Professor of Surgery Icahn School of Medicine New York, NY USA Vice Chair of Surgery Chief of Vascular/Endovascular Surgery Mt Sinai St Luke s and

Transcription

1 Professor of Surgery Icahn School of Medicine New York, NY USA Vice Chair of Surgery Chief of Vascular/Endovascular Surgery Mt Sinai St Luke s and West Hospitals New York, NY USA

2 I m bi-vascular with a strong touch of the cutaneous ie: I do open and endovascular surgery and a lot of wound care; Approximately 4-6 major vascular cases a week; 6-8 in patient wound cases a week and 70 outpatient wound patients and 30 vascular patients a week.. Paid Consultant Acelity, Smith & Nephew, Integra, Pluristem, MediWound, Acton, e-kare, Kerecis Principal investigator Acelity, Integra, Smith & Nephew, TissueTech, Pluristem Grant Support Smith & Nephew, Acelity, Integra

3 Remember when to think of a CTP Understand the characteristics of a (cod) fish skin derived extracellular matrix where it fits with CTPs Understand the translational implications of the basic science research to date Understand the clinical results to date for the use of this fish skin product in treating wounds

4 When to use a CTP

5 Early adoption of advanced or appropriate care may be more costeffective than traditional standard practices for decreasing the incidence of lower extremity amputation and may speed healing Papanas N, et al. Drug Saf. 2010;33: Kirsner RS. The standard of care for evaluation and treatment of diabetic foot ulcers. 5

6 4 months 2 years 5 years % 80% 92% Healed Not healed Nicolaides, A. N. Investigation of chronic venous insufficiency: A consensus statement (France, March 5-9, 1997). Circulation, 102: E126-E163, 2000.

7 Regenerative Medicine and the Chronic Wound Get back to the basics what makes your wound chronic? Proper patient care AND wound bed preparation is vital for healing success all DFU s must be offloaded for 4 weeks all VLUs compressed BUT - If DFU wounds haven t decreased in size by >50% at 4 weeks consider CTP; if VLU haven t decreased in size by >40% at 4 weeks consider CTP Poor predictors of outcome: DFU > 4 sq cm; > 1 year in duration; over boney prominence VLU > 12 sq cm;> 1 year in duration; significant comorbidities

8

9 To boost growth factor populations within wounds To help convert the wound environment from chronic to acute To jump start wound healing in chronic wounds To fill soft tissue defects filling a pothole To help get coverage over exposed deep structures

10 Extracellular matrix (ECM) serves as supporting scaffold for invading cells Cell adhesion to ECM is a prerequisite for maximal bioactivity of growth factors ECM molecules and growth factors regulate together cellular processes during wound healing Fish skin is a xenograft (acellular graft- ADG) that retains the natural ECM structure of the fish dermis Extracellular Matrix-Inspired Growth Factor Delivery Systems for Skin Wound Healing. Briquez PS, Hubbell JA, Martino MM.Adv Wound Care (New Rochelle) Aug 1;4(8): Review.

11 Availability and Support Ease of Use Insurance Effectiveness CTP Wound Parameters

12

13 Fish skin for medical use patented; fish skin consists of cells embedded in a network of non-living tissue Fish skin is CE Marked and FDA cleared Proteins and lipids in their natural state Decellularized and allergenic and bio compatible Source Icelandic Cod

14 Three classes of Cellular and Tissue Based Products (CTPs) Unique Components - Living cell donation with e ECM - Providing an ECM multiple potential mechanisms of action and types - Cyto-attractant therapy Mechanisms of Action - Provide signals OR - - Provide ECM structure

15 Modulatory to Template Mechanisms of Action Provide signals Modulatory (more frequent applications) Not so much incorporated but may; decrease MMP, enhance signaling, rebalance a wound Ex: Piscine skin; Ovine submucosa, Ovine bladder Mechanisms of Action Provide ECM structure Template (less frequent applications) Incorporated into the wound, provide a structural template; while providing mechanical as well other signaling mechanisms Ex: Fetal Bovine Collagen Ex: Dermal regenerative template

16 Two prospective blinded controlled trials in acute wounds

17

18 Acute 4 mm forearm punch biopsies; patients are their own controls Over the 28-day study period, participants had a total of 7 visits. On day three, participants were contacted by phone by the trial physician to collect information on the status of their wounds. The participants were seen on days, 7, 14, 18, 21, 25 and 28 for assessment of wound status. If the treatment material was intact, it was left in the wound. If it had reabsorbed or if treatment material was not visible, new material was applied to the wound. Standardized digital photographs were taken and healing assessment was performed by four wound healing professionals in an independent, blinded manner.

