Medicine, Faculty of
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Medicine, Department of
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Materials 17 (22): 5425 (2024)
Wu, Kevin Y.
Dave, Archan
Daigle, Patrick
Tran, Simon D.
The lacrimal gland (LG) is vital for ocular health, producing tears that lubricate and protect the eye. Dysfunction of the LG leads to aqueous-deficient dry eye disease (DED), significantly impacting quality of life. Current treatments mainly address symptoms rather than the underlying LG dysfunction, highlighting the need for regenerative therapies. Tissue engineering offers a promising solution, with biomaterials playing crucial roles in scaffolding and supporting cell growth for LG regeneration. This review focuses on recent advances in biomaterials used for tissue engineering of the lacrimal gland. We discuss both natural and synthetic biomaterials that mimic the extracellular matrix and provide structural support for cell proliferation and differentiation. Natural biomaterials, such as Matrigel, decellularized extracellular matrices, chitosan, silk fibroin hydrogels, and human amniotic membrane are evaluated for their biocompatibility and ability to support lacrimal gland cells. Synthetic biomaterials, like polyethersulfone, polyesters, and biodegradable polymers (PLLA and PLGA), are assessed for their mechanical properties and potential to create scaffolds that replicate the complex architecture of the LG. We also explore the integration of growth factors and stem cells with these biomaterials to enhance tissue regeneration. Challenges such as achieving proper vascularization, innervation, and long-term functionality of engineered tissues are discussed. Advances in 3D bioprinting and scaffold fabrication techniques are highlighted as promising avenues to overcome current limitations.
Citation: Wu, K.Y.; Dave, A.; Daigle,P.; Tran, S.D. Advanced Biomaterialsfor Lacrimal Tissue Engineering: AReview. Materials 2024, 17, 5425.https://doi.org/10.3390/ma17225425Academic Editor: Javier GilReceived: 30 September 2024Revised: 31 October 2024Accepted: 31 October 2024Published: 6 November 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).ReviewAdvanced Biomaterials for Lacrimal Tissue Engineering:A ReviewKevin Y. Wu 1 , Archan Dave 2, Patrick Daigle 1 and Simon D. Tran 3,*1 Department of Surgery, Division of Ophthalmology, University of Sherbrooke, Sherbrooke, QC J1G 2E8,Canada; yang.wu@usherbrooke.ca (K.Y.W.)2 Department of Medicine, University of British Columbia, Vancouver, BC V6T 1Z3, Canada3 Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC H3A 1G1, Canada* Correspondence: simon.tran@mcgill.caAbstract: The lacrimal gland (LG) is vital for ocular health, producing tears that lubricate and protect theeye. Dysfunction of the LG leads to aqueous-deficient dry eye disease (DED), significantly impactingquality of life. Current treatments mainly address symptoms rather than the underlying LG dysfunction,highlighting the need for regenerative therapies. Tissue engineering offers a promising solution, withbiomaterials playing crucial roles in scaffolding and supporting cell growth for LG regeneration. Thisreview focuses on recent advances in biomaterials used for tissue engineering of the lacrimal gland.We discuss both natural and synthetic biomaterials that mimic the extracellular matrix and providestructural support for cell proliferation and differentiation. Natural biomaterials, such as Matrigel, de-cellularized extracellular matrices, chitosan, silk fibroin hydrogels, and human amniotic membrane areevaluated for their biocompatibility and ability to support lacrimal gland cells. Synthetic biomaterials,like polyethersulfone, polyesters, and biodegradable polymers (PLLA and PLGA), are assessed fortheir mechanical properties and potential to create scaffolds that replicate the complex architectureof the LG. We also explore the integration of growth factors and stem cells with these biomaterials toenhance tissue regeneration. Challenges such as achieving proper vascularization, innervation, andlong-term functionality of engineered tissues are discussed. Advances in 3D bioprinting and scaffoldfabrication techniques are highlighted as promising avenues to overcome current limitations.Keywords: biomaterials; tissue engineering; lacrimal gland; dry eye disease; natural and syntheticbiomaterials; 3D bioprinting; regenerative medicine1. IntroductionThe lacrimal gland is essential for ocular health, as it is responsible for producing theaqueous component of the tear film that lubricates the eye, providing corneal nutrients,and protecting against microbial infections. Dry eye disease (DED) affects approximately344 million people globally, with prevalence estimates varying widely because of differencesin study populations and definitions. General DED prevalence is estimated at 5–50%,with higher rates in populations over 50 years of age and in women due to hormonalfactors [1]. In the USA, it is estimated that about 20 million people suffer from DED, witha higher incidence in urban settings due to environmental factors like air pollution andlower humidity [1]. Dysfunction of the lacrimal gland leads to aqueous-deficient DED, acondition characterized by symptoms such as ocular discomfort, visual disturbances, anda significant reduction in quality of life [2]. Current treatments for DED primarily focuson symptom management—using artificial tears, anti-inflammatory agents, and punctalplugs—without addressing the underlying glandular dysfunction. These approaches oftenoffer only temporary relief and may have side effects that affect patient compliance [3,4].Advancements in tissue engineering present a promising avenue for restoration oflacrimal gland function by regenerating damaged tissue and reinstating natural tear produc-tion. Central to this approach is the development of suitable biomaterials that can serve asMaterials 2024, 17, 5425. https://doi.org/10.3390/ma17225425 https://www.mdpi.com/journal/materialsMaterials 2024, 17, 5425 2 of 21scaffolds to support cell growth, differentiation, and the formation of functional glandularstructures [5]. Both natural and synthetic biomaterials have been explored for this purpose.Natural biomaterials, like Matrigel, decellularized extracellular matrices, chitosan, silkfibroin hydrogels, and human amniotic membrane, have shown potential in mimicking theextracellular matrix and promoting cell viability and differentiation. Synthetic biomate-rials such as polyethersulfone, polyesters, and biodegradable polymers like poly-L-lacticacid (PLLA) and poly(lactic-co-glycolic acid) (PLGA) offer tunable mechanical propertiesand structural versatility but may require modifications to enhance biocompatibility andcellular interactions [6].This review focuses on recent advances in biomaterials used for lacrimal gland tissueengineering. We examine the properties of various natural and synthetic biomaterials, theirapplications in supporting lacrimal gland cell growth, and their roles in facilitating tissueregeneration. Additionally, we discuss the integration of stem cells and growth factors withthese biomaterials to enhance regenerative outcomes. Challenges such as replicating thecomplex architecture of the lacrimal gland, achieving proper vascularization and innerva-tion, and ensuring long-term functionality of engineered tissues are also addressed. Byproviding an overview of the current state of biomaterial-based lacrimal gland engineering,we aim to highlight the potential of these approaches to develop effective regenerativetherapies for patients with lacrimal gland dysfunction.2. Lacrimal Gland Anatomy and DisordersThe aqueous portion of the tear film is secreted by the lacrimal gland (LG), which sitson the anterior, superotemporal portion of the orbit within the lacrimal fossa of the frontalbone [7]. The LG comprises both the main lacrimal gland and smaller accessory lacrimalglands of Krause and Wolfring (Figure 1). The glands of Krause are located in the conjunctivalfornices, and the glands of Wolfring are distributed along the nonmarginal tarsal borders.Tear fluid released by the glands help to nourish, moisten, and protect the ocular surfacethrough its inherent antibacterial activity. Aqueous tears consist mainly of water, electrolytes,and proteins responsible for maintaining the health of the ocular surface [8]. This 2 cm longtubuloacinar gland contains many lobules, which are composed of mixed serous and muci-nous acini, as well as supporting myoepithelial cells and between 8 and 12 main