Abstract Background HC-HA/PTX3 (a complex formed by high molecular weight hyaluronan covalently linked to heavy chain 1 of inter-α-trypsin inhibitor and tightly bound to pentraxin 3) is a unique extracellular matrix from human amniotic membrane that exerts an anti-scarring action and reprograms human corneal fibroblasts (HCF) and myofibroblasts to corneal stromal keratocytes in the absence of transforming growth factor β1 (TGFβ1) by downregulating canonical Smad-mediated signaling and upregulating bone morphogenetic protein (BMP) signaling. It remains unclear whether HC-HA/PTX3 can further reprogram HCF into neural crest (NC) progenitors in the presence of TGFβ1. Methods Human corneal fibroblasts were seeded on plastic, immobilized hyaluronic acid (HA) or HC-HA/PTX3 or on plastic with or without soluble HA and HC-HA/PTX3 in DMEM + 10% fetal bovine serum (FBS), with or without various inhibitors with or without TGFβ1. Transcript expression of NC and signaling markers was determined by RT-qPCR. Immunostaining was performed to monitor cytolocalization of signaling markers and α-smooth muscle actin (α-SMA). Raft separation before Western blot was used to study protein distributions in both rafts. Western blot and ELISA were used to measure relative protein level. Results Herein, we show for the first time that in the presence of exogenous TGFβ1, HC-HA/PTX3 continues to reprogram HCF into NC progenitors by upregulating mRNA expression of NC markers, confirmed by successful induction into human corneal endothelial cells, highlighted by hexagonal shape, mRNA expression of corneal endothelial markers and junctional staining of corneal endothelial markers such as Na-K-ATPase, α-catenin, β-catenin, F-actin, N-cadherin, p120 and ZO-1 but the lack of fibrogenic marker S100A4. Such reprogramming requires suppression of TGFβ1 SMAD-mediated canonical signaling that starts from HC-HA/PTX3 binding with CD44 to sequester type II TGFβ receptor (TβRII) in lipid raft and ends with downregulation of TβRII by nuclear translocation of cyclin D1. Mechanistically, nuclear translocation of cyclin D1 is mediated by activation of transforming growth factor-beta-activated kinase 1-transcription factor Jun (TAK1-cJUN) noncanonical signaling because of type I TGFβ receptor (TβRI) and type III TGFβ receptor (TβRIII) (without TβRII) in non-lipid raft as well as by nuclear translocation of CD44 intracellular domain (CD44ICD) formed by Membrane Type 1 Matrix Metalloproteinase (MT1MMP)/γ-secretase cleavage to facilitate such reprogramming. Conclusions Thus, HC-HA/PTX3 from amniotic membrane can be deployed as a new strategy to reverse scar toward regeneration.

Pain is a hallmark of inflammation and tissue injury, particularly following major surgical procedures. Despite its prevalence and clinical impact, pathological pain, defined as persistent pain that impairs function, remains a major unmet medical need. Human birth tissue, including the amniotic membrane and umbilical cord, has long been recognized for its regenerative properties and its ability to support wound healing. Recent studies have identified heavy chain 1 (HC)-hyaluronic acid (HA)/pentraxin 3 (HC-HA/PTX3) as a key matrix component within these tissues. This complex exhibits anti-inflammatory and anti-scarring activities and helps maintain stem cell quiescence. Emerging evidence, including our own findings, indicates that human birth tissue-derived products may also modulate pain responses in certain settings, such as post-surgical pain, potentially through mechanisms involving neuronal inhibition and regenerative healing. We summarize the molecular and cellular mechanisms by which human birth tissue-derived products and HC-HA/PTX3 may exert anti-inflammatory and analgesic effects. We then highlight preclinical and clinical studies evaluating their potential roles in wound healing and pathological pain. Finally, we discuss translational opportunities, current challenges, and future directions for advancing these biologics within the emerging field of regenerative pain medicine. This review outlines a framework for potential regenerative pain management using birth tissue-derived products, which may serve as a foundation for developing new therapies for certain pathological pain conditions.

Amniotic membrane (AM) is widely used in ophthalmology because of its antiinflammatory, antiscarring, and wound healing promoting properties, which are due in part to a key matrix component, heavy chain-hyaluronan/pentraxin3. Herein, we assess various processing methods used to manufacture AM sheet products and their effect on the biological properties of AM mediated by heavy chain-hyaluronan/pentraxin3.

