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If you are not up on your Marvel Comics movie mythology, you may want to consult the Marvel Cinematic Universe and Marvel Wiki (who knew, right?) as you read Dr. Cooper’s article. (We did while editing.) Or see the Glossary on the next page. Otherwise, we’re not sure we would have known what the good doctor was talking about. Enjoy.
When we treat ocular surface diseases in the 21st century, should scar formation or recalcitrant keratitis be what healing should look like? To be blunt, the answer is a resounding “no.”
Lending credence to this assertion, in his State of the Union Address in January 2015, President Obama unveiled a $215 million investment in the Precision Medicine Initiative.1 The challenge of delivering tailored, customized solutions to patients resonates when we consider the aging baby boom population in the United States, which will face an increased incidence of chronic and inflammatory diseases, traumatic injuries, and emerging pathogens. In the realm of eye disease, the ability to maintain a healthy cornea is at risk when patients are faced with such conditions as filamentary keratitis from dry eye, neurotrophic ulcers from autoimmune or infectious etiologies, and herpetic eye disease.
Researchers are constantly exploring avenues to develop better therapeutic options to alleviate pain and reduce the progression of sight-threatening disease. In the field of wound care, over the past 20 years, amniotic membrane (AM) technology has rapidly evolved. This tool of regenerative medicine may provide a stepping stone toward targeted preventive treatment for patients with challenging corneal disorders. The purpose of this article is to assist eye care specialists in understanding the science and clinical significance of the healing process and the present and future implications of regenerative medicine.
A BRIEF HISTORY
In 1910, J.W. Davis at Johns Hopkins University reported the first known medical use of AM, in the form of a skin graft.2 Although this first application was seen as a failure, the experiment emboldened research in 1913 by Sabella and Stern, who independently and successfully used AM grafts in burn management.3,4
The first report of AM used as a conjunctival graft was by De Rötth, who in 1940 sought a tissue alternative to allograft for symblepharon and pterygium repair. Although this was groundbreaking work, his initial attempt was unsuccessful. It is hypothesized that this was due to the presence of live cells and chorion transplanted with the amnion, which may have induced graft rejection.5
During the 1940s, it was demonstrated that amniotic tissue could be processed and stored in culture medium, thereby allowing increased shelf life. With the use of Dulbecco’s modified Eagle culture medium and glycerol at -80˚C, the amnion was rendered a substrate with no live cells to induce graft rejection. In the mid-1940s, Sorsby et al were relatively successful using prepared, dry AM, or amnioplastin, as corneal grafts.6 They reported treatment of 58 patients with caustic ocular burns.6 The limitation of their work was the need for repeated dry AM application.
The use of AM for corneal graft material fell out of favor for the next 30 years, aside from a mention by Collin in 1975 that manipulating fetal membrane tissue is possible with extreme difficulty.7 Although it almost seemed relegated to antiquity, preserved AM was successfully (and secretly) used as a treatment for traumatic corneal injury in the Soviet Union during the Cold War.7
The subsequent resurrection of AM by Batlle and Perdomo sparked Tseng et al at Bascom Palmer Eye Institute to formulate a new wet cryopreserved AM platform to expand the tissue’s role in ocular reconstruction.8 At the time of this writing, there is a vast amount in research and development accelerating new therapeutics and surgical techniques in eye care and other health care specialties with the use of AM.
WOUNDED SOLDIER: THE PROCESS
Unless you are Captain America, the normal adult wound-healing process is characterized by three phases: an inflammatory phase, a proliferative phase, and a remodeling phase.9
The Infinity Stones are six immensely powerful objects tied to different aspects of the universe, created by the Cosmic Entities. Each of the stones possesses unique capabilities that have been enhanced and altered by various alien civilizations through the millennia. In the comics, they are referred to as Infinity “Gems.”
Captain America (Steve Rogers) received Super Soldier Serum and Vita Ray radiation treatment during World War II emerging with a greatly enhanced physique and superior regenerative capabilities when injured in battle.
The Orb, in the cinematic universe is an artifact that contains one of the six Infinity Stones. In the comics, it is actually considered an Infinity Power Gem giving the owner access to all power and energy that ever has or will exist for increased strength and durability, and can back the other gems and boost their effects.
The Soul Gem is one of the six Infinity Gems. It grants its user control over reality.
Heimdall is a friend to Thor who is an all-seeing and allhearing fellow Asgardian tasked with guarding the Bifrost Bridge. He can see and hear nearly everything that happens in the Nine Realms.
The Tesseract is a crystalline cube-shaped containment vessel for an Infinity Stone possessing unlimited energy representing space. Once again, in the comics, it is considered an Infinity Space Gem allowing its user to manipulate space any way one sees fit. Its most basic powers allow one to teleport themselves.
