In ophthalmology, although transplanting limbal epithelial stem cells (SCs) is effective in restoring vision in eyes suffering from limbal SC deficiency, its success is threatened by nonresolving inflammation in the limbal stroma. Amniotic membrane (AM) transplantation is reintroduced to augment the success of transplantation of limbal epithelial SCs by reducing inflammation and scarring in the limbal stroma and by promoting epithelial growth. Our cumulative research effort has led to the discovery of heavy chain-hyaluronan/pentraxin 3 (HC-HA/PTX3) as one key matrix component responsible for the aforementioned therapeutic actions of AM and umbilical cord (UC). HC-HA/PTX3 is a complex formed by PTX3 tightly with HC-HA of which HA is covalently linked with HC1 derived from inter-α-trypsin inhibitor (IαI) through the catalytic action of tumor necrosis factor-stimulated gene-6 (TSG-6). Besides exerting a broad and extensive anti-inflammatory, anti-scarring, and anti-angiogenic effects, HC-HA/PTX3 also acts as “top soil” to uniquely support the phenotype of niche cells to maintain SC quiescence and retains a multi-potent plasticity of generating progenitors cells or mesenchymal SCs so as to support tissue homeostasis and regeneration. We thus envision that HC-HA/PTX3-containing therapeutics can be formulated from this platform technology to mitigate nonresolving inflammation and reinforce the well-being of SC niches beyond ophthalmology to promote regenerative healing.

Keywords: anti-inflammation, amniotic membrane, anti-scarring, Hyaluronan, Quiescence, regenerative healing, stem cell niche, umbilical cord

6.1 Introduction

Stem cells (SC) with extensive proliferative potential for giving rise to one or more differentiated cell types hold considerable promise for the treatment of a number of diseases in regenerative medicine. SCs are common in early mammalian embryos, but by adulthood, they are dispersed and kept in a unique microenvironment termed “niche” where they continue to maintain quiescence while performing relentless self-renewal to replenish the SC pool that is depleted by differentiation for the fate decision.

Among all adult epithelial tissues, the model of the corneal epithelium is most unique in its ready access and clear anatomic separation from the surrounding conjunctiva. The seminal discovery made by Dr. Tung-Tien Sun in 1986 ¹ taught us that corneal epithelial SCs are located at the basal epithelial layer of the limbus, which marks the junction between the cornea and the conjunctiva. Since then, cumulative studies have led us to conclude that these limbal SCs govern the homeostasis of the corneal epithelium. ^1,​2 This important discovery has also allowed us to use impression cytology to identify a diseased state termed “limbal SC deficiency” (LSCD) in a number of ocular surface diseases with the hallmark of “conjunctivalization” of the corneal surface. ³ Histopathologically, these diseases with LSCD also manifest superficial neovascularization, chronic stromal inflammation, and scarring. ² Because corneal blindness caused by LSCD is due to the loss of limbal SCs, visual rehabilitation cannot be achieved by conventional corneal transplantation. Hence, the aforementioned discovery has also led us to devise transplantation of autologous limbal SCs in 1989 for unilateral LSCD ⁴ and transplantation of allogeneic limbal SCs in 1994 for bilateral LSCD. ⁵ Although many subsequent studies have confirmed their clinical efficacy in patients with LSCD, one case with acute chemical burn was noted to have unsatisfactory outcome as early as 1989 when transplantation of autologous limbal SCs was first reported. ⁴ Late on, we then identified nonresolving inflammation in the limbal stroma as a likely cause of failure in an experimental rabbit model of LSCD caused by a combination of chemical and mechanical insults. ⁶

To examine the hypothesis that severely or chronically inflamed limbal stroma is not suitable for receiving transplanted limbal SCs, we then sought for a means to restore a healthy and supportive limbal stromal niche. Therefore, we tested transplantation of human amniotic membrane (AM) and reported in 1995 that this surgical procedure alone can restore a normal corneal surface in 5 (38%) of 13 rabbits when compared to 0 (0%) of 10 untransplanted rabbits that suffered from acute chemical burns. ⁷ To draw a plausible relationship between AM transplantation (AMT) and limbal SC transplantation, we proposed in 1997 a metaphor, in which the former serves as “top soil” to support the latter, which acts as if we plant the “seed” in a garden. ⁸ This hypothetical viewpoint has since been substantiated by a number of clinical studies. For example, AMT alone can prevent LSCD in acute chemical burns ^9,​10 and acute Stevens–Johnson syndrome ^{11,​12,​13} to restore vision in corneas with partial (i.e., <360° involvement) LSCD ^14,​15 and to augment the success of transplanting autologous ^16,​17 and allogeneic ^5,​18 limbal SCs for corneas with total LSCD. Furthermore, AM as a single ¹⁹ and dual layers ²⁰ is also used to aid in vivo expansion of limbal SCs in a new surgical procedure termed “simple limbal epithelial transplantation” as well as used as a substrate or carrier to promote ex vivo expansion of limbal SCs. ²¹

6.2 Clinical Ophthalmic Indications

Since our first reintroduction of AMT in 1995 in ophthalmology, ⁷ over 1,000 peer-reviewed publications have been published in the last 20 years describing the clinical efficacies of AMT for treating diverse ocular surface diseases. In 2001, through a formal process of Request for Designation, the U.S. Food and Drug Administration (FDA) ruled that transplantation of cryopreserved human AM (manufactured by Bio-Tissue, Inc.) for ocular surface reconstruction is classified as “361 human cell/tissue products (HCT/P)” to exert anti-inflammatory, anti-scarring, and anti-angiogenic actions to promote wound healing. Subsequently, the Centers of Medicare and Medicaid Services (CMS) in the United States of America has granted three level 1 CPT codes to cover the surgical procedures of transplanting cryopreserved AM and umbilical cord (UC) for a number of ophthalmic indications (▶ Fig. 6.1).