The heavy chain (HC)-hyaluronan (HA)/pentraxin 3 (HC-HA/PTX3) complex is formed by tumor necrosis factor-stimulated gene-6 (TSG-6) catalyzing the covalent (ester bond) transfer of HC1 from inter-α-trypsin inhibitor (IαI) to HA followed by tight binding of PTX3. The presence of such a complex has been found in human amniotic membrane (AM) and is considered to be a major matrix component responsible for its anti‑inflammatory and anti‑scarring properties to promote regenerative healing. Because the therapeutic potentials of AM and umbilical cord (UC) are similar, we herein evaluated whether human UC also contains HC-HA/PTX3. Immunostaining of UC cross-sections showed abundant PTX3, HC1, HA, TSG-6, and bikunin. Western blot analysis suggested the presence of HC1 complex bound via a NaOH-sensitive bond and tightly bound to PTX3 multimer in UC and AM extracts but not in chorion and placenta extracts. HC-HA/PTX3 was purified from UC extract by successive runs of density gradient ultracentrifugation and verified the presence of HC1 but not HC2 or HC3 based on western blot analysis. These results suggest the presence of HC-HA/PTX3 complex in UC is similar to AM.

Background: The biologics derived from human amniotic membranes (AMs) demonstrate potential pain-inhibitory effects in clinical settings. However, the molecular basis underlying this therapeutic effect remains elusive. HC-HA/PTX3 is a unique water-soluble regenerative matrix that is purified from human AMs. We examined whether HC-HA/PTX3 can modulate the gene networks and transcriptional signatures in the dorsal root ganglia (DRG) neurons transmitting peripheral sensory inputs to the spinal cord. Methods: We conducted bulk RNA-sequencing (RNA-seq) of mouse DRG neurons after treating them with HC-HA/PTX3 (15 µg/mL) for 10 min and 24 h in culture. Differential gene expression analysis was performed using the limma package, and Gene Ontology (GO) and protein–protein interaction (PPI) analyses were conducted to identify the networks of pain-related genes. Western blotting and in vitro calcium imaging were used to examine the protein levels and signaling of pro-opiomelanocortin (POMC) in DRG neurons. Results: Compared to the vehicle-treated group, 24 h treatment with HC-HA/PTX3 induced 2047 differentially expressed genes (DEGs), which were centered on the ATPase activity, receptor–ligand activity, and extracellular matrix pathways. Importantly, PPI analysis revealed that over 50 of these DEGs are closely related to pain and analgesia. Notably, HC-HA/PTX3 increased the expression and signaling pathway of POMC, which may affect opioid analgesia. Conclusions: HC-HA/PTX3 induced profound changes in the gene expression in DRG neurons, centered around various neurochemical mechanisms associated with pain modulation. Our findings suggest that HC-HA/PTX3 may be an important biological active component in human AMs that partly underlies its pain inhibitory effect, presenting a new strategy for pain treatment.

Pain after surgery causes significant suffering. Opioid analgesics cause severe side effects and accidental death. Therefore, there is an urgent need to develop non-opioid therapies for managing post-surgical pain. Local application of Clarix Flo (FLO), a human amniotic membrane (AM) product, attenuated established post-surgical pain hypersensitivity without exhibiting known side effects of opioid use in mice. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways. We further purified the major matrix component, the heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) from human AM that has greater purity and water solubility than FLO. HC-HA/PTX3 replicated FLO-induced neuronal and pain inhibition. Mechanistically, HC-HA/PTX3-induced cytoskeleton rearrangements to inhibit sodium current and high-voltage activated calcium current on nociceptive DRG neurons, suggesting it is a key bioactive component mediating pain relief. Collectively, our findings highlight the potential of naturally derived biologics from human birth tissues as an effective non-opioid treatment for post-surgical pain. Moreover, we unravel the underlying neuronal mechanisms of pain inhibition induced by FLO and HC-HA/PTX3.