excretoryducts [7]. The polarized lacrimal epithelial cells are organized into distinct acini, whichencircle a central duct lined by ductal epithelial cells. Acinar cells compose approximately80% of the cell population, while duct cells account for approximately 10 to 12% of thetotal [9]. The LG is anchored to the orbital periosteum by connective tissue, receives bloodfrom the lacrimal artery, and is innervated by the lacrimal nerve through the ophthalmicbranch of the trigeminal nerve (CN V). The LG also receives sympathetic innervation, whichworks in conjunction with parasympathetic signals to regulate fluid secretion and maintainhomeostatic tear volumes [9]. Myoepithelial cells around the acini contract in response tonerve signals, allowing for fluid to drain onto the ocular surface through the excretory duct.Neural regulation of the LG is a complex process, mainly governed by the tear–reflex looppathway which secretes tears in response to ocular stimulation [9].The lacrimal gland plays a crucial role in ocular health by producing the aqueouscomponent of the tear film, which is essential for lubricating the eye, providing nutrients tothe cornea, and protecting against microbial infections. Loss of lacrimal gland function leadsto a deficiency in tear production, resulting in dry eye disease (DED). DED is a multifactorialcondition characterized by a variety of non-specific symptoms, including ocular redness,burning sensations, itching (i.e., pruritus), foreign body sensation, and the presence ofstringy or mucous discharge [2]. If left untreated, DED can progress to severe complications,such as corneal epithelial breakdown, ulceration, and corneal melting, which significantlythreaten visual acuity [10]. Additionally, the chronic discomfort and visual disturbancesassociated with DED can markedly diminish an individual’s quality of vision and overallquality of life [11]. There is a high prevalence of DED, at around 12%, depending on sex,age, and location [12]. In short, alterations in the secretory function of the lacrimal glandMaterials 2024, 17, 5425 3 of 21can compromise tear film stability and degrade the quality of tear secretions, leading to adisruption in ocular surface homeostasis. Inflammation (i.e., dacryoadenitis) secondary toautoimmune diseases, such as Sjögren’s syndrome [13], rheumatoid arthritis, and systemiclupus erythematosus, can be associated with LG dysfunction. Sjögren’s syndrome is a rare,T-cell-driven disease characterized by lymphocytic infiltration of the lacrimal and salivaryglands. It should be noted that there is no standardized screening tool to refer to for Sjögren’ssyndrome; therefore, it is often underdiagnosed [14]. Beyond autoimmune conditions, factorssuch as chemotherapy, radiation therapy, and graft-versus-host disease can damage lacrimalgland tissue or provoke immune-mediated attacks on the gland. Physical trauma—whethersurgical or accidental—can disrupt tear secretion pathways. Other causes of LG dysfunctioninclude but are not limited to infectious agents (bacterial, viral, and fungal), neoplasticprocesses (benign or malignant tumors), granulomatous diseases like sarcoidosis, infiltrativediseases such as amyloidosis, and age-related atrophy of the gland [11,15].Figure 1. Lacrimal system anatomy. Created with BioRender.com and Servier Medical Art.Current treatments for dry eye disease, including those caused by LG dysfunction,primarily focus on symptom management rather than addressing the underlying lacrimalgland dysfunction. Conservative approaches include moisture-chamber spectacles to re-duce tear evaporation and the frequent application of artificial tears to supplement tearvolume [16,17]. More advanced therapies involve punctal plugs to decrease tear drainage,topical anti-inflammatory agents, such as cyclosporine A and lifitegrast, to modulate ocularsurface inflammation, and short-term use of topical corticosteroids during acute exacerba-tions [18]. Autologous serum eye drops are also employed in severe cases to provide essentialtear components. However, these treatments often offer only temporary relief, require fre-quent administration, and may have side effects that impact patient compliance [3,4]. Thelimitations of current therapies highlight the need for innovative solutions that restore naturaltear production in patients suffering from LG dysfunction. This has sparked growing interestin lacrimal gland tissue engineering as a potential strategy to restore gland function andprovide long-term relief for patients with aqueous-deficient dry eye disease.3. Biomaterials for Lacrimal Tissue EngineeringMost commonly, tissue engineering consists of manufacturing a biocompatible scaf-fold and using specific progenitor cells and multiple growth factors to ensure effectiveregeneration and reconstruction of tissue. Scaffolds serve as the structural foundationfor cultured cells, enabling them to attach and form the unique morphology of the tissue.Moreover, scaffolds must provide a suitable environment for differentiated cells to expand,Materials 2024, 17, 5425 4 of 21proliferate, migrate, and be resilient enough to endure foreign stressors. The most idealscaffold is one that mimics the extracellular matrix (ECM) of the target tissue [6].However, scaffold-free tissue engineering relies on the self-organization of cells, whichare allowed to produce their own ECM through cell-to-cell interactions. This closely mimicsnatural tissue development, reduces the risk of scaffold-related complications, and offersnatural communication among cells in the tissue. Schrader et al., (2009) demonstratedthe formation of 3D LG spheroids from acinar cells using a rotary-cell-culture system [19].However, apoptosis was observed in the spheroid center, likely due to the lack of vascularsupply, which consequently diminished cell secretory function by day 28. Ackermann et al.,(2015) isolated murine LG stem cells and explored their potential for differentiating intovarious cell types [20]. The group specifically employed the hanging drop culture methodto form 3D organoid bodies; however, the transmission electron microscopy (TEM) resultsshowed that most cells remained undifferentiated with a few exceptions. Lin et al., (2017)demonstrated LG progenitor cell proliferation into a gland-like spheroid and acinotubularstructure when cultured in a 3D environment using laminin gel [21]. The cells showedsigns of acinar and ductal differentiation, expressing markers such as K4, lactoferrin, andaquaporin 5. However, the size, shape, and exact composition were difficult to control,and the 3D cultures lost their structural integrity over time. While the 3D culture inthis study lasted for about 2 weeks, Tiwari et al., (2018) produced lacrispheres lastingfor 21–28 days, during which they retained their stemness, proliferative potential, andsecretory function. The group enzymatically digested human LG and cultivated them in aserum-free, 3D culture system. The group provided a scaffold-free platform that was ableto maintain the long-term survival of LG cells and secrete significant levels of tear protein,including lysozyme, lactoferrin, and sclgA [22]. Despite these advancements, scaffold-free tissue engineering faces challenges, as noted by its inability to maintain long-termstructural stability and proliferative capacity as compared to scaffold-based 3D culturesystems. However, the ability of these systems to self-organize into functional secretoryunits demonstrates the regenerative potential of the LG and encourages future explorationinto scaffold-free LG restoration.Scaffold-based systems use either natural or synthetic biomaterials to support LG cellgrowth and differentiation. These materials are often biocompatible and possess specificmechanical properties and degradation rates, making them adaptable for different tissue-engineering applications. The spatial arrangement of cells, delivery of growth factors, size,and shape can also be controlled through scaffold-based systems. The most popular naturalbiomaterial for organoid development is Matrigel; however, its variability in compositionamong batches, indeterminate degradation rate and immunogenic properties presentschallenges in tissue transplantation applications [5]. Kozlowski et al., (2021) provideextensive alternative solutions, such as decellularized ECMs, synthetic hydrogels, andgel-forming recombinant proteins [5].This section covers both natural and synthetic biomaterials that are used, currentlyinvestigated, or may be considered in LG tissue engineering.3.1. Natural BiomaterialsXenografting refers to the