Scientific & Clinical Compendium - Aberdeen Investment … · 2 EpiFix® Scientific & Clinical...

Scientific & Clinical Compendium March 2013

EpiFix® PRODUCT DESCRIPTION EpiFix® Amniotic Membrane Allograft is a unique material comprised of dehydrated human amnion/chorion membrane (dHAM). The EpiFix® graft is minimally manipulated and made available for transplantation after it has completed the patented PURION® Process. EpiFix® allograft contains active growth factors, specialized cytokines and structural extracellular matrix (ECM) proteins.

EpiFix® CONFIGURATIONS EpiFix® Amniotic Membrane Allograft: This amnion/chorion configuration is delivered as a dry sheet embossed with the monogram “SB”, which enables the user to identify the correct orientation prior to the application of the graft to ensure optimal therapeutic benefit. The allograft may be used as presented upon opening the package, or alternatively by moistening prior to use with normal saline solution. EpiFix® is supplied in multiple sizes and is designed for single use in a single patient.

EpiFix® Micronized Injectable Amniotic Membrane Allograft: This amnion/chorion configuration presents as a powder that may be used topically as powder or mixed with saline for injection. Alternatively, EpiFix® Micronized Injectable can be mixed with sterile saline to create a liquid (micro-grafts are suspended in the saline) for injection into or adjacent to the targeted damaged dermal tissue. EpiFix® Micronized Injectable is available in several sizes and is designed for single use (single or multiple injections sites) in a single patient.

PACKAGING All PURION® Processed amniotic membrane allografts are dehydrated and packaged aseptically into an inner peel pouch and sealed with an outer peel pouch system within a clean room environment. The outer peel pouch is NOT considered sterile. The inner pouch, which contains the graft, is considered sterile unless damaged or compromised.

SOURCE OF AMNIOTIC TISSUE Eligible amnion donors are living mothers that have delivered a live birth through Caesarean section. All tissues are recovered under full informed consent of the donor. Each donor must then answer a series of questions to ensure the donor has not engaged in behaviors to place her at an increased risk for the transmission of infectious diseases and to ensure the donor has not shown signs or symptoms of illnesses. Donor procurement and screening processes were developed to prevent the transmission of infectious diseases from donors to recipients of the material. These processes follow the FDA regulations and American Association of Tissue Banks (AATB) standards.

2 EpiFix® Scientific & Clinical Compendium MiMedx Group, Inc.

- Basement membrane: Collagen III, IV, V, laminin, fibronectin - Compact Layer: Collagen I, III, V, VI, fibronectin

- Fibroblast Layer: Collagen I, III, VI, laminin, fibronectin

- Basement Membrane: Collagen IV, fibronectin, laminin- Fibroblast Layer; Collagen I, III, IV, V, VI, proteoglycans

AMNION

CHORION

Histology cross section of EpiFix® amniotic membrane allograft at 40xFIGURE 1

THE PURION® PROCESS The PURION® Process is a patented process that safely and gently cleans the tissue. The membrane layers are then dehydrated in a way that preserves the key elements associated with healing. The sterilized tissue is packaged and stored at room temperature and has a 5 year shelf life. The tissue may be delivered in a dried sheet configuration using an onlay surgical or clinical technique. Dehydrated human amnion/chorion membrane allografts can also be micronized to create a powder configuration that can be administered as a topical powder or can be mixed with a saline to create an injectable solution. For more information on the PURION® Process ask your account executive for a copy of the “PURION® Processed Dehydrated Human Amnion/Chorion Membrane Allograft” Monograph (SB123.006).

IMMUNE RESPONSE The amnion and chorion layers of human amniotic membrane serve as the principle tissue separation between mother and child. The layers do not have vascularity nor do they present antigens that the mother or child would react with during the pregnancy.1 This lack of antigens enables tissue transplants to take place without the need to match blood types or other indicators that would cause a rejection. Amniotic tissues have shown little to no HLA-A, B, C antigens and β2 microglobulin.2 This is clinically supported by the fact the tissue delivers multiple immunosuppressive cytokines (IL-10 and TGF-b)3 and over 85,000 implants have taken place to date. The company has not received any adverse event reports attributed to the allograft. An extensive literary search was conducted and no peer review papers note amnion or chorion producing an immune response.

