, and the medium in a consistent, “cloudy” mixture.Aspirate all contents of vial into syringe.Always hold syringe vertical to keep an air pocket at the proximal end of syringe. 6 Keeping syringe in upright position (tip pointing down), grip the catheter and syringe at their junction to prevent separation. Slowly extract the catheter from vial while holding plunger in place to avoid drawback of cells. 7 When treating a defect which requires multiple vials of cells, aspirate and inject one vial at a time. 25

Cell Implantation Cell Implantation1 Always hold syringe vertical to keep an air pocket at the proximal end of the syringe.2 Insert catheter tip through superior opening of periosteum. Advance catheter to the most inferior aspect of the defect.3 Slowly inject a cell dose (see Dosage and Administration section of the package insert) while moving the catheter tip from side to side and withdraw the catheter proximally.This will ensure an even distribution of the cells throughout the defect.4 When treating multiple defects with only one vial, cell volume must be distributed proportionately with respect to defect size.5 Complete the implantation by closing the superior opening of the periosteum with additional sutures and fibrin glue. Key Points: Use luer lock twist to avoid separation of catheter from syringe. Always hold syringe with catheter tip facing down. Evenly disperse cells over entire base of defect. To avoid abrading or damaging the periosteum with the catheter, two openings in the periosteum may be necessary to inject the cells. 26

Notes 27

Notes28



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