transplantation of tissue from animals to humans. Thisapproach to bioengineering provides an immediate source for grafting, hence significantlyreducing waiting times. It typically offers fully formed tissues with proper structure andfunctional vascular components in place. For example, Henker et al., (2013) claim that theporcine LG anatomy shares morphological similarities with human glands, albeit largerin size and having more seromucous secretory units compared to humans [23]. In fact,the team proposes that pig-to-human LG transplantation could be feasible, owing to thestraightforward vascular connections and favorable anatomical positioning. However, thestudy describes immunological barriers and the risk of endogenous pig retrovirus transfer asobstacles to pig-to-human transplantation. While rat, mouse, and rabbit models are oftencompared to human LGs, they each possess unique characteristics that make interspeciesMaterials 2024, 17, 5425 5 of 21transplantation challenging. However, exploration of LG models from other species arecritical in understanding LG function, pathologies, and developing bioengineering tools [24].Many natural biomaterials are considered for LG bioengineering [Table 1]. Matrigel isa natural polymer derived from ECM proteins secreted by Engelbreth–Holm–Swarm (EHS)mouse sarcoma cells. Matrigel contains many structural proteins, such as laminin, collagenIV, entactin, and heparin sulfate proteoglycans, hence holding major components of themany tissue basement membranes [25]. Many growth factors, such as epidermal growthfactor (EGF), fibroblast growth factor (FGF), and insulin-like growth factor 1 (IGF-1), areincluded in Matrigel [26]. These activate cellular signaling pathways, including mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways, and, ultimately, stimulatemitosis and cell proliferation. In fact, stem cell differentiation is enhanced in vitro whenplated on the thick basement membrane matrix due to its ability to promote cell survival,differentiation, and vascularization [27]. Early adopters of Matrigel, such as Schechter et al.,(2002), applied its use as a “raft culture” of lacrimal acinar cells enclosed within the gelcoating [28]. These rafts were seeded onto Matrigel-coated culture plates, and growthwas observed for 28 days. Matrigel closely mimicked the natural environment of theLG and allowed the cells to maintain a differentiated state and polarity, enabling betterin vitro study of the acinar physiology. However, the study observed a decrease in theexpression of prolactin (PRL) and MHC-II proteins after 21 days in culture, suggestingthat while Matrigel supports acinar cell development for a significant period, there maybe a gradual loss of functionality over time, limiting clinical translation or long-termuses. Furthermore, Matrigel possess rather poor mechanical properties, compared to othersynthetic and natural biomaterials; hence, it is usually fortified with another material, suchas collagen, if used as a scaffolding material [29]. However, Matrigel’s laminin and collagenIV components enable integrin binding and is critical for mechanotransduction, whichallows cells to sense and response to the matrix and guide differentiation [30]. Tiwari et al.,(2012) cultured human LG tissue on different substrates, such as Matrigel, collagen, andhuman amniotic membrane, and evaluated the expression of stem cell (ABCG2, c-Kit, andALDH1) and differentiation markers [31]. The functionality of cultured cells was alsoassessed through secretion of secretory proteins, such as IgA, lactoferrin, and lysozyme.Matrigel provided an optimal substrate and exhibited higher rates of proliferation whilemaintaining secretory function. However, the study notes that the differences betweenMatrigel, collagen, and human amniotic membrane were not significant. Interestingly,Yoshino (2000) found lower rates of proliferation on Matrigel when compared to type 1collagen gel with and without fibroblasts and plastic as the control. However, Matrigelpromoted higher levels of acinar differentiation [32,33]. The incorporation of fibroblastsin collagen gels also further supported ductal differentiation. Asal et al., (2023) aimedto develop functional LG organoids using human-induced pluripotent stem cells (iPSCs)through a multizonal ocular differentiation approach [34]. iPSCs were seeded onto aMatrigel matrix to support their differentiation over the course of 7 weeks. The studyfound that Matrigel effectively supported the formation of acinar structures and facilitatedbranching morphogenesis. Gleixner et al., (2024) demonstrated the use of Matrigel todevelop immortalized human LG cells lines in a 3D spheroid culture model [35]. The groupclaimed Matrigel’s complex composition of basement membrane components, such aslaminin, collagen IV, and other growth factors, are effective in enhancing differentiationand promoting gland-like structure formation. Zeng et al., (2024) utilized Matrigel todevelop a culture system to support the in vitro expansion of mouse LG epithelial cells [36].Matrigel was used in conjunction with two small molecules, Y27632 and SB431542, topromote high proliferation and maintain cell morphology through multiple passages.Upon removal of the small molecules, LG epithelial cells were differentiated into secretorycells with increased expression of differentiation makers such as AQP5 and lactoferrin(LTF). The authors demonstrate that Matrigel effectively mimics the ECM in vitro and,when combined with small molecules, offers a reliable strategy to promote the proliferationof LG epithelial cells. Overall, Matrigel appears to be the most popular scaffold materialMaterials 2024, 17, 5425 6 of 21for LG bioengineering and current trends seem to point toward fortifying Matrigel andintegrating growth factors, small molecules, cytokines, and activating transcription factorsto enhance cell proliferation and differentiation.Wiebe-Ben Zakour et al., (2024) recently demonstrated the use of decellularized LGhydrogel derived from porcine decellularized LGs as a bioink to study aqueous deficientDED in vitro. The study showed increased cell viability and proliferation compared to thetraditional substrates, such as collagen type 1 and Matrigel. The group suggests that thenewly formed hydrogel maintains the native biochemical composition of regular LG ECM;however, rapid degradation poses challenges for long-term cultivation and limits varioustissue-engineering applications [37]. The biological mechanism by which decellularizedECM supports LG engineering lies in its retention of bioactive molecules, such as gly-cosaminoglycans and growth factors, which help guide native cell behaviors essential forLG function. The group further aimed to enhance the mechanical stability and reduce therapid biodegradation of the hydrogel derived from decellularized porcine LG (dLG-HG).Varying concentrations of genipin was used to crosslink dLG-HG, and the degradation wasquantified over 10 days through the activity matrix metalloproteinases. A 0.5 mM concen-tration of genipin increased the stiffness of dLG-HG by about 46% and significantly delayedthe cell-dependent biodegradation of the biomaterial, without compromising cell viabilityand secretory function. Genipin can be used to control and fine-tune the degradation of thehydrogel to match the requirements of specific tissue-engineering investigations, hence pro-viding sufficient time for tissue modeling and regeneration without the material breakingdown too quickly. Lin et al., (2015) prepared an LG scaffold through decellularization ofadult rabbit LG. Adult rabbit LG progenitor cells were cultured and seeded on the scaffoldresulting in good cell viability and function [38]. Massie et al., (2017) aimed to evaluatedecellularized porcine jejunum (SIS-Muc) as a potential scaffold for the reconstructionof LG tissue [39]. SIS-Muc was found to retain the critical basement membrane proteinssuch as collagen IV and laminin after decellularization. Furthermore, LG epithelial cellscultured on SIS-Muc were found to proliferate more rapidly and remodel the mucosa into athicker cell layer. In addition to the increased metabolic activity, there was evidence of cellpolarization and presence of secretory vesicles; however, acini-like structures were absent.Furthermore, other key tear film proteins, such as lysozymes, lipocalin-1, and lactoferrin,were found in lower levels than naturally produced in tears. Spaniol et al., (2013) alsodemonstrated decellularization of porcine LG to develop scaffolding and noted an intactconnective tissue matrix, with