Biomaterials and tissue engineering strategies for posterior lamellar eyelid reconstruction: Replacement or regeneration?

Abstract Reconstruction of posterior lamellar eyelids remains challenging due to their delicate structure, highly specialized function, and cosmetic concerns. Current clinically available techniques for posterior lamellar reconstruction mainly focus on reconstructing the contour of the eyelids. However, the posterior lamella not only provides structural support for the eyelid but also offers a smooth mucosal surface to facilitate globe movement and secrete lipids to maintain ocular surface homeostasis. Bioengineered posterior lamellar substitutes developed via acellular or cellular approaches have shown promise as alternatives to current therapies and encouraging outcomes in animal studies and clinical conditions. Here, we provide a brief reference on the current application of autografts, biomaterials, and tissue‐engineered substitutes for posterior lamellar eyelid reconstruction. We also shed light on future challenges and directions for eyelid regeneration strategies and offer perspectives on transitioning replacement strategies to regeneration strategies for eyelid reconstruction in the future.


| INTRODUCTION
The eyelids play critical roles in protecting the globe, maintaining ocular surface hydration, and expressing emotions. Anatomically, the eyelids contain two layers, namely, the anterior lamella and the posterior lamella 1 (Figure 1a). The eyelids not only physically provide protective coverage for the cornea and the globe but also serve important roles in maintaining ocular health and preserving visual integrity. Esthetically, the frame of the eye is distinctively defined by the position and shape of the eyelids. Thus, eyelid reconstruction must reconstruct both structure and function of the eyelids and restore an esthetically acceptable appearance with minimal surgical morbidities. To achieve these goals, we previously proposed that eyelid defects should be reconstructed following the "like for like" reconstructive principle and the surgeon should take into consideration the characteristics of the defects, including the thickness, size, and location. 2 By following these principles, numerous well-developed surgical techniques, including free skin grafts and tissue flaps, have been applied to repair skin or myocutaneous defects in anterior lamellar reconstruction, with satisfying functional and cosmetic outcomes in clinical practice. [3][4][5] However, due to the unique structural and functional properties of the eyelid, as well as the lack of histologically ideal donor tissues, current Yuxin   Bioengineering represents the future of reconstructive surgery and is emerging as a promising alternative for eyelid reconstruction ( Figure 3). Ideally, bioengineered posterior lamellar substitutes should restore key structures and functions of the eyelid without additional donor-site morbidities or postoperative complications. To date, many innovative native tissue grafts, 10,11 biomaterials, 12,13 and bioengineered tissues 14,15 have been proposed as posterior lamellar substitutes, some of which have been clinically used for eyelid reconstruction and have yielded promising preliminary results. 11,14,15 The bioengineering of posterior lamellar substitutes can be categorized into two distinct approaches: (1) acellular approaches, in which natural or synthetic acellular biomaterials are transplanted into defect areas to act as bioscaffolds for guiding tis-  Figure 4b). It appears that the presence of functional cells within the bioscaffold before implantation is particularly important for successful eyelid reconstruction, and strategies based on cellular approaches have indeed achieved promising results in preclinical studies. [16][17][18] However, these laboratory technologies are still in the progress of translation from bench to bedside, which will face many challenges. In this review, the eyelid anatomy will be briefly presented first to provide context for the requirements of ideal posterior lamellar substitutes, followed by an overview of current biomaterials and tissue engineering strategies for posterior lamellar eyelid reconstruction. Finally, the ongoing challenges and future directions of eyelid regeneration strategies will be discussed, and some perspectives will be offered on transitioning replacement strategies to regeneration strategies for eyelid reconstruction in the future.