Source: Marvel Cinematic Universe Wiki and Marvel Wiki http://marvelcinematicuniverse.wikia.com/wiki/Marvel_ Cinematic_Universe_Wiki
Inflammation is the hallmark defense mechanism in tissue injury, and it is marked by cellular infiltration by polymorphonuclear neutrophils (PMNs), macrophages, and lymphocytes derived from innate and adaptive immune responses.10 The first responders, PMNs, eventually undergo apoptosis due to their truncated lifespan, and they are naturally removed by macrophages via phagocytosis.11 This results in the restoration and maintenance of antiinflammatory and immune homeostatic balance. If there is a wide extent of injury influencing PMN infiltration, this might lead to a drawn-out series of events, in which a prolonged lifespan facilitates additional collateral damage.11,12
Enter angiogenesis, or new blood vessel formation, as a coping mechanism during the proliferative phase, in an attempt to reestablish the microvasculature network damaged in tissue injury. Impaired angiogenesis delays the flow of nutrients and oxygen to cells, creating an environment rife with hypoxia, in turn leading to unexpected cell death and poor healing at the wound site.13 This dysregulation includes a handoff between M1 and M2 macrophages that significantly increases the presence of epidermal Langerhans cells, activation of T lymphocytes (CD3+, HLA DR, CD25), and a high amount of transforming growth factor (TGF) beta-1, promulgating nonhealing wounds and/or hypertrophic scar tissue.14,15
These chronic wounds illustrate an exudative behavior similar to what is seen in diabetic retinopathy, with increased levels of angiogenic proteins compared with acute wounds. Therefore, supplying growth factors to stimulate angiogenesis is a key feature of wound care therapies in the remodeling phase. This can be accomplished with amniotic tissue and, by extension, stem cell recruitment.
THE ORB: QUALITY OF HEALING WITH VITA RAYS
To return to my earlier question: What should high quality and rapid healing look like if we had the opportunity to observe it in vivo or ex vivo? Characteristically, fetal wound healing is said to be “scarless.” Recently, in the public spotlight, have been several human case reports of spina bifida, a devastating condition in which the vertebral column is exposed, causing spinal cord deformation and ultimately permanent neurological deficit when the child is born. Through arduous work and Expanded Access use granted by the US Food and Drug Administration, Harrison and Tsao have reported promising results with umbilical cord patching in these patients.16,17
Papanna et al provided support for these cases in their work with a sheep model, comparing biocellulose film in underwater adhesive to human umbilical cord (HUC) with suture.18 These researchers surgically created spina bifida in 14 fetuses at 75 days, and one of the two treatments (HUC with suture or biocellulose film) was performed in each at 95 days gestation. In the fetuses that survived to final analysis, all treated with HUC with suture illustrated complete regrowth of epidermal, dermal, and subdermal tissue with no spinal cord exposure. Additionally, there were no observed cases of Chiari II malformation in the HUC with suture group.18
These reports are suggestive of the potential power of AM tissue. When there is an injury to the embryo in utero, the inflammatory response is diminished, likely due to an immature immune system. The response differs from that in adults in terms of inflammatory cells that penetrate the wound space and a reduction in the presence of interleukin (IL)-6 and IL-8.19,20 Due to proinflammatory downregulation, there is a lessened scarring response in fetal wound healing. Furthermore, there is speculation that AM contributes to the fetal immune tolerance state by delivering antiinflammatory and antiscarring action through modulated immune activation.21
Tseng et al strengthened this hypothesis by attempting to identify the molecules responsible for AM’s antiinflammatory action. These Vita Rays were identified as heavy chain hyaluronic acid (HC-HA) and pentraxin 3 (PTX3) in previous studies in which transplanted cryopreserved AM induced apoptosis of neutrophils, monocytes, and macrophages to reduce infiltration of these cellular types and promote polarization of M2 macrophages.21-26
It is important to note that the antiinflammatory action exerted by cryopreserved AM is retained in water-soluble or wet AM extract, secondary to retention of the extracellular matrix. In dehydrated or dry AM, by contrast, this ultrastructure is compromised, leading to a more proinflammatory separation of PTX3 and HA into the low weight conformation by protein denaturation. This impaired ability to modulate the immune response to tissue injury and remodeling calls into question the quality of healing possible with dry AM.