PURPOSE: To assess the extent of inferior fornix shortening in conjunctivochalasis (CCh) and to evaluate whether fornix deepening reconstruction can restore the fornix tear reservoir in patients with CCh. MATERIALS AND METHODS: This was a retrospective review of five patients (3 unilateral and 2 bilateral eyes, total 7 eyes) with CCh who underwent fornix deepening reconstruction with conjunctival recession and amniotic membrane transplantation. Postsurgical outcome measures included changes in fornix depth with correlation to basal tear volumes, symptoms, corneal staining, and conjunctival inflammation. RESULTS: For the three patients with unilateral surgery, both the fornix depth (8.3 ± 1.5 mm) and wetting length (9.3 ± 8.5 mm) of the operative eyes were less than the fellow eyes (10.3 ± 1.5 mm and 10.3 ± 8.5 mm, respectively). At 5.3 ± 2.7 months (range 1.7–8.7) postoperatively, the fornix depth increased significantly by 2.0 ± 1.1 mm (P = 0.02). Deepening of the fornix depth was accompanied by overwhelming symptomatic relief (91.5%) that could be subdivided into complete relief (87.5%) and partial relief (4%) of symptoms, with blurred vision being the most notably relieved symptom (P = 0.03). Furthermore, superficial punctate keratitis and conjunctival inflammation were significantly improved at follow-up (P = 0.008 and 0.05, respectively). CONCLUSION: Deepening of the fornix to restore the tear reservoir is an important surgical objective that may change the tear hydrodynamic state to provide a stable tear film and improve outcomes in CCh.

PURPOSE:To report a successful treatment of chronic rosacea associated ocular demodicosis with lid scrub containing terpinen-4-ol (T4O).OBSERVATIONS:A 72-year old woman presented with recurrent and refractory ocular erythema, irritation, dryness, and photophobia despite conventional medical treatment (artificial tears, hypochlorous acid lid hygiene, doxycycline, and erythromycin) for 5 years. Examination revealed facial erythema, telangiectasias on cheeks, nose and lids, and cylindrical dandruff (CD) on bilateral upper and lower lashes. Epilation sampling confirmed demodicosis. After treatment with lid wipe containing T4O (Cliradex, Biotissue, Miami, FL) over face and lids, ocular discomfort, CD, facial and eyelid erythema, telangiectatic vessels were significantly reduced. Complete eradication of demodex mites and resolution of symptoms and signs lasted 8 months of follow-up.CONCLUSIONS:This case suggests that T4O is effective in treating chronic rosacea associated ocular demodex blepharitis.

Quiescence and self-renewal of human corneal epithelial progenitor/stem cells (LEPC) are regulated by the limbal niche, presumably through close interaction with limbal (stromal) niche cells (LNC). Paired box homeotic gene 6 (Pax6), a conserved transcription factor essential for eye development, is essential for proper differentiation of limbal and corneal epithelial stem cells. Pax6 haploinsufficiency causes limbal stem cell deficiency, which leads to subsequent corneal blindness. We previously reported that serial passage of nuclear Pax6+ LNC resulted in the gradual loss of nuclear Pax6+ and neural crest progenitor status, the latter of which was reverted upon recovery of Pax6. These findings suggest Pax6 plays a pivotal role in supporting the self-renewal of LEPC in limbal niche. Herein, we show that HC-HA/PTX3, a unique matrix purified from amniotic membrane (AM) and consists of heavy chain 1of inter-α-trypsin inhibitor covalently linked to hyaluronic acid and complexed with pentraxin 3, is capable of reverting senescent LNC to nuclear Pax6+ neural crest progenitors that support self-renewal of LEPC. Such reversion is causally linked to early cell aggregation mediated by activation of C-X-C chemokine receptor type 4 (CXCR4)-mediated signaling followed by activation of bone morphogenetic protein (BMP) signaling. Furthermore, CXCR4-mediated signaling, but not BMP signaling, controls recovery of the nuclear Pax6+ neural crest progenitors. These findings not only explain why AM helps in vivo and ex vivo expansion of human LEPC, but they also illuminate the potential role of HC-HA/PTX3 as a surrogate matrix niche that complements stem cell-based therapies in regenerative medicine.