expression of critical basement component proteins such ascollagen IV and laminin [40]. Decellularized LG hydrogel is a promising biomaterial for LGreconstruction; however, unpredictable degradation rates remain a significant challenge,and more mechanical fine-tuning is required before clinical translation.Chitosan is a natural polysaccharide and is derived from chitin. Deacetylation in-creases the water solubility of the biomaterial, which allows it to be used in bioengineeringapplications. The polymer is linked through β-(1→4) glycosidic bonds and allows forthe formation of hydrogen and ionic interactions, hence creating scaffolds for growth,attachment, and differentiation of LG cells. Chitosan has a positively charged surface,allowing for better facilitation with negatively charged cell membranes, which promotescell adhesion. In addition, Chitosan’s antimicrobial effects help reduce inflammation andcreate a favorable environment for tissue regeneration [41]. Most significantly, chitosanis highly biocompatible, nontoxic, biodegradable, and possess low immunogenicity [42].Hsiao et al., (2017) utilized chitosan biomaterials to facilitate the regeneration of LG tis-sue by promoting branching morphogenesis [43]. The group demonstrated that chitosanenhances the temporal and spatial expression of hepatocyte growth factor (HGF)-relatedmolecules and increasing branching of LG tissue. Chitosan was also shown to promoteincreased binding affinity between HGF and c-MET, activating downstream signalingpathways (MAPK and Akt/PKB) that are vital for branching morphogenesis. The grouppostulates that the structural similarity of chitosan to glycosaminoglycans allows for itto interact effectively with endogenous growth factors, hence removing the need for ex-Materials 2024, 17, 5425 7 of 21ternal supplements or bioincompatible agents. However, the study indicates that othermorphogens may also be involved, and the exact identity and roles of these additionalfactors was not explored. Further morphogenetic effects of chitosan were also found tobe dependent on the molecular weight and integrity of its glycosidic linkages, suggestingthe effectiveness of chitosan may be influenced by its specific preparation and chemicalproperties, hence complicating its standardization for clinical use.Dai et al., (2022) developed an innovative in situ-forming injectable hydrogel as adegradable lacrimal plug for DED [44]. The hydrogel plug was synthesized based on amethacrylate-modified silk fibroin (SFMA) that was photo-crosslinked in situ using visuallight. Silk fibroin is a natural protein derived from silk-producing arthropods and possessstrong biocompatible, biodegradable, and mechanical properties [45]. Silk fibroin containsArg-Gly-Asp (RGD) sequences that bind to integrins on the cell surface and may initiatesignaling pathways that promote cell attachment and proliferation [46]. Photo-crosslinkingallowed for precise spatial and temporal control, ensuring the hydrogel formed at thedesired location within the lacrimal passage and that solidification triggered on demandwhen the material was exposed to light [47]. This may allow clinicians to create a custom-fit,bioengineered solution for each patient, reducing the risk of plug migration and surgicalcomplications. Moreover, mechanical properties can be controlled by adjusting the durationand intensity of light to suit different clinical needs. The SFMA hydrogel demonstratedexcellent biocompatibility both in vitro and in vivo, noting no significant inflammatoryresponses and improved lacrimal fluid retention [44]. In addition, indocyanine greenfluorescence tracer nanoparticles (FTNs) were incorporated into the hydrogel to allow forlong-term non-invasive tracking when exposed to near-infrared light. This has large clinicalimplications in patient monitoring and long-term management of dry eye, enabling theability to make timely adjustments and reducing complications.Human amniotic membrane is a natural collagen-based biomaterial derived from theinnermost layer of the placenta, specifically from the amnion [48]. The membrane is widelyused in medical applications, such as wound healing [49] and tissue engineering for eye, skin,vascular system, urethral, cartilage, bone, nerve, heart, and ENT applications [50]. Amnioticmembrane contains a plethora of growth factors and nutrients, which enable intracellularsignaling cascades that promote wound healing and epithelization. Schrader et al., (2007)evaluated the growth pattern and secretory function of LG acinar cells cultured on amnioticmembrane in rabbit models [51]. The cells were analyzed over 28 days, and the secretory func-tion was tested through β-hexosaminidase activity. Secretory response diminished significantlyat the end of 28 days, and the cells showed a reduction in secretory granules, and the formationof flattening and spindle-shaped cell morphologies was observed. Interestingly, cells in directcontact with the amniotic membrane retained their acinar morphology. The group suggeststhat amniotic membrane has the capacity to support cell viability, differentiation, and secretoryfunction, highlighting its use in regenerative medicine, specifically for the reconstruction ofLG tissue. However, Singh et al., (2022) suggest that human amniotic membrane may notadequately support the formation of new blood vessels, which could hinder the growth ofacinar cells and lead to cell death. Additionally, amniotic membrane can only grow on surfaces,hence restricting its ability to serve as a true 3D scaffold. Hence, further optimization andinvestigation are required to elucidate the ability of human amniotic membrane to maintainlong-term cell functionality and support the growth and differentiation of many cells in acomplex, natural structure [52]. Interestingly, Ogawa et al., (2017) demonstrated that the heavy-chain-hyaluronan/pentraxin 3 (HC-HA/PTX3) protein, which was purified from humanamniotic fluid, preserved tear secretion and maintained conjunctival goblet cell density in micewith chronic graft-versus-host disease (cGVHD) [53]. Furthermore, it significantly reducedinflammation through suppression of immune cells and mitigated fibrosis in the lacrimal gland.This study underscores the potential of HC-HA/PTX3, suggesting that the human amnioticmembrane could be effectively combined with other scaffolds to promote therapeutic effects.Chen et al., (2014) used xenogeneic (bovine) acellular dermal matrix combined withautologous conjunctival tissue to reconstruct the lacrimal duct in patients with partial orMaterials 2024, 17, 5425 8 of 21total absence of the lacrimal duct [54]. The method involved creating a lacrimal duct byrolling a conjunctival petal attached to an acellular dermal matrix into a tube and surgicallyimplanting the structure to form a functional tear drainage system. Epiphora symptomswere alleviated in all human patients over approximately 7.2 months. The acellular dermalmatrix produced a strong, stable framework, which prevented collapse and was essentialfor ensuring the duct’s patency and position. In addition, the matrix was biocompatibleand capable of degrading at an optimum pace, allowing the surrounding natural tissues totake over the support function through minimal scarring. However, long-term outcomesare not yet known, so it is unclear how the biomaterial will maintain duct patency andavoid complications such as blockage or fibrosis.Natural hydrogels, such as collagen, fibrin, and gelatin, may also be used in LG re-construction. Collagen is one of the most abundant proteins in the body and forms thestructural basis of many tissues. Hirayma et al., (2013) utilized collagen gel to encap-sulate the bioengineered glands during transplantation into mice with LG defects [55].The bioengineered LG glands developed proper 3D structures, discharged essential tearproteins, and the harderian glands secreted lipids for tear film stability. This study andNakamura et al., (1996) highlight collagen I as an important biomaterial in facilitatingbranching and overall morphogenesis during LG development [56]. In fact, Rusch et al.,(2021) investigated the role of freshly isolated mesenchymal stem cells (MSCs) in the LGof mouse models and found that 85% of the MSCs produced collagen type I [57]. How-ever, during allogeneic transplantation, collagen production appeared to contribute topathogenic fibrosis, which may impair LG function and contribute to DED in the contextof chronic graft-versus-host disease. Hence, while MSCs have the potential to aid in tis-sue repair through collagen secretion, the