MPR488-INTEGRA-AMNIOEXCELPLUS whitepaper-V01-10 …ECM Degradation Bioburden (Bioﬁlm) Senescent Fibroblasts Impaired Cell Migration MMPs TIMPs CHRONIC ... endothelial cells, and

2 AmnioExcel® Plus AmnioExcel® Plus 3

Since its inception in 1989, Integra LifeSciences has forged a reputation as a world leader in medical technology and is dedicated to limiting uncertainty for healthcare practitioners, so they can focus on providing the highest standard of patient care. Integra off ers a broad range of innovative solutions for the care of complex wounds to help improve patient outcomes.

There are four distinct phases of wound healing; these are the hemostasis, infl ammatory, proliferative, and maturation/remodeling stages (fi gure 1).1

INTRODUCTION

Proliferation• Wounds transition from an infl ammatory phenotype to a constructive

phenotype

• Newly secreted ECM, including Type III collagen, fi lls and covers the wound

• Initially, the wound contracts through the alignment of myofi broblasts, thereby drawing the wound margins towards the center.

• Epithelial cells originate from the wound bed or margins, and close the wound

Remodeling• Collagen fi bers re-model from type III to type I with an increase in tensile

strength

• Fibroblasts contribute to the remodeling of newly deposited collagen once the wound has closed

• Remodeling usually begins 21 days post-injury and can last for up to two years

Proliferation

Hemostasis• Platelet aggregation stops bleeding and seals the wound opening

• Fibrinogen is catalyzed to fi brin to create a fi brin mesh, which strengthens the platelet plug and leads to the formation of a clot

Infl ammation• Neutrophils migrate into the wound to secrete antibacterial substances

and produce chemotactic factors to att ract cells necessary for the orderly progression of wound healing

• Neutrophils also remove bacteria, debris, and devitalized tissue

• Once neutrophils exit the wound, macrophages enter, and continueto remove debris and dead cells

• Heat, swelling, pain, and erythema (redness of the skin) are associated with this phase Remodeling

Figure 1: Four stages of wound healing: hemostasis and infl ammation1 Figure 1: Four stages of wound healing: proliferation and remodeling1

Hemostasis

Inflammation

Dynamic ReciprocityDynamic reciprocity is a process where cells detect, interpret, and respond to the signals from its microenvironment.

Wound healing involves interactions between cells and their microenvironment that consists primarily of

extracellular matrix (ECM). These interactions are bidirectional and continuously changing in response to signals

from the microenvironment. Dynamic Reciprocity is the interplay between all components of the system

(mechanical forces, growth factor signaling, etc.) and not just the ECM, that directs cell behavior (e.g., migration)

and the ability of the wound to progress through each stage of wound healing.1,2


CHRONIC WOUND HEALING

DAYS1 2 3 4 5 6 7 8 2221 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

O W D L

Infl ammation

Hemostasis

NORMMAL WOOUND HEALIING

DAYS1 2 3 4 5 6 7 8 2221 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Infl ammation

Hemostasis

Proliferation Remodeling

CHRONIC WOUNDS

Figure 2: Stages of wound healing over time in acute, typical (a) and non-healing or chronic wounds (b)3,4

a

b

Figure 3: Physiologic factors leading to non-healing or chronic wounds4

Normal wound healing, characterized by a well-coordinated progression through the phases of healing, is intended to restore the skin's barrier function and integrity.1 In chronic wounds, the infl ammatory phase is sustained and prevents the orderly progression of wound healing (fi gure 2a,b).1,3

There has been a concerted eff ort over the past two decades to examine the therapeutic eff ectsof various growth factors in the clinical management of non-healing wounds (e.g., pressure ulcers, chronic venous ulcers, and diabetic foot ulcers).5 Growth factors and cytokines are chemical messengers that regulate wound repair:

• Growth hormones promote cell migration, diff erentiation and proliferation, as well asECM production and remodeling5

• Cytokines att ract and activate cells, modulate cell growth, regulate infl ammation,and promote proliferation5

Non-healing or chronic wounds contain high levels of matrix metalloproteinases (MMPs) and reduced levels of cytokines, growth factors, and tissue inhibitors of metalloproteinases (TIMPs) (fi gure 3) that destabilize the ECM. The resulting tissue damage disturbs normal dynamic reciprocity, which delays or halts wound healing.1,4

Prolonged Inflammation

MMPs Destroy GFsFew GF Receptors

ECM Degradation

Bioburden(Biofilm)

Senescent FibroblastsImpaired Cell Migration

MMPsTIMPs

CHRONIC WOUND


The human placental membrane encircles the amniotic fl uid and the fetus as a protective barrier. The fetal

components of the placenta are composed of the amnion and chorion (fi gure 4).6 The amnion is the

innermost of the two placental membranes, nearest the fetus. Within the ECM that comprises the amnion and chorion membranes, is an abundance of growth factors and cytokines (table 1).6 The benefi cial wound management properties of the human amniotic membrane has been recognized for over a century, which has led to its common use in wound care.7