| EYELID ANATOMY: FOCUSING ON THE POSTERIOR LAMELLAR
The eyelid is essentially a bilamellar structure that composed of the anterior and posterior lamella (Figure 1a). The former consists of the skin and the orbicularis oculi muscle, providing blood supply to the lamellar structures. The eyelid skin is thin and lacks subcutaneous fat. It is highly elastic and able to facilitate eyelid movement. The orbicularis oculi muscle, consisting of the preseptal, orbital, and pretarsal subunits, is responsible for eyelid closure. By using the laxity of adjacent tissues, local skin or myocutaneous flaps are used to reconstruct skin and muscle defects of the anterior lamella. 2 The posterior lamellar comprises the palpebral conjunctiva and the tarsal plate, which form histological foundations of the two primary functions of the posterior lamellar, that is, structural support and corneal protection via a mucosal surface. The tarsal plate is a semilunar-shaped structure approximately 25 mm in length and 1 mm in thickness, while the height of the tarsal plate ranges from 8 to 12 mm in the upper eyelid and 4 to 5 mm in the lower eyelid. 19,20 The tarsal plate is a distinct transitional connective tissue which possesses the characteristics of both cartilage and dense fibrous connective tissue. 21,22 Histologically, the tarsal plate is not a truly fibrocartilaginous structure 23 (Figure 1c). The meibum, an essential composition of the tear film, is spread out when meibomian gland ducts are compressed by the transverse muscle fibers during blinking. Meibomian gland dysfunctions can result in ocular and eyelid discomfort, ocular surface diseases such as evaporative dry eye. 28 The conjunctiva, which arises from the corneoscleral limbus and overlies the inner surface of the eyelid, comprises a nonkeratinized stratified columnar and stratified squamous epithelium interspersed with goblet cells and a vascularized basement membrane composed of laminin and Col IV. 29 The epithelium is composed of stratified squamous nongoblet cells (90%-95%), goblet cells (5%-10%), and occasional lymphocytes and melanocytes 30 (Figure 1b). Soluble mucins can be secreted by the goblet cells, which serve as a crucial component of the tear film to bathe the ocular surface ( Figure 1d). Conjunctival loss or scarring that can result from conjunctival damage or ocular surface disease may lead to eyelid misalignment, limited eye movement, diplopia, and dry eye symptoms. 31 In addition, it should be noted that the conjunctiva can spontaneously re-epithelialize upon injury, as both stratified squamous nongoblet and goblet cells can be regenerated continuously by conjunctival stem cells, 32 inspiring tissue engineering strategies using conjunctival stem cells to repair conjunctival defects. F I G U R E 3 The three basic elements in eyelid tissue engineering.
Collectively, the posterior lamella is a delicate bilamellar structure that serves two primary functions: mechanical support for the eyelid and sufficient lubrication of the cornea. Specifically, the tarsal plate provides mechanical support, maintains the appearance of the eyelid, and prevents abnormalities such as corneal exposure and eyelid retraction, making it an essential part of physical appearance and eyelid functions.
The conjunctiva provides a mucosal surface that allows smooth globe movement; additionally, the goblet cells within the conjunctiva and the meibomian glands within the tarsal plate secrete mucins and lipids, respectively, which stabilize the tear film to maintain ocular surface homeostasis. To achieve functional eyelid reconstruction, an ideal posterior lamellar substitute should have the following characteristics:  Tarsomarginal graft is a composite graft, which is composed of the tarsal plate, conjunctiva, lid margin, and eyelashes ( Figure 5a). Currently, tarsomarginal grafts of One-fourth and even one-third of the whole eyelid width can be obtained by primary closure. 44 Tarsomarginal grafts are suitable for eyelid defect reconstruction in various areas, including medial, central, or lateral defects in both lower and upper eyelids, particularly, the defects involving the eyelid margin, as these grafts preserve the native structures of the lid margin. As a free graft, the tarsomarginal graft should be covered with a local myocutaneous flap or a distal axial flap to ensure sufficient blood supply, which allows simultaneous transplantation of not only one but also two even three tarsomarginal grafts harvested from different donor eyelids. The main limitations of these autologous grafts include small graft size, eyelid retraction, scar formation, and donor eyelid morbidity, 45 which hinder their clinical application. 44,45 The postoperative complication rate for free tarsoconjunctival graft implantation has been reported to be as high as 43% in the East Asian population 35 and 84% in the Caucasian population. 46 Ectropion and entropion are the most common complications in the lower and upper eyelids, respectively.

| Oral mucosa grafts
The oral mucosa consists of vascular connective tissue named the lamina propria and nonkeratinized stratified squamous avascular epithelium 56 (Figure 5b). Oral mucosal grafts obtained from different parts of the oral cavity have variable features. Oral mucosal grafts from the lip and bucca are rich in elastin, which confers resistance to shearing and compression, and are highly vascularized, which facilitates integration of the graft once transplanted. Thus, buccal and labial mucosal grafts are typically used to replace the conjunctiva or as a lining for other tarsal substitutes, 57 such as auricular cartilage grafts 49,58 and amniotic membrane grafts. 59,60 Oral mucosal grafts can be harvested in forms of oral mucosal membrane grafts and minor salivary gland grafts, the latter one has been used to treat severe dry eye disease. The mucosal membrane can serve as a bioscaffold to support epithelial cells proliferation and growth. In addition, as shown by in vitro studies, epithelial stem/progenitor cells that exist in oral mucosal grafts can re-epithelialize the tarsal plate and stabilize the ocular surfaces. 61,62 However, the mucous membranes do not contain goblet cells, which may lead to corneal irritation and dry eye symptoms. 63 Hence, simultaneous minor salivary gland grafting may be warranted to solve this problem. 64