FDA REGULATION OF HUMAN AMNIOTIC TISSUE PURION® Processed dehydrated human amniotic tissue is regulated under Section 361 of the Public Health Service Act by the United States Food and Drug Administration (FDA). PURION® Processed dehydrated allografts are minimally manipulated and intended for homologous use.

EpiFix® Scientific & Clinical Compendium MiMedx Group, Inc. 3

FDA Regulatory Classifications for Tissue and Cell Based ProductsTABLE 1

361 HCT/Ps (Human Cell, Tissue and Cellular and Tissue based Products)

510(k) Clearance

Premarket Approval (PMA)

Biologic License Application (BLA)

New Drug Application (NDA)

Human Tissue (Allograft)

Medical Device (Example: decellularized human dermis, xenografts, collagen dressings, bone void filler)

Medical Device (Example: human living skin substitutes, bone substitute)

Biologic product (Example: Cell products, such as those containing hematopoietic progenitor cells, vaccines, and blood components)

Drug product (Example: living stem cells non-autologous, second degree relative, or autologous stem cells that are expanded in the laboratory)

Minimally manipulated, intended for homologous use. No clearance or pre-market approval required. Requires FDA Good Tissue Practices (GTP)

Requires FDA Substantial Equivalence, shorter submission and less required verses PMA. Based on predicate device. Requires FDA Current Good Manufacturing Practice (cGMP)

Requires extensive FDA Pre-Market approval process, including comprehensive clinical trials. Requires FDA Current Good Manufacturing Practice (cGMP)

Requires extensive FDA Pre-Market approval process, including comprehensive pre-clinical and clinical trials. Requires compliance to FDA Current Good Manufacturing Practice (cGMP)

Requires extensive FDA Pre-Market approval process, including comprehensive clinical trials. Requires FDA Current Good Manufacturing Practice (cGMP)

SCIENTIFIC EVIDENCEGROWTH FACTORS FOUND IN EpiFix® The following growth factors are known to be present within EpiFix®: epidermal growth factor (EGF), transforming growth factor alpha and beta (TGF-α&β), basic fibroblast growth factor (bFGF), platelet derived growth factors (PDGF AA & BB), and vascular endothelial growth fact (VEGF).4

SPECIALIZED CYTOKINES FOUND IN EpiFix® Some of the known specialized cytokines and proteins found in dHAM include: Interleukin I receptor antagonist (IL-1ra), Interleukin 4 (IL-4) and Interleukin 10 (IL-10), which are immuno-supporessive cytokines that could contribute to the immuno-privileged properties of tissue.8

UNIQUE ENZYME INHIBITORS Matrix Metalloproteases (MMPs) are one of the contributing factors to non-healing wounds. MMPs are produced by cells to degrade ECM components to assist with cell migration and debris clean-up after tissue has been damaged. In chronic wounds, MMPs can go unchecked, due to a patient’s inability to produce enough tissue inhibitors of metalloproteases (TIMPs) which are responsible for inhibiting these MMPs. Excessive MMPs can cause major degradation of newly formed granulations tissue which, in turn, causes a delay in healing. Some of the TIMPs in EpiFix® are TIMP-1, 2 and 4.