19 Blinded Randomized Clinical Trial 81 Healthy voulunteers 162 wounds fish skin vs SIS 4 mm Forearm punch model Significantly (p=0.041) faster healing No autoimmune reactions ELISA tests on RF, ANA, ENA, anti ds-dna, ANCA, anti-ccp, and anticollagen I and II. Healing Rate and Autoimmune Safety of Full-Thickness Wounds Treated With Fish Skin Acellular Dermal Matrix Versus Porcine Small-Intestine Submucosa: A Noninferiority StudyBaldur Tumi Baldursson, MD, PhD Hilmar Kjartansson, MD, Fífa Konrádsdóttir, MSc, Palmar Gudnason, MSc, Gudmundur F. Sigurjonsson, MEng Sigrún Helga Lund, PhD; The International Journal of Lower Extremity Wounds2015, Vol. 14(1) 37 43

20 85 healthy volunteers 170 Wounds 85 treated with Fish Skin v. 85 treated with dhacm No loss of follow up. Their average age was 24.1, with a standard deviation of 4.6 years. 66.7% of the cohort were women. Primary end point Wounds treated with fish skin healed significantly faster with a hazard ratio of 2.37 (95% CI: ) at a p-value of over dhacm allograft treated wounds. JC Lantis II MD, FACS; K Petursdottir, MD; B Baldursson; RS. Kirsner, MD, PHD, FAAD; D Weir, RN, CWON; Double-Blind, Prospective, Randomized Clinical Trial on 170 Acute Wounds Shows Significantly Faster Healing Rate with Intact Fish Skin Compared to Human Amniotic Membrane; APWCA September 6-8, Baltimore MD

21 100% 90% 80% Proportion Healed 70% 60% 50% 40% 30% 20% 10% 0% Days dhacm Fish skin JC Lantis II MD, FACS; K Petursdottir, MD; B Baldursson; RS. Kirsner, MD, PHD, FAAD; D Weir, RN, CWON; Double-Blind, Prospective, Randomized Clinical Trial on 170 Acute Wounds Shows Significantly Faster Healing Rate with Intact Fish Skin Compared to Human Amniotic Membrane; APWCA September 6-8, Baltimore MD

22 However, the immediate question is: What is the applicability of a study on acute wounds on the treatment decision for chronic wounds? The aggressive debridement (wound excision or ulcerectomy), which, along with offloading is the centerpiece of the care of diabetic foot ulcers is explicitly or implicitly attempts or is meant to return the wound dynamics from a chronic to an acute phase. Therefore the results on acute wounds may have implications for debrided chronic wounds. Also advantageous in this controlled experiment is that removes some biases inherent with clinical wound studies

23 Specifically the Hard to Heal Wounds

24 German Case study on 7 patients Marine Omega3 wound matrix for the treatment of complicated wounds Phlebologie 2016 Pain reduction in all patients Complete wound closure in all participants Complicated wounds-exposed bony segments Marine Omega3 wound matrix for the treatment of complicated wounds Experiences associated with amputations in the lower limb in diabetic patients T. T. Trinh; F. Dünschede; C.-F. Vahl; B. Dorweiler; Phlebologie 2016; 45: 93 98

25 Retrospective antimicrobial all comers wound study 54 non healing wounds were treated with fish skin grafts* for a period of four weeks and 2 applications on average. Improvement is defined as >20% reduction of wound area. Data was extracted from the patient records database at the Landspitali University Hospital in Reykjavik, Iceland. Study was approved by the National Bioethics Committee of Iceland (VSN ). Antibiotics therapy was not a predetermined endpoint since this was a retrospective study. Lantis J, Baldursson B, Magnusson S, Kjartansson, Sigurjonsson G. Acellular Fish Skin Graft for Surgical, Trauma, Venous and Arterial and Diabetic Wounds: A retrospective clinical study Oral session 17th Congress of the Asian Society for Vascular Surgery, October 20-23, 2016 Singapore