optimization of collagen secretion is critical toavoid fibrotic outcomes. Moreover, Lee et al., (2016) noticed that collagen type II 1a-basedpeptides possess anti-inflammatory properties and can improve the pathogenesis of ocularsurface disorders by increasing tear volume and stabilizing the corneal epithelium [58].Fibrin, typically sourced from human plasma, is a less explored biomaterial for LGreconstruction. However, fibrin hydrogels have been used to promote salivary gland regen-eration, showing effectiveness in improving epithelial tissue organization while facilitatingthe development of vascularization and nerve formation [59]. Fibrin actively promotes celladhesion, migration, and differentiation; however, it often produces insufficient mechanicalstrength and very fast degradation rates which hinder long-term tissue support [60]. Acommon theme among natural hydrogels is that they are prone to breaking down byproteases such as matrix metalloproteinases (MMPs); hence, they are often altered withcrosslinkers that are able to fine-tune degradation rate [61].Table 1. Natural biomaterials suitable for lacrimal gland tissue bioengineering.Biomaterial Derivation Features Disadvantages ReferencesMatrigel ECM proteins of EHSmouse sarcomaSupports cell proliferation,acinar differentiation, mimicsnatural basement membrane,and promotes gland-likestructure formationVariability between batches andanimal-derived, hence limitingclinical applications; decreasedexpression of proteins after sometime, hence limiting long-termuse; indeterminate degradationrate; and may be immunogenic[28,29,31,34–37]DecellularizedLacrimal GlandHydrogelPorcinedecellularizedlacrimal gland,most commonlyMaintains the nativebiochemical composition ofthe lacrimal gland ECMRapid degradation limitslong-term use; requires genipincrosslinking for enhancedmechanical stability; limitedavailability; and incompletedecellularization can lead toimmune response[37–40]Materials 2024, 17, 5425 9 of 21Table 1. Cont.Biomaterial Derivation Features Disadvantages ReferencesChitosan Polysaccharidefrom chitinPromotes branchingmorphogenesis; interactswith endogenous growthfactors; biocompatible;nontoxic; and biodegradableLimited mechanical strength [43]Silk FibroinHydrogelSilk fibroin fromsilk-producingarthropodsCustomizable mechanicalproperties;photo-crosslinkable forcontrolled solidification insitu; and excellentbiocompatibilityLong-term in vivo outcomesunknown and complexpreparation process[44]HumanAmnioticMembraneInnermost layer ofplacenta (amnion)Supports cell viability anddifferentiation and reducesinflammation and fibrosisLacks the ability to supportvascularization and only growson surfaces, hence not suitablefor true 3D scaffolds[51–53]CollagenMultiple, typicallyderived fromanimal sourcesPromotes 3D structureformation and facilitatesbranching morphogenesisRapid degradation [55–58]Fibrin Fibrinogen, usuallyfrom human plasmaPromotes cell adhesion,migration, vascularization,and nerve formation;supports epithelialtissue organizationLow mechanical strength andfast degradation rates, hinderinglong-term tissue support[60]3.2. Synthetic BiomaterialsIn addition to natural biomaterials, synthetic biomaterials also considered for LGbioengineering [Table 2]. Long et al., (2006) first explored the feasibility of using polyether-sulfone (PES) as a scaffold for LG bioengineering [62]. PES was fabricated using a phaseinversion technique, carefully ensuring the material was semipermeable to allow for trans-fer of nutrients, such as ascorbic acid and glucose, while blocking rat IgG immunoglobulinsto prevent immune reactions. Overall, PES tubes supported the attachment, growth, andproliferations of lacrimal acinar cells and allowed for selective permeability. However, thegroup explains that number of lacrimal acinar cell growth on PES was lower compared toother endothelial cells and had poor ability to proliferate in vitro when cultured over fivetimes. However, the group suggests increasing the porosity of the material to increase cellgrowth. Moreover, the study conducted limited immunogenicity testing, and the overallimmune response was not thoroughly examined because of the lack of in vivo testing.Polyesters are also widely used in various medical and biological applications be-cause of their biocompatibility, biodegradability, and good mechanical strength. Polyestersare mainly found in sutures, surgical meshes [63], orthopedic and ophthalmological im-plants [64], and drug delivery applications [65]. Selvam et al., (2007) developed a tissue-engineered tear secretory system by investigating cultured rabbit lacrimal acinar cell mono-layers on polyester membrane scaffolds [66]. The functionality was assessed through activetransepithelial ion fluxes across cell monolayers. The microporous polyester membranesupported proper cell polarity, tight junctions, functional protein secretion, and acinar cellproliferation. The authors highlighted the capability of polyester membrane scaffolds toform continuous monolayers, that exhibited transepithelial resistances characteristic of“leaky” epithelia observed in vivo. The group further suggests that these models will beuseful in studying fundamental LG physiology. However, future research should prioritizein vivo studies to assess the biocompatibility and long-term stability of polyester.Schrader et al., (2010) explored different scaffolds such as collagen, Matrigel, andpolymeric material to determine the ideal substrate for cell adhesion, growth, and differen-Materials 2024, 17, 5425 10 of 21tiation [67]. PLLA (poly-I-lactic acid) showed the best results for growth and morphologicaldevelopment of LG cells, and amniotic fluid proved to be a suitable carrier for LG cellsfor up to 21 days in vitro. While the study is a significant step toward demonstratingthe potential of PLLA polymer for tissue repair, challenges such as maintenance of cellfunction, circumventing apoptosis, and ensuring the long-term viability of transplantsremain. Selvam et al., (2007) also explored materials such as silicone, collagen I, copolymersof poly-D,L-lactide-co-glycolide (PLGA; 85:15 and 50:50), poly-L-lactic acid (PLLA), andThermanox® plastic cell culture coverslips for developing a bioengineered tear secretorysystem [68]. The group proposes that PLLA, with Matrigel coating, provided the bestsupport for acinar cell-like morphology and contributed to the healthy formation of mi-crovilli, secretory granules, and junctional complexes. PLLA is a synthetic biopolymer withmodifiable mechanical properties derived from renewable resources such as cornstarchand sugar cane. PLLA is a slow, biodegradable scaffold material that turns into lacticacid, which may lead to local pH changes and inflammation. While PLLA’s biocompatibleand mechanical properties are advantageous for creating a stable structure to support cellgrowth and tissue formation, PLLA’s hydrophobic nature may limit cell adhesion andprotein absorption [69]. Selvam et al.’s (2007) group optimized PLLA’s properties througha Matrigel coating; however, they noted some loss in the mechanical integrity over anextended period of time. In contrast, silicon promoted the formation of 3D acinus-like struc-tures and remained stable over an extended period of time. The group also demonstratedenhanced secretory functions with PLLA and, to a lesser extent, with PLGA, as indicatedby β-hexosaminidase secretion, when compared to collagen I and plastic [68].Table 2. Synthetic biomaterials suitable for lacrimal gland tissue bioengineering.Biomaterial Features Disadvantages ReferencesPolyethersulfone (PES)Excellent mechanical stability;semipermeable and supports nutrienttransfer while blockingimmunoglobulins; and promotes acinarcell attachmentHydrophobic surface; lower acinar cellproliferation compared to otherendothelial cells; limitedimmunogenicity testing; and lack ofvivo studies[62]PolyesterSupports cell polarity, tight junctions,protein secretion, and acinarcell proliferationLack of in vivo studies to assess thelong-term biocompatibilityand stability[66]Poly-I-lactic acid (PLLA)Biodegradable; good mechanicalproperties; and supports acinarmorphology, secretory granules, andjunctional complexesVery slow degradation, acidicbyproducts may affect lacrimal glandcells; and a hydrophobic nature[67,68]Poly-D,L-lactide-co-glycolide (PLGA)Biodegradable scaffold with goodbiocompatibilityDegrades into acidic byproducts andsome loss in the mechanical integrityover time, with lower acinar secretoryfunctions compared to PLLA[67,68]Silicon Biocompatible and remains stable overan extended