| Hard palate mucoperiosteal grafts
The hard palate mucosa that composed of a mucosal surface and a dense fibrous connective tissue layer has a histological structure similar to that of the posterior lamella ( Figure 5c). Hard palate mucoperiosteal grafts, taken from the center of the hard palate, can provide both rigid structural support and a moist mucosal surface for the eyelid, making them an optimal substitute for the reconstruction of the posterior lamella. 53,65,66 However, the hard palate mucosa is a firm tissue featuring keratinized stratified squamous epithelium. Furthermore, long-term observation revealed that the persistence of orthokeratosis and/or parakeratosis may last for years following transplantation, which may result in irritation of the cornea and cause pain. 67 Additionally, the donor area of hard palate mucoperiosteal grafts is usually left alone for secondary healing, which may cause possible hemorrhage, donor-site discomfort, and difficulties in eating.
The gingival mucosa is a firm, thick, and keratinized mucosa that extends to the nonkeratinized alveolar mucosa, which is relatively loose and mobile. Grafts harvested from the gingival alveolar region consist of a segment of gingival mucosa 2 mm in height, with the remainder comprising alveolar mucosa. This transitional structure allows gingival alveolar mucosa grafts to be suitable for repairing posterior lamellar defects that involve the eyelid margin. The marginal palpebral area can be replaced by the tight gingival pyart of the graft to achieve a rigid and stable structure, and the nonkeratinized alveolar part can ensure a nonirritating posterior lamellar surface. 51

| Nasal mucosal grafts
Nasal mucosal grafts harvested from the nasal septum, 68,69 turbinate, 67 or ala 55 bear a close histological resemblance to the tarsoconjunctiva. Furthermore, the nasal mucosa contains not only a large number of goblet cells but also subepithelial mucin glands; therefore, it is able to provide substitute mucin. 67 Through a tight connection to the eyelid, septal mucochondral grafts can provide firmness as a tarsal substitute and a stable eyelid margin ( Figure 5d). After thinning of the cartilage, grafts will curl up toward the mucosa side, matching the contour of the tarsal plate and achieving satisfying eyelid support and cosmetic outcomes. 70 Nasal mucosal grafts harvested from the inner part of the nasal ala can avoid the risk of septal perforation and hemorrhage. 55 In addition, composite mucosal grafts should be harvested from beyond the nasal hair area because conjunctival or corneal irritation may be caused by any remaining nasal hair. 71

| Auricular cartilage grafts
Auricular cartilage grafts are thin elastic cartilage with suitable flexibility and appropriate physical strength ( Figure 5e). Due to their lower metabolic rate and fewer vascular requirements, auricular cartilage grafts remain viable for many years after implantation. Thus, reconstruction of the posterior lamella with auricular cartilage grafts leads to a cosmetic contour and adequate support without significant graft absorption or shrinkage. 72,73 When auricular cartilage grafts are used for tarsoconjunctiva replacement, the conjunctival surface is missing and usually left for reepithelization, but the rough surface of the grafts may result in corneal irritation. 74 Adding an oral mucosal graft for coverage could help solve this problem. 49