FACTOR PRIMARY ACTIVITY5

TABLE 2

EGF

TGF-α

TGF-β

bFGF

PDGF

VEGF

The best documented activity of EGF is its ability to promote proliferation and differentiation of mesenchymal and epithelial cells. In vitro, EGF is a proliferation factor for fibroblasts, epithelial and endothelial cells, and promotes colony formation of epidermal cells in culture. In vivo, EGF induces epithelial development and promotes angiogenesis (new blood vessel growth).Important for normal wound healing, TGF-alpha shows a similar potency to EGF as a mitogen (stimulated cell division) for fibroblasts and as an inducer of epithelial development in vivo. TGF-alpha is reportedly more potent than EGF as an angiogenic factor in vivo and as a stimulator for keratinocyte migration.TGF-beta is proposed to act as cellular switches that regulate processes such as immune function, proliferation, epithelial-mesenchymal transition, and wound healing.Promotes proliferation of many cells; The Fibroblast Growth Factors (FGFs) constitute a large family of proteins involved in many aspects of development including cell proliferation, growth, and differentiation. They act on several cell types to regulate diverse physiologic functions including angiogenesis, cell growth, pattern formation, embryonic development, metabolic regulation, cell migration, neurotrophic effects, and tissue repair. Platelet-derived growth factor (PDGF) is a major mitogen (promotes cell division) for connective tissue cells and certain other cell types. In-vitro, activation of PDGF receptors leads to stimulation of cell growth, but also to changes in cell shape and motility; PDGF induces reorganization of the actin filament system and stimulates chemotaxis, i.e., a directed cell movement toward a gradient of PDGF. In vivo, PDGF has important roles during the embryonic development as well as during wound healing.6

In vitro, VEGF is a potent endothelial cell mitogen (promotes cell division). VEGF has been shown to stimulate von Willebrand factor release from endothelial cells and induce expression of tissue factor activity in endothelial cells as well as in monocytes. VEGF has also been shown to be chemotactic. In vivo, VEGF can induce angiogenesis as well as increase micro-vascular permeability. The modified extracellular matrix subsequently promotes the migration of macrophages, fibroblasts and endothelial cells. Based on its in vitro and in vivo properties, VEGF is expected to play important roles during normal and pathological angiogenesis, a process that is associated with wound healing and embryonic development.5


ECM FOUND IN EpiFix® Major structural proteins found in amnion/chorion and its anchoring basement membranes include: collagen types (I7, III7, IV7, V7, and VII8), fibronectin7, laminins7, and proteoglycans9.

GROWTH FACTORS RELEASE PROFILE The growth factor release profiles have been tested in EpiFix®. This list is a sample of growth factors MiMedx® has determined to be present within EpiFix®: platelet derived growth factor (PDGF AA & BB), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), epidermal growth factor (EGF), and vascular endothelial growth factor.

PURION® Processed dehydrated human amnion/chorion was micronized into a powder-like material. Neutral saline was used to rehydrate the micronized amniotic tissue and then centrifuged for ten minutes to create a supernatant liquid and solid material pellet. The supernatant liquid was tested for PDGF-AA, PDGF-BB, bFGF, TGF-β, and bFGF using ELISA protocols. The results showed that less than 50% of the TGF- β, bFGF, PDGF-AA, and PDGF-BB were found to be released into solution, except for EGF. EGF released 63% in to solution (Table 3).

As the remaining solid is absorbed over time, it has been proposed that the growth factors still bound into the extracellular matrix are released into the surrounding tissue, providing a continual release of growth factors during the tissue regeneration process10 (Figure 2).

THE ROLE OF THE CHORION

The amniotic layer is the inner lining of the amniotic sac and is derived from the embryo. The adjacent layer, the chorion, surrounds the amniotic layer and is also derived from the embryo. The proprietary PURION® Processed EpiFix® grafts include both membranes. The chorion is included because of the substantial amount of ECM, growth factors and cytokines within the layer4, and it does not contain antigens1.

While some tissue processors have decided to exclude the chorion layer, MiMedx® has chosen to include it. This is made possible by the proprietary processing technology. No published data supports presence of an immunologic effect on a patient from the chorion. Single layered and amnion only membrane grafts are typically thinner, less durable, and faster resorbing.

Native Growth Relative Amount Release Profile (n=5) Factors Present Determined by ELISA 24 hr incubation period at 4˚C Assay (n=5) Remaining in Tissue

PDGF-AA +++++

PDGF-BB +

bFGF ++

TGF-b1 ++

EGF +

*Growth factors were measured by ELISA in the incubation fluid and in the tissue.

13%

4%

44%

31%

63%

87%

96%

56%

69%

37%


TABLE 3

FIGURE 2

FIGURE 3

GROWTH FACTOR ACTIVITY In vitro experiments were performed to determine whether dHAM affects human cell proliferation and migration. The experiments confirm that growth factors and cytokines found in EpiFix® stimulate cell migration and proliferation. These results were confirmed by outside laboratory testing.