Conversely, the HC-HA/PTX3 complex is significantly anti-inflammatory and antiscarring in nature, synchronizing apoptosis of freshly isolated neutrophils by M2 macrophages to resolve inflammation.26 In this active process found in mouse and human cell models, fibroblasts are blocked from differentiating into myofibroblasts by the suppression of TGF-beta 1 promoter activity, which downregulates Th1 and Th17 lymphocytes from invading epithelial cells in order to initiate tissue regeneration.27-30
HEIMDALL’S SOUL GEM: CLINICAL REALITY
Translating this experimental science to a clinical perspective, there have been numerous case reports comparing the advantages of wet versus dry AM. Rodriguez recently reported on an investigator-initiated retrospective chart review of 39 patients treated with Prokera (Bio-Tissue) cryopreserved AM for dry eye disease. After 294 days of mitigated success with therapeutic and palliative treatments, a single cryopreserved wet AM produced complete resolution of keratopathy in 94.9% of these patients within 5.3 days.31
These rapid results are not isolated. A recent blog by Cooper and Pereira described a case of a patient with a significant fluoride-based acid chemical burn measuring 4 mm in size and encompassing approximately 35% of epithelial and intrastromal cornea (Figure 1). The patient received a Prokera Slim corneal bandage and was given Besivance (besifloxacin ophthalmic suspension 0.6%; Bausch + Lomb) antibiotic drops as a preventive measure on the day of presentation and was followed daily. It took 3 days to achieve complete resolution of the lesion (Figure 2), a notable quality and speed of healing.32
By contrast, Moore et al conducted a retrospective review of the use of AmbioDisk (Katena), a dehydrated AM, in 59 treatment episodes for 44 eyes of 39 consecutive patients with persistent keratoepithelial defect or epitheliopathy over a 545-day period. The cases represented a diverse range of disease, including alkaline burn, herpes simplex, epithelial basement membrane dystrophy, Sjögren syndrome, keratoconus, and persistent keratoepithelial defect after phototherapeutic keratectomy for corneal opacity or pterygium removal. These authors used 83 AMs in these episodes, with successful closure or healing of the lesions in 84.7% and treatment times of 2.8 to 5.7 weeks. Although healing occurred in the vast majority of cases, there was a need for retreatment with an average of 1.1 to 2.3 AMs used per patient.33
THE TESSERACT’S WINDOW OF OPPORTUNITY
Healing alone is no longer state of the art in eye care. From a health care economics perspective, a value-based reimbursement system is set to launch in the United States by the end of this decade rewarding those whom are more efficient and effective in delivering care. Subsequently, there may be a distinct advantage in the use of cryopreserved rather than dehydrated AM tissue, with regard to the amount of durable material needed and the treatment time required to achieve desired results.
By 2050, regenerative medicine will potentially have moved far beyond today’s treatment protocols through advancements in recombinant or designer DNA synthetics integrated directly into our bodies and handheld precision laser technology to mend wounds in a scarless fashion taking mere moments. The call to action is to embrace AM tissue now as the vanguard in the delivery of truly preventive medical care. n
1. National Institutes of Health. About the Precision Medicine Initiative Cohort Program. https://www.nih.gov/precision-medicine-initiative-cohort-program. Accessed August 31, 2016.
2. Davis JW. Skin transplantation with a review of 550 cases at the Johns Hopkins Hospital. Johns Hopkins Med J. 1910;15:307-396.
3. Sabella N. Use of fetal membranes in skin grafting. Med Rec N Y. 1913;83:478-480.
4. Stern M. The grafting of preserved amniotic membrane to burned and ulcerated surfaces, substituting skin grafts: a preliminary report. JAMA. 1913;60(13):973-974.
5. de Rötth A. Plastic repair of conjunctival defects with fetal membranes. Arch Ophthalmol. 1940;23(3):522-525.
6. Sorsby A, Haythorne J, Reed H. Further experience with amniotic membrane grafts in caustic burns of the eye. Br J Ophthalmol. 1947;31(7):409-418.
7. Collin JR. Peritoneal autografts in conjunctival replacement. Br J Ophthalmol. 1975;59(5):288-293.
8. Tseng SC, Prabhasawat P, Lee SH. Amniotic membrane transplantation for conjunctival surface reconstruction. Am J Ophthalmol. 1997;124(6):765-774.
9. Clark RAF. Overview and general considerations of wound repair. In: Clark RAF, Henson PM, eds. The Molecular and Cellular Biology of Wound Repair. New York: Springer;1988: 3-33.
10. Fadok VA, Bratton DL, Konowal A, et al. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J Clin Invest. 1998;101:890-898.
11. Koh TJ, DiPietro LA. Inflammation and wound healing: the role of the macrophage. Expert Rev Mol Med. 2011;13:e23.
12. Khanna S, Biswas S, Shang Y, et al. Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice. PLoS One. 2010;5:e9539.
13. Sen CK. Wound healing essentials: let there be oxygen. Wound Repair Regen. 2009;17:1-18.
14. Niessen FB, Schalkwijk J, Vos H, et al. Hypertrophic scar formation is associated with an increased number of epidermal Langerhans cells. J Pathol. 2004;202:121-129.