period of timeNon-biodegradable and requiresfurther testing for long-term stabilityand potential immune responsesin vivo[68]4. Cell Sources and Growth FactorsStem cells serve as the repair system during injury and help replenish other cells,hence maintaining the structure and function of the LG. This regenerative property ofLG, which is mediated by natural LG progenitor cells, are being investigated and in-tegrated with bioengineered tissues to help facilitate advances in scaffold design anddevelop functional glandular tissues. Currently, limited research has been conductedon LG regeneration, and few cell sources have been explored. LG progenitor cells areMaterials 2024, 17, 5425 11 of 21found within the LG itself and are often isolated and expanded in vitro before being trans-planted into an injured or diseased LG. Gromova et al., (2017) isolated and characterizedepithelial cell progenitors from the LG of adult mice, specifically selecting a populationwith certain hallmark characteristics [70]. The isolated progenitor population expressedc-kit+dim/EpCAM+/Sca1−/CD34−/CD45− and pluripotency factors Runx1 and EpCAM.During in vitro analysis, cells differentiated in structures resembling acini and ducts of theLG and were transplanted into both injured and diseased mouse models. Engraftmentand integration into the LG were successful in the acute injury model, but, most notably,progenitor cell transplantation led to significant improvements in the LG structure of thechronic disease model, and tear production was significantly increased. Delcroix et al.,(2023) further explored novel LG progenitor lines using the first transcriptomic atlas ofLG in mice [71]. The group discovered Sox10+ cell populations, which are important forthe creation of secretory units and contribute to the development of acinar, ductal, andmyoepithelial lineages. Exploring novel progenitor cells expands the range of optionsfor LG tissue engineering and enables the customization of therapeutic approaches to fitpatient needs.Regenerative tissue therapy has also been investigated through the use of mesenchy-mal stem cells (MSCs) in the repair and growth of injured LG [72]. MSCs are multipotentstromal cells that are able to differentiate into many cell types and are capable of self-renewal [73]. Jaffet et al., (2023) produced the first evidence of an MSC population withinthe human lacrimal gland [74]. These LG-MSCs possessed characteristics similar to MSCsfrom other tissues, notably expressing a higher level of IL-1β, which is involved in angio-genesis and LG development. This suggests that MSCs may be critical for maintaining thehealth of the LG and could play a role in its regeneration, opening new avenues for researchat developing cell-based therapies. While these multipotential cells can support the growthof acinar and epithelial cells within the LG, it may be difficult to isolate a colony of thesecells for clinical purposes [75]. Interestingly, MSCs derived from patients with Sjögren’ssyndrome lacked certain colony-forming-unit efficiency and adipogenic differentiationpotential compared to healthy controls [76]. This could offer valuable insight into thespecific protocols for selecting MSCs for tissue regeneration.Induced pluripotent stem cells (iPSCs) can also differentiate into multiple cell types,including acinar and ductal cells of the LG. Hayashi et al., (2022) developed a lacrimal-gland-like organoid using iPSCs and embryonic stem cells and demonstrated organoid maturationin rats as it developed lumina, expressed lactoferrin and lysozyme proteins, and contributedto tear-film production [77]. In addition, organoids showed an upregulation in genesassociated with LG development such as BARX2 transcriptional factor. Asal et al., (2023)also utilized iPSCs to demonstrate its differentiation into acinar, ductal, and myoepithelialcell types, indicating its utility in LG bioengineering as a promising cell source [34]. Thisgroup employed a multizonal ocular differentiation strategy that efficiently producedLG-specific cells in just 4–7 weeks, achieving the shortest timeframe currently reported andpaving the way for a more robust outlook.Current studies explore new avenues of in situ LG regeneration, which include com-bining many novel strategies, such as adding growth factors and cytokines, gene therapy,and stem cell therapy. Lin et al., (2019) characterized human LG tissue through immunos-taining progenitor markers [78]. Precursor cell markers C-Kit, K15, Nestin, and P63 weredemonstrated to be involved in the differentiation of lacrimal epithelial cells into mini-glands. This exploration further advances stem-cell-based research on LG reconstructionand regeneration. However, the study reveals a rapid loss in differentiation in later cellpassages, highlighting the need for research to maintain the differentiation capacity ofthese cells. Basova et al., (2017) investigated the role of Pannexin-1 in inflammation andthe engraftment of epithelial progenitor cells in LG [79]. The authors demonstrated thatcontrolling Panx1 activity can significantly improve engraftment and enhance the efficacyof regenerative treatments for LG dysfunction. Biomaterials that can effectively modulatethe activity of such markers are crucial to producing localized anti-inflammatory effects andMaterials 2024, 17, 5425 12 of 21supporting transplant of LG tissue. Voronov et al., (2013) further investigated the role ofspecific Runt-related (Runx) transcription factors involved in the regulation, proliferation,and differentiation of stems cells in lacrimal bodies [80]. Specifically, the group revealedRunx1 and Runx2 are essential epithelial markers for LG morphogenesis, while Runx3 is ex-pressed in both epithelial and mesenchymal compartments. As a result, the downregulationof such transcription factors significantly impairs LG growth, branching and development,highlighting the importance of research into specific factors required for LG tissue regen-eration and biosynthesis in vivo. Recently, Finburgh et al., (2023) identified the role offibroblast growth factor 10 (FGF10) in the development, homeostasis, and regeneration ofLG [81]. In fact, the group found that the injection of FGF10 into damaged adult LG of micesignificantly increased cell proliferation, cell viability, and accelerated the repair process ininflamed glands. FGF10 is found to be highly expressed in postnatal LG and to decrease inadulthood. Future research should conduct a large transcriptome analysis on the neonataldevelopment of LG in mice and investigate growth factors largely active during this activestage in life. Growth factors implicated in LG regeneration should be added to scaffoldsduring the tissue-engineering process to allow for the optimal growth of damaged LG.Ueda et al., (2009) investigated the growth factors involved in postnatal LG developmentand demonstrated that EGF and HGF increased cell survival and proliferation, while theaddition of FGF10 did not significantly stimulate epithelial cell proliferation in vitro [82].However, FGF10 was more intensely expressed in mesenchymal cells than in epithelialcells. Figure 2 highlights the overall regeneration and reconstruction process of lacrimalgland restoration.Figure 2. Regeneration and reconstruction of lacrimal gland involve the extraction of stem cells andsubsequent spheroid development with added growth factors. Cultured spheroids may be integratedwith scaffolds and combined with 3D bioprinting technology to reconstruct atrophic lacrimal gland.MSCs: mesenchymal stem cells; iPSCs: induced pluripotent stem cells; EPCs: endothelial progenitorcells; HGF: hepatocyte growth factor; EGF: epidermal growth factor; FGF: fibroblast growth factor.Created with BioRender.com.Materials 2024, 17, 5425 13 of 215. Advances in Tissue-Engineering TechniquesTissue engineering utilizes diverse fabrication techniques, each with unique advan-tages and limitation. Although these techniques are widely discussed in other literature,lacrimal gland and soft tissue fabrication is frequently achieved using methods like electro-spinning, 3D bioprinting, and electrospray techniques, and advancements in these areasare discussed.Electrospinning applies a high voltage to a polymer solution, stretching the poly-mer into ultrafine fibers that are deposited onto a substrate to form a structured mesh(Figure 3) [83]. Certain polymers are more suited to electrospinning because of their abilityto form stable jets and continuous fibers when subjected to an electric field. Syntheticmaterials such as polyethylene glycol (PEG) and