| Decellularized matrixes
Studies have demonstrated that the amniotic membrane can support epithelialization by offering a basement membrane and an avascular stromal matrix similar to the native conjunctiva. Furthermore, amniotic membrane possesses anti-angiogenic, anti-inflammatory, and antiscarring properties, making it the most widespread substitute for conjunctival replacement. 75,76 Clinically, decellularized amniotic membrane grafts are placed directly over the bare tarsus up to the eyelid margin 77,78 or applied in combination with other techniques for posterior lamellar reconstruction. 79,80 As a scaffold, the implanted decellularized amniotic membrane can support the growth of surrounding conjunctiva-derived cells, including conjunctival nongoblet epithelial cells and goblet cells, 78,81 and is then absorbed within 6 months without scarring. 82 Derived materials, such as freeze-dried amniotic membrane, 75 air-dried dehydrated amniotic membrane, 83 and ureade-epithelialized amniotic membrane, 84 have been developed to optimize the processes of amniotic membrane sterilization and storage and to better preserve the basement membrane structures, growth factor contents, and stroma matrix components. Although good functional and esthetic outcomes have been reported, the limited availability, variable standards for preparation, inconsistent tissue properties, and risk of infectious disease transmission continue to make the use of decellularized amniotic grafts problematic. 84 In addition to decellularized amniotic membranes, decellularized porcine and human conjunctival grafts have also been tested for conjunctival defect reconstruction in animal experiments 85,86 and clinical studies. 85 In clinical cases, transplanted decellularized porcine conjunctival grafts were integrated with autologous conjunctiva without graft disintegration or fibroplasia and were completely epithelialized at 2 weeks. 85 In addition, other decellularized biomembranes, such as decellularized bovine or porcine pericardium 10,87 and decellularized adipose-derived mesenchymal stromal cell matrix, 88 have also shown promising results in facilitating the adhesion of goblet cells and conjunctival epithelial cells in conjunctival substitute engineering. Acellular dermal matrixes originated from human (AlloDerm ® , BellaDerm ® ), [89][90][91] porcine (Endurage ® ), 11,[92][93][94] and bovine (Surgimen ® ) 94,95 dermis provide off-the-shelf materials to replace the tarsal plate because of relatively low immunogenicity and good histocompatibility. 96 Structurally, the ADM is a sturdy but flexible flat sheet compris-  101 However, the main drawback limiting the widespread use of ADM grafts is graft contraction and resorption, especially for grafts originating from humans. 102 The mean graft contraction rate has been reported to be 57% for acellular dermal matrix grafts versus 16% for hard palate mucosal grafts. 103 Another clinically available acellular membrane for tarsal plate reconstruction is TarSys ® , which is decellularized porcine small intestinal submucosa containing Col I, Col III, Col IV, and associated glycosaminoglycans. 104,105 However, as TarSys ® is a xenogeneic product, concerns due to the risk of infectious disease transmission and reported immunogenic inflammatory-related complications remain. 106-108

| Natural polymers
Natural polymer scaffolds sourced from ECM components have been widely used for conjunctival defect repair due to their high degree of biocompatibility; such scaffolds include Col I, 109 chitosan, 110 and keratin. 111 Among them, Col I, the most abundant protein in the conjunctival matrix, is the most commonly used. It has been demonstrated to successfully support not only conjunctival epithelial cell proliferation but also differentiation in vitro. 112 In vivo, collagen-based substitutes have been shown to promote conjunctival regeneration with complete epithelization, minor fibrosis, and minimal wound contracture. 109,113 However, untreated collagen hydrogels are opaque and exhibit poor mechanical stability because they are made of loosely packed collagen fibers and thus differ from the native conjunctival stroma, which limits their surgical applicability. Therefore, plastic compressed collagen gels 109,112 and vitrified collagen membranes 113 have been developed to simulate collagen fibril arrangement in normal conjunctiva and provide enhanced mechanical strength. In vitro studies have demonstrated that plastic compressed collagen gels and vitrified collagen membranes support conjunctival epithelial cell proliferation and phenotype development. 112,113 Once implanted into the conjunctival defect area in rabbit models, these conjunctival equivalents engineered from both collagen-based materials promoted conjunctival regeneration by supporting rapid re-epithelization as well as sufficient goblet cell repopulation while minimizing wound contracture and fibrosis. 109,113 Other natural ECM proteins, such as gelatin, 114,115 elastin, 116 and their combinations, 81  Additionally, synthetic polymers, such as porous polyethylene (PE), 13,104,123 poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), 124 poly(propylene fumarate) (PPF), 125,126 poly(lactic-coglycolic) acid (PLGA), 18 and PCL, 16 have also been used in tarsal plate reconstruction in recent studies. Among them, only porous high-density PE (Medpor ® ) has been used as an eyelid spacer for treating lower eyelid retraction in clinical practice. 123 However, this material should be used sparingly in eyelid surgery, due to high postoperative complication rates, such as poor stability, implant exposure, abnormal skin contours, and unexplained pain. 127 Other synthetic scaffolds are available and have been investigated for tarsal reconstruction in animal models. However, poor tissue tolerance impedes their clinical translation, and inflammatory tissue responses have been observed around the implants. 7 The common advantages of these polymers are good biodegradability, rigidity, and moldability. However, compared with scaffolds derived from natural tissues, synthetic polymers have relatively low biocompatibility and usually have hydrophobic features because of the lack of natural cell recognition sites, 7,128 which limits their clinical translation.
These limitations have prompted attempts to modify their biological characteristics by combining several differential biomaterials to make scaffolds with properties more closely resembling those of the natural tarsal plate. For example, with its excellent mechanical integrity and cytocompatibility, the biodegradable biopolymer PLA is widely used in the biomedical field. However, this material is hydrophobic, which is unfavorable for cell adhesion and growth. 128 It has been considered a novel direction of tissue engineering to blend PLA with other biomaterials with better wettability, which could result in better tissue recovery.