Dear Editor, In the January–March 2020 editorial in the Taiwan Journal of Ophthalmology, I briefly discussed the use of PROKERA® as an amniotic membrane (AM) corneal bandage to reduce inflammation, promote healing, and restore ocular surface health in patients suffering from dry eye disease (DED) and/or neurotrophic keratitis (NK).[1,2] PROKERA® is the only medical device designated by the U. S. Food and Drug Administration (FDA) for eye conditions involving damage to the ocular surface cells or underlying stromal inflammation or scarring. Cleared by the FDA in 2003, PROKERA® has been the U. S. market leader for >15 years and is used by thousands of eye care professionals worldwide. Herein, I wish to provide supplementary information regarding a subclass of PROKERA® called PROKERA® Slim and expand upon its use for various clinical indications.

The trabecular meshwork (TM) is composed of TM cells and beams of the extracellular matrix, together contributing to aqueous humor (AH) outflow resistance. Herein, we validated that our culture system on 2D Matrigel expressed putative TM markers and myocilin, of which the latter was upregulated by dexamethasone. Continuous passage of these cells on 2D Matrigel resulted in a gradual loss of expression of these markers. However, such a loss was restored by seeding cells in 3D Matrigel where expression of TM markers was further upregulated upon continuous passage. In contrast, TM cells seeded on fibronectin, collagen I/IV, or laminin lost expression of these markers and turned into myofibroblasts with expression of αSMA, which were dose-dependently upregulated by TGF-β1/TGF-β2. TM cells in 3D Matrigel also expressed TGF-β1/TGF-β3 despite challenge of TGF-β1. The maintenance of TM phenotype by 3D Matrigel was linked to inhibition of canonical TGF-β signaling and activation of pFAK-pSrc-pP190RhoGAP-P120RasGAP signaling. These findings indicate that basement membrane matrix with low rigidity plays an active role in maintaining TM phenotype in the presence of TGF-β1 and shed light on its physiological role. Furthermore, abnormal matrices may perpetuate the pathological TM phenotype when the level of TGF-β2 is elevated in glaucoma patients.

Purpose To evaluate the clinical outcomes of self-retained cryopreserved amniotic membrane (cAM) for the treatment of corneal ulcers. Methods This was a single-center, retrospective review of consecutive patients with non-healing corneal ulcers that underwent treatment with self-retained cAM (PROKERA® Slim). The primary outcome measure was time to complete corneal epithelialization. Ocular discomfort, corneal staining, corneal signs, and visual acuity were assessed at 1 week, 1 month, 3 months, and 6 months. Complications, adverse events, and ulcer recurrence were also recorded. Results A total of 13 eyes (13 patients) with recalcitrant corneal ulcers were included for analysis, 9 (69%) of which progressed from neurotrophic keratitis (NK). Prior to cAM application, patients used conventional treatments such as artificial tears (n = 11), antibiotics (n = 11), ointment (n = 11), steroids (n = 6), and antivirals (n = 3). Self-retained cAMs (n = 1.5 ± 0.8) were placed for 6.8 ± 3.4 days, during which time antibiotics were continued. Four cases (31%) were subsequently treated with bandage contact lens (n = 3) and tarsorrhaphy (n = 1). All corneal ulcers healed in a median of 14 days (range: 4–43). This was accompanied by a significant improvement in ocular discomfort, corneal staining, and corneal signs at 1 week, 1 month, 3 months, and 6 months (P<.05). Recurrence was noted in one case. No adverse events were observed. Conclusion Self-retained cAM may be a valuable, in-office treatment option for healing recalcitrant corneal ulcers of various etiologies, especially those with underlying NK. Further prospective, controlled studies are warranted.