26 RESULTS Omega3 Fish Skin* for Chronic Wounds Treatment with fish skin grafts* showed a 38% reduction in antibiotics use. Overall, 87% of non healing wounds treated with fish skin grafts* either improved or healed after 4 weeks of treatment. No wound became worse in the course of the 4 week treatment with the acellular fish skin*. Wounds that either improved or healed completely: 83% (10) of surgical or traumatic wounds 92% (25) of venous leg ulcers 71% (5) of arterial ulcers 87% (7) of diabetic foot ulcers Diabetic Foot Ulcers Not responding 13% Healed 50% Improved 37% Surgical/Trauma Wounds Venous or mixed venous/arterial Not responding 8% Healed 22% Improved 70% Arterial Ulcers % Not responding 17% Healed 42% Improved 41% Not respondin g 25% Healed 13% Improved 62% Antibiotics-Before Antibiotics-After Lantis J, Baldursson B, Magnusson S, Kjartansson, Sigurjonsson G. Acellular Fish Skin Graft for Surgical, Trauma, Venous and Arterial and Diabetic Wounds: A retrospective clinical study Oral session 17th Congress of the Asian Society for Vascular Surgery, October 20-23, 2016 Singapore

27

28 Percentage of wound closure area from baseline after 5 weekly ECM applications in 18 patients with at least one hard to heal criteria. Application of the ECM for 5 sequential weeks, followed by 3 weeks of standard of care; Secondary wound dressings were applied per their underlying etiology. Wound area, signs of infection were collectedof infection and pain were assessed weekly visits. A Prospective, Postmarket, Compassionate Clinical Evaluation of a Novel Acellular Fish-skin Graft Which Contains Omega-3 Fatty Acids for the Closure of Hard-to-heal Lower Extremity Chronic Ulcers. Yang CK, Polanco TO, Lantis JC 2nd. Wounds Apr;28(4):112-8

29 18 patients 40% decrease in wound surface area was seen with 5 weekly applications (P<0.05) o o Notable reduction pain and drainage Lack of closure once product discontinued A Prospective, Postmarket, Compassionate Clinical Evaluation of a Novel Acellular Fish-skin Graft Which Contains Omega-3 Fatty Acids for the Closure of Hard-to-heal Lower Extremity Chronic Ulcers. Yang CK, Polanco TO, Lantis JC 2nd. Wounds Apr;28(4):112-8

30

31 A cost savings model more products should use Table 1. Liklihood of healing by prognostic factors. The table shows the likelihood that a patient with a given prognostic factor or combination of prognostic factors will heal after 20 weeks. 1 Table 2. Ulcer related health care cost per episode. The table displays the ulcer-related pharmacy, medical and total costs per week. 2 The cost-simulation study was based on a prognostic model for identifying diabetic foot ulcers that are not likely to heal. The model was generated by data from 27,630 patients with diabetic foot ulcers. 1 The model predicts the likelihood of a wound not healing after 20 weeks based on some of all of the following parameters being met: (1) Size > 2 cm 2 (2) Previous duration of wound > 2 months (3) The ulcer grade 3 The cost per week was then assigned to the two scenarios 2 with the additional cost of using the acellular fish skin graft product. 1. Margolis, D. J., Allen-Taylor, L., Hoffstad, O. & Berlin, J. A. Diabetic neuropathic foot ulcers: predicting which ones will not heal. Am. J. Med. 115, (2003). 2. Stockl K. et al. Costs of Lower-Extremity Ulcers Among Patients With Diabetes. Diabetes Care Sep;27(9): Lantis J, Magnusson S, Margolis D Baldursson BT, Kjartansson, G Sigurjonsson.G Cost Saving Potential of Acellular Fish Skin Graft: A Cost Simulation Study on Diabetic Foot Ulcers. 17th Congress of the Asian Society for Vascular Surgery, October 20-23, 2016 Singapore

32 DFU Treated with fish skin versus Predictive Model Unhealed Wounds Week Acellular Fish Skin* Standard Care Reduction in wounds with acellular fish skin* compared to modeled standard of care. At 20 weeks, 24 of the acellular fish skin graft patients had healed compared to 11 patients with the modeled standard of care group. 27 DFU patients Lantis J, Magnusson S, Margolis D Baldursson BT, Kjartansson, G Sigurjonsson.G Cost Saving Potential of Acellular Fish Skin Graft: A Cost Simulation Study on Diabetic Foot Ulcers. 17th Congress of the Asian Society for Vascular Surgery, October 20-23, 2016 Singapore