The effects of dehydrated human amniotic membrane on cell proliferation were assessed in vitro using human dermal fibroblasts (HDF).10 Test media was generated through the incubation (37°C for 16 hours) of 2 mL serum free culture media with varying amounts of dehydrated human amniotic membrane (1 and ½ of a 16 mm diameter membrane disk). In addition, serum free and 10% serum culture media were also incubated to provide negative and positive controls respectively. The HDF cells were incubated (37°C, 5% CO2) with test media, negative and positive controls for 72 hours. Cell proliferation was then measured using XTT cell proliferation assay.

Samples of dHAM were incubated in normal saline at 37°C for 1, 8, 24, 48, or 96 hours. At each time point, the incubation fluid and tissue were collected and the amount of the indicated growth factors was measured by ELISA assay. For one set of samples, after 96 hours, the tissue was digested with bacterial collagenase and the amount of growth factors in the digestion was measured by ELISA. The trend line illustrates the means for five replicate samples at each time point.

The time release properties of PDGF-AA, bFGF, and TGF-β.

Cell proliferation amounts based on membrane size (n=3)

ABSO

RBAN

CE


CULTUREMiMedx® Internal Report “The effects of EpiFix on cell proliferation In-Vitro” Research conducted by third party.

In all cases, the PURION® Processed dehydrated human amniotic membrane material showed statistically significant increase of cellular proliferation when compared to serum free control (Figure 3). The effects of dehydrated human amniotic membrane on human cell migration were evaluated in vitro in a trans-well culture apparatus according to established methods (Figure 4). Human cells were cultured in the upper chamber and PURION® Processed dehydrated human amniotic membrane was placed in the lower chamber.11

** = P <0.01 and * = P <0.05 in relation to serum free media treatment.

Serum free 10% Serum 1 disk 1/2 disk

1.4

1.2

1

0.8

0.6

0.4

0.2

0

Dehydrated Human Amniotic Membrane Dehydrated Human Amniotic Membrane

*

FIGURE 4 Trans-well culture apparatus


The number of human cells migrating through the porous membrane was counted after 24 hours of culture. Cell counts of migrating cells per micrograph relative to the FBS positive control are depicted in Figure 5.

Statistically higher cell counts were observed in High (12x13mm or 156mm2) and Medium-sized (4mm diameter disk or 12.6mm2) samples relative to the Low (1.5mm diameter disk or 1.77mm2) counterpart and the No Serum negative control.

FIGURE 5 Percent of cell migration relative to positive control (n=6)

% m

igra

tion

MiMedx® Internal Report - SBR -100005.00 “Amnion-influenced cell migration” Research conducted by third party.

* indicates significantly higher (p ≤ 0.05) migration rate than both No Serum and Low group

*

High156mm2

100

90

80

70

60

50

40

30

20

10

0Low

1.77mm2Medium12.6mm2

No Serum

Figure 5 demonstrates that increased cell migration correlates with higher amounts of PURION® Processed dehydrated human amniotic membrane.

CLINICAL DATAEnhancing Soft Tissue HealingA number of studies demonstrate the clinical effectiveness of using dehydrated human amniotic membrane allografts. Areas of clinical work include treatment of diabetic foot ulcers, venous leg ulcers, Mohs surgery, and radiation desquamation, among others. One prospective, randomized, single-center clinical trial compared the proportion of diabetic foot ulcers completely healed by use of EpiFix® every other week, plus standard of care (SOC) versus a SOC protocol of advanced wound care dressings in patients with a nonhealing diabetic foot ulcer with adequate arterial perfusion. Following surgical debridement, all patients underwent weekly dressing changes and the graft was applied under a non-adherent dressing. In the EpiFix® group, 92 percent of patients healed completely in 6 weeks compared to 8 percent of the SOC group.12

In a case series on the treatment of chronic wounds, Forbes and Fetterolf found that the use of dehydrated human amniotic membrane demonstrated improved healing over the use of previously ineffective traditional treatment modalities.13 Wounds included chronic as well as acute traumatic injuries, and the patients treated responded over the course of several weeks after application of EpiFix® where a non-healing wound was observed.