1 T ocular surface, consisting of both the cornea and conjunctiva, is the only wetted body surface that is directly exposed to the outside environment. This mother‐nature design was developed through evolution and is essential to maintain ocular surface health so that one may enjoy clear vision without suffering from discomfort due to dryness in the open‐eye state. In this Issue, Mead et al.,[1] points out that the neuroanatomic integration of the ocular surface epithelia with the external adnexae, i.e., eyelids, lacrimal glands, and meibomian glands, is the operating mechanism to ensure ocular surface health. These diverse components are integrated into one unit by the first branch of the trigeminal nerve, which triggers tearing (compositional) and blinking (hydrodynamic) reflexes to maintain a stable preocular tear film. The concept of neuroanatomic integration explains why corneal pathologies are overlapped in two seemingly different diseases, i.e., neurotrophic keratitis and dry eye disease, once we realize that there is a progressive loss of subbasal corneal nerve density with increasing severity of the latter. For the rest of the body, taking diabetic foot ulcers as an example,[2] ischemia is the primary cause of nonhealing ulcers. As the cornea is avascular and already setup for ischemia, its source of oxygen depends on a stable precorneal tear film when the eye is open. To further compensate for this avascular “ischemic” state, the cornea is endowed with the most highly innervated tissue in the body to drive the aforementioned neuroanatomic integration. Therefore, the neuroanatomic integration also explains why neurotrophic keratitis causes the worst form of dry eye and is the prime cause of persistent epithelial defect and nonhealing ulcers for the cornea.

An open label, multicenter 16-week trial of cryopreserved human umbilical cord (TTAX01) was previously undertaken in 32 subjects presenting with a Wagner grade 3 or 4 diabetic foot ulcer, with 16 (50%) of these having confirmed closure following a median of one product application (previous study). All but two subjects (30/32; 94%) consented to participate in this follow-up study to 1-year postexposure. No restrictions were placed on treatments for open wounds. At 8-week intervals, subjects were evaluated for adverse events (AEs) and wound status (open or closed). Average time from initial exposure to end of follow-up was 378 days (range 343-433), with 29 of 30 (97%) subjects completing a full year. AEs were all typical for the population under study, and none were attributed to prior exposure to TTAX01. One previously healed wound re-opened, one previously unconfirmed closed wound remained healed, and nine new wound closures occurred, giving 25 of 29 (86.2%) healed in the ITT population. Three of the new closures followed the use of various tissue-based products. Three subjects whose wounds were healed required subsequent minor amputations due to osteomyelitis, one of which progressed to a major amputation (1/29; 3.4%). One additional subject underwent two minor amputations prior to healing. Overall, the study found TTAX01 to be safe in long-term follow-up and associated with both a low rate of major amputation and a higher than expected rates of healing.

OBJECTIVE:Despite significant improvement in spinal cord function after in utero spina bifida (SB) repair compared with traditional postnatal repair, over half of the children who undergo this procedure do not benefit completely. This lack of benefit has been attributed to closure methods of the defect, with subsequent spinal cord tethering at the repair site. Hence, a regenerative patch or material with antiinflammatory and anti-scarring properties may alleviate comorbidities with improved outcomes. The authors' primary objective was therefore to compare cryopreserved human umbilical cord (HUC) versus acellular dermal matrix (ADM) patches for regenerative repair of in utero SB lesions in an animal model. METHODS:In vivo studies were conducted in retinoic acid-induced SB defects in fetuses of Sprague-Dawley rats. HUC or ADM patches were sutured over the SB defects at a gestational age of 20 days. Repaired SB defect tissues were harvested after 48-52 hours. Tissue sections were immunofluorescently stained for the presence of neutrophils, macrophages, keratinocytes, meningeal cells, and astrocytes and for any associated apoptosis. In vitro meningeal or keratinocyte cell coculture experiments with the ADM and HUC patches were performed. All experiments were scored quantitatively in a blinded manner. RESULTS:Neutrophil counts and apoptotic cells were lower in the HUC-based repair group (n = 8) than in the ADM patch repair group (n = 7). In the HUC patch repair group, keratinocytes were present on the outer surface of the patch, meningeal cells were present on the inner surface of the patch adjacent to the neural placode, and astrocytes were noted to be absent. In the ADM patch repair group, all 3 cell types were present on both surfaces of the patch. In vitro studies showed that human meningeal cells grew preferentially on the mesenchymal side of the HUC patch, whereas keratinocytes showed tropism for the epithelial side, suggesting an inherent HUC-based cell polarity. In contrast, the ADM patch studies showed no polarity and decreased cellular infiltration. CONCLUSIONS:The HUC patch demonstrated reduced acute inflammation and apoptosis together with superior organization in regenerative cellular growth when compared with the ADM patch, and is therefore likely the better patch material for in utero SB defect repair. These properties may make the HUC biomaterial useful as a "meningeal patch" during spinal cord surgeries, thereby potentially reducing tethering and improving on spinal cord function.