33 Total Cost (USD) Model of Potential Cost Savings Omega 3 Fish skin* vs Modeled Standard 160, , , ,000 80,000 60,000 40,000 20, Week Acellular Fish Skin* Standard Care Cost per week at 20 weeks was 28,576 USD for the fish skin* group compared to 93,575 USD for the modeled standard of care group. *ONLY CLOSED PATIENTS ANALYZED of Care Cost saved with the acellular fish skin graft* per healed wound was 62,5% Lantis J, Magnusson S, Margolis D Baldursson BT, Kjartansson, G Sigurjonsson.G Cost Saving Potential of Acellular Fish Skin Graft: A Cost Simulation Study on Diabetic Foot Ulcers. 17th Congress of the Asian Society for Vascular Surgery, October 20-23, 2016 Singapore

34

35 ECM what and why Histology does it matter? Are looks enough? What we know about composition Omega 3 fatty acids what we know so far; balancing inflammation? Bacterial resistance

36 Human dermis Fish dermis SEM pictures show the structure of human and fish dermis in high magnification. Bar 1µm. Figures: Regenerative and Antibacterial Properties of Acellular Fish Skin Grafts and Human Amnion/Chorion Membrane: Implications for Tissue Preservation in Combat Casualty Care Skuli Magnusson, BSc* ; Baldur Tumi Baldursson, MD, PhD* ; Hilmar Kjartansson, MD* ; Ottar Rolfsson, PhD ; Gudmundur Fertram Sigurjonsson, MEng* MILITARY MEDICINE, 182, 3/4:383, 2017

37 Fish Skin and Human Skin Comparison Fish Skin Human skin. Regenerative and Antibacterial Properties of Acellular Fish Skin Grafts and Human Amnion/Chorion Membrane: Implications for Tissue Preservation in Combat Casualty Care Skuli Magnusson, BSc* ; Baldur Tumi Baldursson, MD, PhD* ; Hilmar Kjartansson, MD* ; Ottar Rolfsson, PhD ; Gudmundur Fertram Sigurjonsson, MEng* MILITARY MEDICINE, 182, 3/4:383, 2017

38 The acellular fish skin has been compared to dehydrated human amnion/chorion membrane (dhacm) allograft, with respect to structure and ability to sustain fibroblast ingrowth in culture

39 Fibroblast seeding on Omega3 fish skin graft (left) and a human amnion/chorion membrane allograft (right). Results show that cells infiltrate and proliferate in the Omega3 Fish graft but not into the human amnion/chorion membrane allograft. Negative controls showed no cell staining. Regenerative and Antibacterial Properties of Acellular Fish Skin Grafts and Human Amnion/Chorion Membrane: Implications for Tissue Preservation in Combat Casualty Care Skuli Magnusson, BSc* ; Baldur Tumi Baldursson, MD, PhD* ; Hilmar Kjartansson, MD* ; Ottar Rolfsson, PhD ; Gudmundur Fertram Sigurjonsson, MEng* MILITARY MEDICINE, 182, 3/4:383, 2017

40 H&E images of cell infiltration into Fish Skin from in vitro experiments From human implants showing cell infiltration Regenerative and Antibacterial Properties of Acellular Fish Skin Grafts and Human Amnion/Chorion Membrane: Implications for Tissue Preservation in Combat Casualty Care Skuli Magnusson, BSc* ; Baldur Tumi Baldursson, MD, PhD* ; Hilmar Kjartansson, MD* ; Ottar Rolfsson, PhD ; Gudmundur Fertram Sigurjonsson, MEng* MILITARY MEDICINE, 182, 3/4:383, 2017

41 Cellular ingrowth Negative control Regenerative and Antibacterial Properties of Acellular Fish Skin Grafts and Human Amnion/Chorion Membrane: Implications for Tissue Preservation in Combat Casualty Care Skuli Magnusson, BSc* ; Baldur Tumi Baldursson, MD, PhD* ; Hilmar Kjartansson, MD* ; Ottar Rolfsson, PhD ; Gudmundur Fertram Sigurjonsson, MEng* MILITARY MEDICINE, 182, 3/4:383, 2017