In addition to growth factors and other proteins, cytokines, and peptides, human

placental membrane also provides an intact collagen matrix, which plays an essential role in

wound management (table 1).6,8 Together, these factors have been shown to be critical in restoring

the balance necessary to promote the progression of healing of stalled wounds.4,6

Human placental membrane can also serve as a protection from the external environment by adhering to the wound

surface, thus reducing the risk of infection.8

HUMAN PLACENTAL MEMBRANE BENEFITS

The placenta is a protective barrier enclosing the fetus during development6

AM

NIO

NC

HO

RIO

N

Figure 4: The placental membrane. Consists of the amnion and chorion, which are each composed of several distinct layers

Epithelium

Compact layer

Fibroblast layer

Spongy layer

Cellular layer

Reticular layer

Trophoblast layer

Basement membrane

Basement membrane


HUMAN PLACENTAL MEMBRANE BENEFITSGrowth factors6,9 + bFGF

+ EGF + HGF + IGF-1 + KGF + PDGF-AA + PDGF-BB + PLGF + TGF-alpha + TGF-beta + VEGF + ANG

9 Stimulate migration, proliferation, and differentiation of fibroblasts, endothelial cells, and epithelial cells6

9 ECM production6

9 Promote re-epithelialization6

9 Promote angiogenesis6

9 Anti-scarring6

9 Anti-inflammatory10

Tissue inhibitor of metalloproteinases11

+ TIMP-1 + TIMP-2 + TIMP-4

9 MMP inhibition11

9 Reduces rate of ECM degradation12

9 Stimulates cell proliferation12

9 Promotes angiogenesis12

Anti-inflammatory/ immunomodulatory interleukins6

+ IL-1RA + IL-4 + IL-6 + IL-8 + IL-10 + IL-13

9 Anti-inflammatory6,10

9 Stimulates cell proliferation10

9 Induce cell migration10

9 Promotes angiogenesis10

9 Anti-scarring6

Collagen matrix6 + Fibril-forming collagen types I, III, V

+ Network-forming collagen type IV

+ Collagen type VI + Collagen type VII

9 Physical barrier8,13

9 Induce cell migration and proliferation1,13

9 Promote cellular differentiation13

9 Facilitate angiogenesis1

The following biological properties of human placental membrane makes it a highly-valued source of structural tissue for wound care:

9 Anti-inflammatory11,13

9 Low immunogenicity13

9 Low antigenicity13

9 Promotes re-epithelialization6

9 Anti-scarring13

9 Angiogenic6,14

9 Antimicrobial13

Table 1: Components of amniotic membranes and their function in wounds

10 AmnioExcel® Plus

THE SCIENCE BEHIND AMNIOEXCEL® PLUSHuman placental membrane consists mainly of structural collagen and other ECM components, and contains high concentrations of growth factors and cytokines. Donated placental tissue is processed to produce an implantable translucent membrane, AmnioExcel® Plus, suitable for a variety of uses, including biostructural covering to revitalize the process of wound repair in a complex sett ing.

AmnioExcel Plus is a human amnion-derived membrane, donated from consenting women and retrieved at the time of planned cesarean section during live childbirth. In accordance with FDA and AATB guidelines, placental tissues are screened for communicable diseases before processing. AmnioExcel Plus material treatment begins within hours aft er collection to preserve each component of the amnion membrane, with its distinct biochemical composition and in vivo functionality. This process minimizes the biologically destructive nature of dehydration and terminal sterilization associated with many modern processing technologies.

Figure 5: Structure of AmnioExcel Plus. AmnioExcel Plus H&E stain(a); AmnioExcel Plus H&E stain magnifi cation (amnion-chorion-amnion tri-layer) (b); confocal microscopy of AmnioExcel Plus top epithelium layer/surface (c1), compact layer (c2), and bottom epithelium layer/surface (c3) at 20x magnifi cation.

a

b

A focus on the DryFlex® process

The membrane is dehydrated using the

proprietary DryFlex® process, which

preserves the quality of the growth

factors, cytokines, and ECM found in

native placental tissue.

The tissue allograft that results provides

structural tissue (fi gure 5) to advance soft

tissue repair, replacement and

reconstruction. AmnioExcel Plus is

terminally sterilized and can be stored at

room temperature for up to fi ve years.

Cellular and ECM structures are

maintained in AmnioExcel Plus.