In a clinical report on EpiFix® application on lower extremity wounds, Shah conducted a review to determine whether soft tissue enhancement is achieved within a wound bed after application of amniotic membrane allograft to select patients with non-healing lower extremity ulcers. The cases confirmed the applicability of the use of EpiFix® Human Amniotic Membrane Allograft to the treatment of lower extremity ulcers with significant wound healing progress over one to four week period.14

Pre-Operative Observation: 34 year-old insulin independent diabetic. The total surface area of the wound was 18.76 cm2. The wound was extended to the muscles but no bone was exposed.


Prior to Treatment Post-Debridement - Day 0 Post-EpiFix® Treatment - 6 weeks

Diabetic Foot Wound

Mohs Surgery is microscopically controlled surgery used to treat common types of skin cancer. Closure techniques include suture, autograft, reconstructive surgery, and closure by secondary intent. Through a series of case studies, EpiFix® used post-Mohs surgery has been shown to cause less inflammation, reduce scarring and enhance healing.


First EpiFix® application 1 week post-application 3 weeks post-applicationRadiation Desquamation

EpiFix® Application 10 months after EpiFix® 2 Weeks post-application

Mohs Surgery

Reducing ScarringDehydrated human amniotic membrane allografts help reduce scarring, as demonstrated in a retrospective study that reviewed the use of amnion-based allograft membrane to prevent post-operative scarring between the tendon, peritendonous structures, and overlying skin.15 Patients were evaluated at an average of 1.7 years post-surgery. Of 14 patients, 86 percent were clear of scarring around the surgery site and 93 percent were scar tissue-free at the tendon repair site. Of the patients with signs of scar tissue, the effects were reported as mild or moderate. The findings were statistically significant with p=0.012.

Photos courtesy of Janice Warner, MD

EpiFix® VS OTHER AMNIOTIC MEMBRANE MATERIALS

Processor Product Amnion Chorion Ease of Storage Shelf Sterilized Storage Temp Life

MiMedx® EpiFix® X X Dry Room 5 years Yes

Bio-Tissue AmnioGraft® X Must be frozen -80° F 2 years Not Sterilized

BioDlogics BioDfence X Wet Room 2 years Glutaraldehyde

BioDlogics BioDfence DryFlex X Dry Room ? YesMusculoskeletal

AmnioClear® X X Dry Room 2 years Not SterilizedTransplant Foundation®

Comparison of amniotic tissue16

PURION® Processing AdvantageEpiFix® vs. Cryopreserved Amniotic Membrane16

There are many ways to process and store amniotic tissue. Studies show that EpiFix® contains multiple active growth factors. An internal study shows that the cryopreserved amniotic tissue allografts from competitors yielded less growth factor content than EpiFix® (n=3).

EpiFix® vs. Other Amniotic Membranes Stored at Room Temperature16

PURION® Processing of amniotic tissues clearly outperforms other amnion processing techniques by preserving the natural growth factors present in amniotic tissue. EpiFix® and three competitive room temperature stored membranes were tested for relative quantities of growth factors. In the study, EpiFix® out measured all three competitive products. EpiFix® had a higher quantity and variety of growth factors preserved in the tissue (Figure 6).

FIGURE 6 Normalized Growth Factor Values of EpiFix® Compared to Competitive Room Temperature Stored Grafts

PDGF-AA

PDGF-BB

EGF

BFGF

TGF-β1

100%

80%

60%

40%

20%

0%EpiFix®* (n=3) AmnioClear® (n=5) BioDfence (n=1) BioDfence DryFlex (n=1)

* EpiFix® growth factor values are equal to 100% to compare the relative amounts of growth factors present in each of the competitive products.