42 Inflammation s role in the chronic wound And does adding fatty acids to the wounds work

43 /PIIS (17) /fulltext

44 Cell signaling for differentiation and migration Environment for tissue regeneration Anti-inflammatory effects Macrophage switch from M1 to M2 Decreased NF-kB and COX expression Bacterial barrier Cell membrane integrity 1, Adv Wound Care (New Rochelle) Nov 1; 3(11): doi: /wound PMCID: PMC Role of Arginine and Omega-3 Fatty Acids in Wound Healing and Infection J. Wesley Alexander 1,,2,* and Dorothy M. Supp 1 2. Wound Repair Regen May-Jun;16(3): doi: /j X x. Omega-3 fatty acids effect on wound healing. McDaniel JC 1, Belury M, Ahijevych K, Blakely W

45 /full 1. Metabonomics reveals drastic changes in anti-inflammatory/pro-resolving polyunsaturated fatty acids-derived lipid mediators in leprosy disease. Amaral JJ, Antunes LC, de Macedo CS, Mattos KA, Han J, Pan J, Candéa AL, Henriques Md, Ribeiro-Alves M, Borchers CH, Sarno EN, Bozza PT, Finlay BB, Pessolani MC.PLoS Negl Trop Dis Aug 15;7(8 2. Biochem Soc Trans Oct 15;45(5): doi: /BST Epub 2017 Sep 12. Omega-3 fatty acids and inflammatory processes: from olecules to man. Calder PC

46 Bone Marrow concentrat e Whole Bone Marrow Mesenchymal Stem Cells Negative Control Fatty acid rich group Fatty acid rich group Rodriguez-Menocalet al. Stem Cell Res Ther. 2015; 6(1): 24.

47 20% faster healing Wound model with lipid extract Speeds up the migration of cells in the wound bed Lantis J,Magnusson S, Baldursson B, Kjartansson H, Rolfsson O, Sigurjonsson G. Fish skin omega-3 PUFAs induce cell migration and transcription of ALOX15, a specialized pro-resolving mediator forming lipoxygenase. SAWC Fall, October 20-22, 2017, Las Vegas NV

48 Omega3 fatty acids activate specialized pro-resolvin pathways End product is reduction of prostaglandins and less inflammation Same downstream effects as NSAID drugs In this study they measured the activation of the pathway with the ALOX-15 gene expression. Lantis J,Magnusson S, Baldursson B, Kjartansson H, Rolfsson O, Sigurjonsson G. Fish skin omega-3 PUFAs induce cell migration and transcription of ALOX15, a specialized pro-resolving mediator forming lipoxygenase. SAWC Fall, October 20-22, 2017, Las Vegas NV

49 Lantis J,Magnusson S, Baldursson B, Kjartansson H, Rolfsson O, Sigurjonsson G. Fish skin omega-3 PUFAs induce cell migration and transcription of ALOX15, a specialized pro-resolving mediator forming lipoxygenase. SAWC Fall, October 20-22, 2017, Las Vegas NV Omega 3 naturally distributed in the skin scaffolding Bovine pericardium Fish Skin Human Skin Amnion/Chorion DHA and EPA types of Omega 3 Fatty acids Low in Omega 6 Omega- 6 Omega- 6 Omega- 3 Omega- 3

50

51 In the two-chamber assay the fish skin graft performed as a bacterial barrier for close to 60 hours compared to 5 45 hours for human amnion/chorion membrane products and several collagen matrix products. Spiking (>10%) the fish skin with more Omega3 fatty acids enhanced its bacterial barrier function by roughly 80% Regenerative and Antibacterial Properties of Acellular Fish Skin Grafts and Human Amnion/Chorion Membrane: Implications for Tissue Preservation in Combat Casualty Care Skuli Magnusson, BSc* ; Baldur Tumi Baldursson, MD, PhD* ; Hilmar Kjartansson, MD* ; Ottar Rolfsson, PhD ; Gudmundur Fertram Sigurjonsson, MEng* MILITARY MEDICINE, 182, 3/4:383, 2017

52 In this model mice were burned for 30 sec with brass blocks to create 3 burn. The wounds were infected 5 minutes post burn with x106 of P. mirabilis strain ATCC51393.