poly-lactic-co-glycolic acid (PLGA) arelargely utilized because of their relatively high molecular weights and dissolution in volatilesolvents; however, a large variety of materials have been used because of the versatility ofthe electrospinning process [84]. Natural polymers, such as gelatin [85], cellulose [86], andsilk [87], exhibit better biocompatibility; however, they are subject to faster degradationrates and fabrication difficulties. Synthetic materials are better able to withstand tensionand shear forces and can be tailored to adapt to the required breakdown rate. However,they are limited by their low biocompatibility. Bosworth et al., (2021) incorporated decellu-larized ECM derived from porcine small intestinal submucosa and urinary bladder matrixwith synthetic poly(ε-caprolactone) (PCL) to produce electrospun bioactive scaffoldingthat is used to support the stratification of large conjunctival defects [88]. The authors’approach to creating the hybrid scaffolding combines the bioactivity of natural polymerswith the structural integrity of synthetic polymers, allowing for the creation of fine-tunedfibrous structures. PCL nanofibers can be arranged randomly or with a straight, parallelorientation to exhibit unique tissue properties characteristic of natural ECM (Figure 4).Soscia et al., (2013) introduced a curvature to the electrospun PLGA nanofibers to supportthe growth and differentiation of salivary gland epithelial cells [89]. The authors proposethat this setup enhanced the functional organization and polarity of epithelial cells andachieved a more realistic mimicry of the basement membrane environment in glandu-lar structures. Other advancements in electrospinning, such as drug delivery methodsutilizing multi-compartment nanofibers, have also been explored. Zhang et al., (2024)further advanced the electrospinning technique by generating the tri-chamber side-by-side electrospinning method, which allowed for the creation of distinct layers within thenanostructure and was capable of simultaneously delivering multiple drugs with distinctrelease profiles [90]. LG tissue engineering can leverage this nanostructural design toenhance the release of antioxidants and antibacterial agents, as well as to support tearduct system functions. Dong et al., (2024) developed a new type of core–shell nanofiberusing coaxial electrospinning to enable the controlled release of ferulic acid, which hasantioxidant and anti-inflammatory properties [91]. The structure allows for the preciseand controlled release of the drug product. The group illustrates that this specific designeffectively prevents the “negative tailing-off effect”, a phenomenon whereby the drug’srelease from the carrier matrix gradually slows, resulting in sub-therapeutic drug levelsand inconsistent dosing. Such scaffolds may help gradually deliver bioactive moleculesessential for tissue repair and improve the effectiveness of LG-tissue-engineered implants.Materials 2024, 17, 5425 14 of 21Figure 3. Electrospinning instrument setup. The bibliography consulted [92] is licensed underCC BY 2.0. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (ac-cessed on 28 October 2024).Figure 4. (A,B) Macroscopic view of a 2D poly-ε-caprolactone (PCL) scaffold fabricated by theelectrospinning technique; (C,D) microscopic view of the scaffold under scanning electron mi-croscopy; (C) nanofibers oriented randomly; (D) nanofibers with a straight, organized orientation.The bibliography consulted [93] is licensed under CC BY 2.0. To view a copy of this license, visithttp://creativecommons.org/licenses/by/4.0/ (accessed on 28 October 2024).Three-dimensional bioprinting has revolutionized tissue engineering by enabling theprecise layering of biomaterials and cells to create complex scaffolds. Hydrogels, suchas collagen, alginate, and decellularized porcine ECM solution, are commonly used asbioinks for gland tissue engineering. For example, Grumm et al., (2023) recently designedan alginate-porcine LG ECM scaffold that enables ion-based crosslinking through 3D bio-printing methods [94]. This further enabled the bioink to sustain a higher cell viabilityand density, while demonstrating larger sheer strength than control alginate bioink. Rod-boon et al., (2022) introduced a novel protocol to engineer LG organoids that simulate agingand dysfunction using a magnetic 3D bioprinting platform (M3DB). The authors proposethat the M3DB-based protocol is a consistent and scalable method for manufacturing LGMaterials 2024, 17, 5425 15 of 21organoids and demonstrates a reproducible approach to create functional models. Thisallows for further application in high-throughput platforms for testing drug formulationsand gene therapies for DED [95]. Similarly, Ferreira et al., (2024) designed a study to createfunctional and aging models of LG organoids using M3DB platform [96]. Specifically, theteam aimed to model DED by inducing cellular senescence and, subsequently, testing theefficacy of HMGB1-Box A gene therapy. Traditional models with Matrigel were reportedto not be ideal for clinical translation because of its variability in models; however, theM3DB platform allowed for a user-friendly, scaffold-free, and homogeneous approach tohigh-throughput applications. Yin et al., (2023) demonstrated that microfluidics-basedbioprinting provided superior spatial and temporal control when compared to traditionalextrusion-based printing systems [97]. Traditional bioprinting methods face limitationsin resolution and case shear stress damage, which is especially problematic for complexepithelial structures. In contrast, coaxial microfluidics allows for intricate structures usingalginate-based microfibers and microtubes. Alginate enables rapid crosslinking, and theteam highlights the usefulness of coaxial printheads to create branching structures andhollow tubes that mimic salivary gland features. As previously mentioned, crosslinking is acrucial technique for stabilizing hydrogels and other biomaterials in tissue engineering. Infact, genipin crosslinking has already shown to increase the mechanical stability and stiff-ness, as well as to optimize the degradability, in in vitro models of porcine decellularizedLG [98].Electrospraying is a popular technique in tissue engineering that uses an electric fieldto create a fine aerosol of small droplets containing cells, biomaterials, drug components,or other biomolecules that can be deposited on a scaffold to form tissues [83]. The precisedroplet size and distribution may be controlled, allowing researchers to create complextissue architectures with desired spatial arrangements and densities of compounds. Elec-trospraying is widely used in drug delivery systems because of its ability to sustain releaseover extended periods and enable targeted delivery, thereby enhancing therapeutic effi-cacy [99]. However, this technique requires the use of biomaterials in liquid form, whichcan restrict the range of biomaterials suitable for effective spraying. Additionally, certaincells and biomolecules may be sensitive to the preparation process, potentially affectingtheir overall activity and viability.6. Future DirectionsLG bioengineering is a nuanced and complex field that requires multiple consid-erations before developing viable therapies. The greatest challenge is mimicking nativelacrimal gland architecture, as the LG is a highly specialized and intricate structure compris-ing multiple unique cells. The scaffold must support the appropriate spatial organization ofdifferent cell types to allow for correct tear production and flow. Furthermore, this involvesthe right stiffness, degradation profile, and porosity of the scaffold. A common difficultyamong current studies is the ability to maintain cell function over time, and LG cell functionseems to decrease as the distance from supporting cells or the scaffold increases. This lossof function may be due to the lack of a microenvironment similar to a natural LG away fromthe scaffold that would normally provide the necessary factors to sustain differentiatedcells. There must also be further development in refining decellularization protocols tominimize residual antigens that may trigger immune reactions.Furthermore, there seems to be no correct natural or synthetic biomaterial used forLG bioengineering. The native LG structure requires biomaterials with specific mechanicalproperties, which include optimal elasticity, mechanical strength, and degradation rate.Natural polymers offer good biocompatibility and cell viability profiles but lose structuralintegrity. Hence, more research