15. Castagnoli C, Trombotto C, Ondei S, et al. Characterization of T-cell subsets infiltrating post-burn hypertrophic scar tissue. Burns. 1997;23:565-572.
16. Farmer DL, von Koch CS, Peacock WJ, et al. In utero repair of myelomeningocele: experimental pathophysiology, initial clinical experience, and outcomes. Arch Surg. 2003;138(8):872-878.
17. Lake, DM. UTHealth researchers report umbilical cord patch could be novel method for fetal spina bifida repair. Media Relations. The University of Texas Health Science Center at Houston (UTHealth), July 11, 2016. https://www.uth.edu/media/story.htm?id=7d5aaa46-9c83-476b-bd93-9db5dc53f413. Accessed October 6, 2016.
18. Papanna, R, Moise, KJ, Mann, LK, et al. Cryopreserved human umbilical cord patch for in-utero spina bifida repair. Ultrasound Obstet Gynecol. 2016;47(2):168-176.
19. Liechty KW, Adzick NS, Crombleholme TM. Diminished interleukin 6 (IL-6) production during scarless human fetal wound repair. Cytokine. 2000;12:671-676.
20. Liechty KW, Crombleholme TM, Cass DL, et al. Diminished interleukin-8 (IL-8) production in the fetal wound healing response. J Surg Res. 1998;77:80-84.
21. Park WC, Tseng SC. Modulation of acute inflammation and keratocyte death by suturing, blood and amniotic membrane in PRK. Invest Ophthalmol Vis Sci. 2000;41:2906-2914.
22. Wang MX, Gray TB, Parks WC, et al. Corneal haze and apoptosis is reduced by amniotic membrane matrix in excimer laser photoablation in rabbits. J Cataract Refract Surg. 2001;27:310-319.
23. Shimmura S, Shimazaki J, Ohashi Y, et al. Anti-inflammatory effects of amniotic membrane transplantation in ocular surface disorders. Cornea. 2001;20:408-413.
24. Bauer D, Wasmuth S, Hermans P, et al. On the influence of neutrophils in corneas with necrotizing HSV-1 keratitis following amniotic membrane transplantation. Exp Eye Res. 2007;85:335-345.
25. Heiligenhaus A, Bauer D, Meller D, et al. Improvement of HSV-1 necrotizing keratitis with amniotic membrane transplantation. Invest Ophthalmol Vis Sci. 2001;42:1969-1974.
26. Bauer D, Wasmuth S, Hennig M, et al. Amniotic membrane transplantation induces apoptosis in T lymphocytes in murine corneas with experimental herpetic stromal keratitis. Invest Ophthalmol Vis Sci. 2009;50:3188-3198.
27. He H, Li W, Tseng DY, et al. Biochemical characterization and function of complexes formed by hyaluronan and the heavy chains of inter-a-inhibitor (HC_HA) purified from extracts of human amniotic membrane. J Biol Chem. 2009;284:20136-20146.
28. Li W, He H, Chen YT, et al. Reversal of myofibroblasts by amniotic membrane stromal extract. J Cell Physiol. 2008;215:657-664.
29. Espana EM, He H, Kawakita T, et al. Human keratocytes cultured on amniotic membrane stroma preserve morphology and express keratocan. Invest Ophthalmol Vis Sci. 2003;44:5136-5141.
30. K awakita T, Espana EM, He H, et al. Keratocan expression of murine keratocytes is maintained on amniotic membrane by downregulating TGF-beta signaling. J Biol Chem. 2005;280:27085-27092.
31. Rodriguez B. Quality healing in chronic dry eye patients. Bio-Tissue website. October 21, 2015. http://info.Bio-Tissue.com/blog/quality-healing-in-chronic-dry-eye-patients. Accessed August 31, 2016.
32. Cooper MS, Pereira K. Successfully treating a chemical corneal burn. Bio-Tissue website. August 23, 2016. http://info.Bio-Tissue.com/blog/successfully-treating-a-chemical-corneal-burn. Accessed August 31, 2016.
33. Moore M, Stojanovic A, Kugler LJ, Wang M. Corneal scarring after refractive surgery. CRST. April 2014. http://crstoday.com/2014/04/corneal-scarring-after-refractive-surgery. Accessed August 31, 2016.
Michael S. Cooper, OD
• Private practice, Windham Eye Group, Willimantic, Connecticut
• Financial disclosure: member of the advisory board, consultant to, or speakers bureau for Alcon Surgical, Allergan, Bausch+Lomb, Bio-Tissue, inVentiv Health, JJVC, and Shire