Amnion

Chorion

Amnionc3: Amnion epithelium

c2: Compact layer

c1: Amnion epithelium

AmnioExcel® Plus 11


Cellular Migration/ Proliferation & Differentiation

ECM Production/Remodeling

AngiogenesisImmune

Modulation

GROWTH FACTORS

bFGF + + +

EGF + +

HGF + +

IGF-1 +

KGF +

PDGF-AA + + +

PGDF-BB + +

PLGF +

TGF-alpha + +

TGF-beta + +

VEGF +

ANG + +

INTERLEUKINS

IL-1RA +

IL-4 +

IL-6 + +

IL-8 + +

IL-10 +

IL-13 +

TISSUE INHIBITOR OF METALLOPROTEINASES

TIMP-1 + +

TIMP-2 + +

TIMP-4 + +

Table 2 . Key protein molecules retained in AmnioExcel Plus

Figure 6. Enhanced cell migration by AmnioExcel Plus in an in vitro assay. Scratch test imaged at 20x with bright-field and Hoechst stain at t=0, t=6, and t=24 hrs (a); Number of migrated cells (b). Adult human dermal fibroblasts (plated at 260,000 cells/ml for 24 hrs) in chambers of an Ibidi 2-well scratch assay (Fisher Scientific, Atlanta, GA) in complete fibroblast growth media.

AmnioExcel® Plus Placental Allograft Membrane promotes enhanced cell migration. As shown in an in vitro scratch assay (figure 6a,b), cell migration into the acellular space was over five times greater when AmnioExcel Plus was applied versus no placental membrane.

THE SCIENCE BEHIND AMNIOEXCEL® PLUS

Basal Media

T = 0hr

Bright-field (20x magnification)

Fluorescent (Hoechst stain)

T = 6hr T = 24hr T = 24hr

AmnioExcel® Plus

0

50

100

150

200

250

300

41Mig

rate

d ce

lls a

t 24

hour

s (ce

lls/m

m2 )

Basal Media AmnioExcel® Plus

215

a

b


THE SCIENCE BEHIND AMNIOEXCEL® PLUS

0%

20%

40%

60%

80%

100%100%

91% *

98% **94% *

50% *

Perc

enta

ge re

duct

ion

in in

flam

mat

ion

com

pare

d to

con

trol

TNF-alpha IL-1beta IL-6

AmnioExcel Plus

Reduction in pro-inflammatory cytokines

IL-8Cytokines(positive control)

AmnioExcel Plussuppresses

pro-inflammatorycytokines

AmnioExcel Plus membrane retains potent anti-inflammatory properties as shown in an in vitro inflammatory response assay. With AmnioExcel Plus, concentrations of pro-inflammatory mediators are reduced by more than 90% for TNF-alpha, IL-1beta, and IL-6, and by 50% for IL-8 (figure 8).

Figure 8. AmnioExcel Plus possesses anti-inflammatory properties. *p<0.001; **p<0.01

AmnioExcel Plus membrane promotes fibroblast cell proliferation and ECM deposition and matrix formation as demonstrated in figure 7. As shown by day seven, fibroblast cells are highly proliferative and have laid down a significant ECM network of type I collagen supported by AmnioExcel Plus membrane.

AmnioExcel® Plus Placental Allograft Membrane promotes cell proliferation and ECM deposition.

Figure 7. AmnioExcel Plus promotes cell proliferation and ECM deposition. Adult human-derived fibroblasts were seeded on AmnioExcel Plus membranes for t=4 and t=7 days. Fluorescent stains for collagen type I (red) and actin cytoskeleton (green) and nuclei (blue) counterstained with DAPI (4',6-diamidino-2-phenylindole), a DNA-specific fluorescent probe.

4 days 7 days


CONCLUSIONAmnioExcel® Plus Placental Allograft Membrane is the next generationof amniotic tissue:

+ Tri-layer membrane contains higher amounts of bioactive† growth factors*

+ Aids in creating the environment to help close complex wounds

+ Thicker graft with non-side specifi c application, is easy to handle and conformsto the wound surface*

AmnioExcel® Plus membrane:

+ Placental tissue allograft composed of three layers: amnion, chorion, amnion

+ DryFlex® processing preserves the quality of the growth factors, cytokines andECM (extracellular matrix) found in native placental tissue*

+ Dehydrated and terminally sterilized with a fi ve year shelf life at room temperature

+ Designed for use as a wound covering

Chronic wounds exhibit impaired healing that have failed to progress through normal stages of healing.15

Such wounds frequently enter a state of pathologic infl ammation due to a postponed, incomplete, or uncoordinated healing process (i.e., loss of dynamic reciprocity).15

Human placental membrane contains growth factors, cytokines, ECM components, and an intact collagen matrix.5

Together, these elements have been shown to be critical in restoring the balance necessary to promote the progression of healing stalled wounds.4

The following biological properties of human placental membrane make it a highly-valued sourceof structural tissue for wound care:

9 Anti-infl ammatory11,13

9 Low immunogenicity13

9 Low antigenicity13

9 Promotes re-epithelialization6

9 Anti-scarring13

9 Angiogenic6,14

9 Antimicrobial13

We thank the mothers who donated for the benefi t of the product development. AmnioExcel Plus membrane is derived from donated placental tissue from healthy mothers during planned C-sections. Integra takes pride in having certifi cation from the AATB to ensure product quality.