must be conducted in developing hybrid biomaterialsthat involve both natural and synthetic polymers, as well as crosslinking and bioactivemolecules to enhance cell adhesion and proliferation.Another challenge lies within achieving proper vascularization and innervation of theimplanted biomaterial. Multiple techniques can be employed to facilitate optimal bloodMaterials 2024, 17, 5425 16 of 21supply throughout the LG. Lacrimal cells can be combined with endothelial cells that arederived from adult and human pluripotent stem cells [100]. Three-dimensional printing canbe utilized as a scalable method for fabricating hydrogels with functional and hierarchicalvascular architectures [101]. Furthermore, current tissue-engineering approaches have notaddressed nervous system integration, which is crucial to control tear secretion. Furtherresearch must address methods on mimicking this neural control in vitro, possibly throughthe inclusion of neural cells or neurotrophic factors. Moreover, the engineered gland shouldrespond appropriately to both sympathetic and parasympathetic neural signals whentransplanted. This involves connecting the gland correctly to existing nerves and ensuringgland cells carry the required receptors and signaling pathways to respond to the releaseof neurotransmitters.In addition, the manufacturing of scaffolds with precise micro- and nanostructures istechnologically demanding. Three-dimensional printing techniques are a new emergingtechnology that offers significant advantages over conventional methods for many tissue-engineering applications. For example, stereolithography and selective laser sinteringallows for the creation of highly complex and patient-specific scaffolds with precise controlover scaffold parameters, such as porosity, size, and customizable cell placement [102].Also, 3D printing techniques provide high repeatability and can be scaled to meet demandin the medical industry once the design and process are optimized. However, there arelarge regulatory hurdles in using scaffolds as biomedical devices by governing bodies.This is usually a costly and lengthy process in the commercialization of newly developedscaffolds. Rodboon at al., (2022) recently introduced a novel magnetic 3D bioprintingplatform to rapidly and consistently biosynthesize lacrispheres from porcine primary LGcells for organoid creation [95]. The authors propose that the development of viable LGorganoids through magnetic 3D printing has significant potential in LG transplantation, aswell as drug discovery and screening, ultimately leading to new therapies for DED andother LG-related disorders. The magnetic 3D printing platform is described as xenogenic-free and highly scalable, using biocompatible magnetic nanoparticles to label cells andmanipulate them using magnetic fields. This offers better control over the size and shapeof the organoids, as well as the precise spatial organization of cells into 3D structures.Lastly, tissue-engineered LGs must produce lacrimal fluid and successfully transportthis fluid to the ocular surface. There is a lack of research addressing the connection of atissue-engineered LG to a natural tear drainage system. New studies should investigate thecreation of a synthetic duct that can be integrated in bioengineered LG or innovative micro-surgical procedures to enhance the ability to connect engineered ducts to existing ocularanatomy. For example, Holtmann et al., (2022) developed a proof-of-concept describing thefeasibility of micro-anastomosis for transplantation of a human LG [103].LG regeneration is also constrained by the challenges of proliferating cells in vitro.Advances in biomaterials that can provide a supportive environment and identify suitablelong-term culture systems are crucial for successful cell transplantation. For example,Zeng et al., (2024) developed a serum-free culture strategy that utilizes a 2C small moleculecombination consisting of both Y27632 and SB431542, which enhances and maintainsproliferative capacity in vitro and provides a stable source of cells for further tissue engi-neering [36].One of the key emerging trends in the field of LG bioengineering is the use of decellu-larized porcine ECM due to its high biocompatibility, ability to support cellular growth, andoptimal features of natural ECM composition [98]. Although it offers some improvementover whole porcine lacrimal gland xenotransplantation, the decellularization process canstill leave behind residual porcine antigens, potentially leading to an immune response.In addition, although rare, there is a risk of transmitting zoonotic pathogens from porcinetissues [104].The new wave of tissue bioengineering is moving toward the addition of stem cells,such as MSCs and iPSCs, which can differentiate into acinar and ductal cells in the LG. Stemcells may be genetically modified to enhance their regenerative potential. For example,Materials 2024, 17, 5425 17 of 21delivering proteins that encode for growth factors, anti-inflammatory proteins, or ECMcomponents may improve the therapeutic outcome of LG regeneration. This possibility hasbeen demonstrated, by Trousdale et al., (2005), through the expression of the TNF inhibitorgene in the LG, which led to the recovery of tear production and reduced inflammation inrabbits with induced autoimmune dacryoadenitis [105].7. ConclusionsAdvancements in biomaterials for lacrimal gland tissue engineering have openedpromising pathways toward restoring natural tear production in patients with aqueous-deficient dry eye disease. Both natural and synthetic biomaterials have demonstratedsignificant potential in mimicking the extracellular matrix, supporting lacrimal gland cellproliferation and differentiation, and facilitating the formation of functional glandularstructures. Natural biomaterials, such as Matrigel and decellularized lacrimal gland hy-drogel, have shown particular efficacy in promoting acinar differentiation, while syntheticbiomaterials like poly-I-lactic acid (PLLA) provide structural stability and support for long-term cell viability. The integration of stem cells and growth factors with these biomaterialsfurther enhances regenerative outcomes, bringing us closer to viable therapeutic solutions.While challenges remain in replicating the complex architecture of the lacrimal gland,achieving proper vascularization and innervation, and ensuring long-term functionalityof engineered tissues, the rapid progress in biomaterial science and tissue-engineeringtechniques offers an optimistic outlook. Advances in electrospinning, microfluidics, and3D bioprinting allow for the precise creation of scaffolds that can replicate the nativetissue environment more closely, supporting cell organization and functional integration.Advances in 3D bioprinting and scaffold fabrication are particularly promising, providinginnovative tools to overcome current limitations. With continued interdisciplinary researchand collaboration, the development of effective biomaterial-based regenerative therapies forlacrimal gland dysfunction is within reach, holding the potential to significantly improvethe quality of life for patients affected by this condition.Author Contributions: Conceptualization, K.Y.W.; writing—original draft preparation, K.Y.W. andA.D.; writing—review and editing, K.Y.W., A.D. and P.D.; supervision, P.D. and S.D.T. All authorshave read and agreed to the published version of the manuscript.Funding: This research received no external funding.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: No new data were created or analyzed in this study.Acknowledgments: The authors would like to acknowledge that all figures were created withpermission, using software from BioRender.com (accessed on 23 October 2024).Conflicts of Interest: The authors declare no conflicts of interest.References1. Chen, H.; McCann, P.; Lien, T.; Xiao, M.; Abraham, A.G.; Gregory, D.G.; Hauswirth, S.G.; Qureshi, R.; Liu, S.-H.; Saldanha,I.J.; et al. Prevalence of Dry Eye and Meibomian Gland Dysfunction in Central and South America: A Systematic Review andMeta-Analysis. BMC Ophthalmol. 2024, 24, 50. [CrossRef] [PubMed]2. Messmer, E.M. The Pathophysiology, Diagnosis, and Treatment of Dry Eye Disease. Dtsch. Ärztebl. Int. 2015, 112, 71–82.[CrossRef] [PubMed]3. 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biomaterials
tissue engineering
lacrimal gland
dry eye disease
natural and synthetic biomaterials
3D bioprinting
regenerative medicine
Advanced Biomaterials for Lacrimal Tissue Engineering: A Review
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