| Tissue-engineered conjunctival equivalents
As far as we know, no study has investigated cellular approaches to repair palpebral conjunctival defects so far. Notably, numerous studies on bulbar conjunctival tissue engineering have demonstrated encouraging results in the promotion of reepithelialization and reduction of scarring in both animal models 17,32,120 and clinical trials 59,129 (Table 1).
Considering that the palpebral conjunctiva has structures and functions similar to those of the bulbar conjunctiva, 30 strategies for engineering bulbar conjunctival equivalents are translatable for engineering palpebral conjunctiva equivalents.
T A B L E 1 Cellular approaches for conjunctival reconstruction.

Conjunctival source
Wu et al.
-Large conjunctiva defect model in rabbits. -Conjunctiva epithelium engineered from rabbit p75 ++ CjECs achieved more complete reepithelization and more goblet cells than that engineered from control cells. (Continues) To meet the needs of conjunctival repair, a tissue-engineered con-  inexpensive, but high rates of complications have also been reported, such as graft contraction and shrinkage, 102 graft exposure, 98 and cyst formation. 14 Most importantly, due to the lack of posterior lamellar cellular components, these grafts barely provide a smooth epithelialized surface and restore secretory function. Although some studies have reported that implanted decellularized matrixes could support cell growth both in vitro 84 and in vivo, 85 concomitant fibrosis rather than complete reepithelization was observed on the surface of the biomaterials upon implantation in animal models. 10,109 Cellular approaches aim to restore the full function of the posterior lamella by implanting a tissue-engineered tarsal or conjunctival equivalent into the defect area, which can be referred to as "regenerative surgery" and represents a future direction in this field. For example, by seeding immortalized human SZ95 sebocytes onto a scaffold, a tarsal plate substitute capable of secreting neutral lipids both in vitro and in vivo was successfully engineered. 16 Moreover, by culturing conjunctival epithelial cells on vitrified collagen membrane, conjunctival equivalents that could greatly promote goblet cell repopulation as well as minimize fibrosis and wound contracture was successfully developed. 113 These results, at least partially, reproduced the secretory function of the human eyelid. However, the field remains in its infancy, and extensive investigation is required in the future.  81 Compared to the amniotic membrane, the 3Dprinted membrane had a higher density of goblet cells on the regenerated epithelium and had a more predictable degradation pattern, less scar formation, and minor inflammatory responses in vivo. 81 Another study demonstrated the successful application of bioprinted hydrogel microconstructs loaded with rabbit conjunctival stem cells for conjunctival regeneration. 159 The bioprinted microconstructs favorably

| Focusing on secretory function
The restoration of secretory function remains another great challenge in eyelid reconstruction. Goblet cells within the conjunctiva and meibomian glands within the tarsus are responsible for secreting lipids and mucins, respectively, which participate in tear film formation and maintain ocular surface homeostasis. 160,161 Engineering tarsal plates and conjunctival equivalents able to secrete lipids and/or mucins presents a potential strategy to solve this problem. Stem cells involved in the development of meibomian glands have not been well demonstrated thus far, posing an obstacle to meibomian gland regeneration.
Recently, a stem/progenitor cell population expressing Krox20, a zinc finger transcription factor, was identified as a crucial driver of the development and homeostasis of the meibomian gland, 162 shedding light on the identification of meibomian gland stem cells and the subsequent construction of meibomian gland substitutes. In addition to regenerating the meibomian glands, engineering tarsal equivalents by directly seeding lipid-secreting sebocytes onto scaffolds presents another feasible approach. 16 However, sebocytes are terminally dif- project administration (equal); resources (equal); funding acquisition (equal); writing-review and editing (equal).