In brief, these indications can be categorized into the following two modes of AMT, that is, as a graft and as a biological bandage (▶ Fig. 6.1). ²² Intuitively, when AMT is used as a graft, it is meant to fill in the tissue defect. Upon healing, the membrane is integrated into the host tissue as host cells grow over or into the membrane to restore the tissue integrity and function. In this mode, AM can be used as a single or multiple layers and secured to the host tissue by sutures or fibrin glue in a sutureless manner. In the second mode, AM is used as a bandage to cover both the site of interest and the healthy host tissue at the same time by sutures or via placement of “sutureless” ProKera (Bio-Tissue, Inc., Miami, FL). This mode is usually considered when there is no/minimal stromal loss and the host epithelium heals underneath the membrane.

ProKera is classified as a type II medical device by the FDA via 510(k) clearance and contains a polycarbonate ring system that fastens cryopreserved AM in between. The sutureless approach via ProKera shortens the surgical time, permits topical anesthesia, and eliminates suture-induced inflammation. It can be applied in the office, the emergency room, and the hospital bedside to facilitate “early intervention” and reduces the overall medical cost. This is particularly important for managing acute chemical burns ^9,​10 and acute Stevens–Johnson syndrome/toxic epidermal necrolysis ^{11,​12,​13} that require acute intervention. Upon healing, ProKera can be removed in the practitioner’s office. If the AM dissolves sooner than expected, one may like to look into “exposure” issue caused by infrequent blinking with incomplete closure. In this regard, ProKera may facilitate blinking because of the polycarbonate ring. This likelihood is supported by accelerated restoration of the corneal surface not only in the eye with the placement of ProKera but sometimes also in the fellow eye without. ²³ Occasionally, AM can be used as both a graft and a patch in one patient to further augment the therapeutic benefit of the transplanted AM graft. ^14,​20 Self-retained AM also may attract inflammatory cells out of the ocular surface, become cloudy, lose its potency, and may require replacement to achieve continuous effectiveness.

6.3 Mechanism of Action of Amniotic Membrane and Umbilical Cord

The aforementioned clinical efficacies observed in ophthalmology let us wonder what can be the underlying mechanism of action cryopreserved AM/UC. Anatomically, the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane to the UC. Developmentally, both the AM and UC share the same cell origin as the fetus. The traditional view of AM and UC is their protective function of the fetus during pregnancy. However, the fact that AM/UC can serve as a graft and as a bandage strongly suggests that this birth tissue can be a “structural” scaffold (as a graft) as well as a “biological” bandage. It also suggests that the biological factor(s) in this birth tissue may be releasable from the tissue to exert colossal anti-inflammatory, anti-scarring, and anti-angiogenic actions.

To search for the relevant biological characteristic of these birth tissues, we have embarked on a 12-year journey from 2002 to 2014. We first verified that the aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM. Specifically, we have shown that human AME can induce apoptosis of IFN-γ, lipopolysaccharide (LPS), and IFN-γ/LPS-activated, but not resting macrophages. ^24,​25 AME also downregulates expression of M1 macrophage markers, such as TNF-α, IL-6, CD86, and MHC II while upregulating M2 macrophage markers such as cytokine IL-10. ²⁵

Following the work of identifying the HC-HA/PTX3 complex as the key component in the cumulus-oocyte complex surrounding the ovulated oocyte to ensure fertilization, ^26,​27 our laboratory was the first reporting that the same biosynthetic pathway used for ovulation also takes place in the AM. In short, we have purified the HC-HA/PTX3 complex from AME by two successive runs of ultracentrifugation in a cesium chloride (CsCl) gradient in the presence of 4M guanidine hydrochloride (HCl). ^28,​29 The biosynthetic process of HC-HA/PTX3 involves two steps (▶ Fig. 6.2): the first is to form HCHA complex via the catalytic action of tumor necrosis factor-stimulated gene-6 (TSG-6) resulting in the covalent (ester bond) transfer of HC1 from inter-α-trypsin inhibitor (IαI) to high molecular weight HA (>3,000 kDa). The source of IαI, which contains two HCs (i.e., HC1 and HC2) and a light chain termed bikunin, is the serum following secretion from the liver for ovulation. However, we discovered that IαI is endogenously produced by AM epithelial cells and stromal cells to circumvent the shortcoming of this avascular tissue. The expression of TSG-6 and PTX3 is constitutive (i.e., without relying on proinflammatory cytokines). The second step is to form the HC-HA/PTX complex by tight association of the HC-HA complex with PTX3.

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