KEY BENEFITS

* Data on fi le, all comparisons are vs. AmnioExcel † As detectable through ELISA testing


REFERENCES1. Schultz GS, Davidson JM, Kirsner RS, Bornstein P, Herman IM. Dynamic reciprocity in the wound microenvironment. Wound Repair Regen.

2011;19(2):134-148.

2. Thorne JT, Segal TR, Chang S, Jorge S, Segars JH, Leppert PC. Dynamic reciprocity between cells and their microenvironment in reproduction. Biol Reprod. 2015;92(1):25.

3. Gurtner GC, Wong VW. Chapter 2: Wound healing: normal and abnormal. In: Thorne CH, Chung KC,‎ Gosain AK, et al, eds. Grabb and Smith's Plastic Surgery. 7th ed. Wolters Kluwer; 2013:42-57.

4. Widgerow AD. Deconstructing the Stalled Wound. Wounds. 2012;24(3):58-66.

5. Barrientos S, Brem H, Stojadinovic O, Tomic-Canic M. Clinical application of growth factors and cytokines in wound healing. Wound Repair Regen. 2014;22(5):569-578.

6. Brantley JN, Verla TD. Use of placental membranes for the treatment of chronic diabetic foot ulcers. Adv Wound Care (New Rochelle). 2015;4(9):545-559.

7. Davis JW. Skin transplantation with a review of 550 cases at the Johns Hopkins Hospital. Johns Hopkins Med. 1910;15:307-396.

8. Parolini O, Solomon A, Evangelista M, Soncini M. Human term placenta as a therapeutic agent: from the first clinical applications to future perspectives. In: Berven E, ed. Human Placenta: Structure and Development. Hauppauge, NY: Nova Science Publishers; 2012:1-48.

9. Koob TJ, Lim JJ, Massee M, Zabek N, Denozière G. Properties of dehydrated human amnion/chorion composite grafts: Implications for wound repair and soft tissue regeneration. J Biomed Mater Res B Appl Biomater. 2014;102(6):1353-1362.

10. Owen JA, Punt J, Stranford SA, Jones PP, Kuby PP. Kuby Immunology. 7th ed. New York, NY: W.H. Freeman, 2013.

11. Hao Y, Ma DH, Hwang DG, Kim WS, Zhang F. Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane. Cornea. 2000;19(3):348-352.

12. Murphy G. Tissue inhibitors of metalloproteinases. Genome Biol. 2011;12(11):233.

13. Fetterolf DE, Snyder RJ. Scientific and clinical support for the use of dehydrated amniotic membrane in wound management. Wounds. 2012;24(10):299-307.

14. Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev. 2003;83(3):835-870.

15. Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res. 2010;89(3):219-229.

General Use

AmnioExcel® Plus membrane is intended for use as a wound covering. This product is an allograft tissue intended for homologous use for the repair, reconstruction and replacement of skin at the discretion of a physician.

Clinical Applications Include:• Chronic and acute wounds• Diabetic ulcers• Venous & arterial ulcers• Pressure ulcers• Traumatic injuries• Burns• Surgical wounds

AmnioExcel Plus Membrane is regulated as a Human Cellular and Tissue-Based Product (HCT/P) under Section 361 of the Public Health Service Act and is governed by the FDA Center for Biologics Evaluation and Research (CBER).

Availability of these products might vary from a given country or region to another, as a result of specific local regulatory approval or clearance requirements for sale in such country or region.

• Non contractual document. The manufacturer reserves the right, without prior notice, to modify the products in order to improve their quality.

• Warning: Applicable laws restrict these products to sale by or on the order of a physician.

• Consult product labels and inserts for any indication, contraindications, hazards, warnings, precautions, and instructions for use.

AmnioExcel, DryFlex, Integra and the Integra logo are registered trademarks of Integra LifeSciences Corporation or its subsidiaries in the United States and/or other countries. ©2018 Integra LifeSciences Corporation. All rights reserved. Printed in USA. 0925243-1-EN

For more information or to place an order, please contact:United States 1-800-654-2873 • 888-980-7752 faxInternational +1 609-936-5400 • + 1 609-750-4259 fax

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MPR488-INTEGRA-AMNIOEXCELPLUS whitepaper-V01-10 …ECM Degradation Bioburden (Bioﬁlm) Senescent Fibroblasts Impaired Cell Migration MMPs TIMPs CHRONIC ... endothelial cells, and