53 Supportive of use in the OR setting

54 Evaluation of Omega-3 Acellular Fish Skin Dermis on Porcine Deep Partial and Full Thickness Burn Wounds U.S. Army Institute of Surgical Research Combat Trauma and Burn Injuries Research Randolph Stone II, PhD

55 Dr. Christy s Lab Burn Injury Research Task Area Robert J. Christy, PhD Shan Natesan, PhD Christine Kowalczewski, PhD Nicholas Clay, PhD Ryan Clohessy, PhD Nicole Wrice, MS David Larson, MS John Wall, BS Andrew Kowalczewski, BS Kyle Florell, BS Sergio Garcia, BS Hannah Dillon, BS Sean Christy Office of Research Anthony Pusateri, PhD CPT Kottke, PhD USAISR Veterinary Support Branch Comparative Pathology Branch Battlefield Pain Research Funding Sources US Army Medical Research and Materiel Command Oak Ridge Institute for Science and Education Military Burn Research Program

56 The objective of this project is to evaluate decellularized fish skin graft on deep partial (DPT) and full thickness (FT) burn wounds in a preclinical porcine model. Evaluate fish skin graft as an acellular dermal matrix and its ability to integrate and improve healing of DPT burn wounds. Assess fish skin graft use as a temporary dressing prior to grafting and its ability to improve healing of highly meshed autografts on FT burn wounds. U.S. Army Institute of Surgical Research Combat Trauma and Burn Injuries Research, Randolph Stone II, PhD

57 Group number Burn Depth (Day -1) Primary treatment after excision of dead tissue (Day 0) mstsg Application (Day 7) 2nd Application of Kerecis (Day 7) 1 Full thickness Kerecis Yes (1.5:1) No 2 Full thickness Kerecis Yes (3:1) Yes 3 Full thickness Cadaver Skin Yes No 4 Deep partial thickness Primatrix No No 5 Deep partial thickness Kerecis No Yes 6 animals 12 wounds per treatment group

58 Images of Model Day 0 Post - Debridement

59 Day 0 Day 3 Day 7 Day 7 Day 10 Before After Grafting Grafting Day 14 DPT - Primatrix DPT - Primatrix DPT - Kerecis DPT - Kerecis

60 Day 21 Day 28 Day 35 Day 45 Day 60 DPT - Primatrix DPT - Primatrix DPT - Kerecis DPT - Kerecis

61 Day 14 Laser Speckle Blood Flow Day 14 Day 21 Day 28 Day 45 Day 60 DPT - Primatrix DPT - Primatrix DPT - Kerecis DPT - Kerecis

62 % R e e p ith e lia liz a tio n # # * 0 Day 0 Day 7 Day 10 * Day 14 Day 21 Day 28 Day 35 D a y 4 5 Day 60 D P T - D 0 (K e re cis) - D 7 (K e re cis) D P T - D 0 (P rim a trix) No Treatm ent = p<0.05 between treatments * # = p<0.05 vs. other groups

63 Primatrix Day 7 Day 14

64 Kerecis Day 7 Day 14

65 Day 21 Day 28

66 Day 21 Day 28

67 Kerecis product was easy to apply to wounds and not conducive to bacterial colonization Kerecis allowed faster re-epithelialization of DPT burn wounds when compared to a commercial comparator (Primatrix) The quicker re-epithelialization did not result in higher contraction rates as no treatment Hydration levels correlate with the re-epithelialization with a quicker return to normal skin levels with the Kerecis treatment Kerecis integrates quicker and a perfused wound bed is obvious at day 14 vs. Primatrix that takes ~21 days to fully integrate

68 Upcoming papers Large cohort of diabetic foot ulcers Chris Winters, DPM Analysis / comparison of Fish Skin to multiple other CTPs in a known military burn animal model Research needs Comprehensive analysis of content collagen, proteoglycan, glycosaminoglycan components of Fish Skin Tissue biopsies of human wounds at one week and one month post implantation histology Inflammatory marker analysis in wounds treated with Fish Skin and wounds not treated with Fish skin Trial of dosing strategy 2x week then 1 x week, then every other week versus 1x week (aka a leading amniotic)

69 A very efficient ECM Resistant to bacteria Readily available Anti-inflammatory Omega 3; Potential added pain reduction Can be grafted early at 7 days In the OR / Burn Resect the wound apply and skin graft early In the clinic Apply to prep and then close the wound