SUMMARYThe use of human amniotic membrane over the past 100 years has produced a significant amount of data in multiple areas of medicine. Based upon relevant immuno-histochemisty, ELISA, cell migration and proliferation studies conducted by MiMedx® and independent laboratories on PURION® Processed dehydrated human amniotic membrane, it is clear the patent protected PURION® Process provides a minimally manipulated and carefully preserved amniotic tissue that contains essential growth factors and extracellular matrix within the membrane. The sterilized dehydrated human amniotic membrane is processed to provide an easy to use, safe option for multiple surgical applications while providing a 5 year shelf life at room temperature. Many of the key components present in natural amniotic membrane are preserved during the gentle PURION® Process, which accounts for the advantageous clinical properties observed when the allografts are used in the clinical applications described. Known to decrease inflammation, reduce scar tissue formation, and support soft tissue regeneration, EpiFix® is proven to reduce wound-closure time, overall cost to treat wounds, and scarring when compared to the use of other biologic skin substitute products.


REFERENCES:1. Sherwood, Lauralee. Human Physiology, From Cells to Systems, 7th Ed. Belmont , CA: Cengage Learning, 2010.

2. Baradaran-Rafii A; Aghayan H; Arjmand B; and Javadi M. “Amniotic Membrane Transplantation.”

3. Morisaki T, et al. Immunosuppressive cytokines (IL-10, TGF-beta) genes expression in human gastric carcinoma tissues. J Surg Oncol. 1996 Dec:63(4):234-9.

4. Kay H; Nelson D; Wang Y. “The Placenta: From Development to Disease.” Wiley-Blackwell. 2011.

5. http://www.rndsystems.com

6. Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev. 1999 Oct;79(4):1283-316.

7. Fukuda K, Chikama T, Nakamura M, Nishida T. Differential distribution of subchains of the basement membrane components type IV collagen and laminin among the amniotic membrane, cornea, and conjunctiva. Cornea. 1999 Jan;18(1):73-9.

8. Hassan Niknejad, Habibollah Peirovi, Masoumeh Jorjani, Abolhassan Ahmadiani, Jalal Ghanavi, Alexander, M. Seifalian. Properties of Amniotic Membrane for Potential Use in Tissue Engineering. European Cells and Materials Vol. 15 2008 (pages 88-99)

9. Dietrich-Ntoukas T, Hofmann-Rummelt C, Kruse FE, Schlötzer-Schrehardt U. Comparative analysis of the basement membrane composition of the human limbus epithelium and amniotic membrane epithelium. Cornea. 2012 May;31(5):564-9.

10. Koob T, Zabek N, Massee M, Waite A. Bioactive Properties of a Dehydrated Human Amniotic Membrane for Tissue Regeneration. Poster Presentation, SAWC 2012.

12. Zelen C, Serena, T, Fetterolf D. Human Amniotic Membrane in the Treatment of Non-Healing Diabetic Foot Ulcers: A Randomized Controlled Trial. Poster presentation Clinical Symposium on Advances in Skin and Wound Care 2012

13. Forbes, J and Fetterolf, D. Dehydrated amniotic membrane allografts for the treatment of chronic wounds; a case series. Journal of Wound Care. June 2012; 21(6):290-296

14. Shah, Clinical Report – The Application of EpiFix® Human Amniotic Membrane Allografts to Lower Extremity Wounds

15. Jay R, Landsman A. A Retrospective Study of a Novel Allograft Membrane to Prevent Post-Operative Adhesions in the Repair of Peroneal and Posterior Tibial Tendons. Poster presentation, Desert Foot, 2012

16. MiMedx® Internal Report - “Amnion Competitive Products Analysis”

60 Chastain Center Blvd., Suite 60, Kennesaw, GA 30144866.477.4219 • www.mimedx.com

Patents and patents pending see: www.mimedx.com/patentsEpiFix®, PURION®, and MiMedx® are registered trademarks of MiMedx Group, Inc. EpiFix® is processed by Surgical Biologics, a MiMedx Group Company.©2013 MiMedx Group, Inc. All rights reserved.

®

EP158.002

Scientific & Clinical Compendium - Aberdeen Investment … · 2 EpiFix® Scientific & Clinical...

Documents

Transcript of Scientific & Clinical Compendium - Aberdeen Investment … · 2 EpiFix® Scientific & Clinical...