The lymphatic system is an integral part of the human body and is involved in various physiologic processes, including circulatory, metabolic, and immune functions. The many functions of the lymphatic system allow the body to maintain homeostasis. One primary function of the lymphatic system is to collect and return protein-rich, solute-rich interstitial fluid back to blood circulation. Another important function of the lymphatic system is to aid in a physiologic immune response by transporting foreign antigens and immune cells to the lymph nodes (Sung et al., 2022). The lymphatic system consists of several key components. The lymphatic capillaries are small, highly permeable vessels that are structured differently from circulatory vasculature. Unlike vascular endothelial cells, the lymphatic endothelial cells (LECs) line the lymphatic capillaries in a single layer with discontinuous, button-like junctions. These gaps in the basement membrane contribute to the highly permeable nature of the lymphatic capillaries and permit the uptake of “lymph” or interstitial fluid (Jia et al., 2021). Specialized anchoring filaments attach the LECs to the surrounding extracellular matrix, while also keeping the capillaries open to allow for permeability (Butler et al., 2009). The lymphatic capillaries merge to form larger collecting lymphatic vessels, characterized by a continuous basement membrane with smooth muscle cells and semilunar-shaped valves. Together, these structures allow for the unidirectional flow of lymphatic fluid (Sung et al., 2022). The collecting vessels transport lymphatic fluid to the lymph nodes, an encapsulated organ that serves to filter lymph and house immune cells. Lymph nodes are categorized into two groups based on their location within the body: peripheral lymph nodes, which collect lymph from all regions of the body, and mesenteric lymph nodes, which are involved in the immune response of the intestines (Nosenko et al., 2016). The collecting lymphatic vessels also converge to form larger lymphatic trunks and ducts, of which the main thoracic duct returns the lymphatic fluid back into venous circulation via an anastomosis through the cardinal vein (Butler et al., 2009). The thoracic duct, the largest lymphatic vessel in the body, originates from the cisterna chyli in the abdomen and ascends through the thorax before draining into the venous system at the junction of the left subclavian and internal jugular veins (Ilahi et al., 2025). This return of lymphatic fluid is critical for maintaining fluid homeostasis and immune function, as disruptions in this process can lead to conditions such as lymphedema.

The unique and delicate architecture of the lymphatic system makes it particularly prone to disease when its structure is compromised. Lymphedema is a chronic, progressive condition, characterized by the accumulation of lymphatic fluid in the extracellular space due to dysfunction of the lymphatic system. This build-up of fluid leads to tissue swelling, often in the limbs, and chronic deposition of fibroadipose tissue (Brown et al., 2023), which hardens affected tissue, and further impedes lymphatic drainage. This fluid stasis also triggers an inflammatory response. Figure 1 illustrates the disrupted lymphatic flow and tissue swelling seen in lymphedema patients. The innate and adaptive immune responses play critical roles in the pathophysiology of lymphedema. Innate immune mechanisms are activated following lymphatic injury, leading to chronic inflammation. Danger-associated molecular patterns (DAMPs) are released from damaged lymphatic endothelial cells, activating Toll-like receptors (TLRs) and initiating pro-inflammatory cascades. Macrophages are then heavily recruited to the affected tissue, where they produce inflammatory cytokines such as IL-6 and TGF-β, contributing to fibrosis and tissue remodeling (Kataru et al., 2019). While macrophages initially promote lymphangiogenesis through VEGF-C secretion, they also contribute to impaired lymphatic function by releasing nitric oxide, reducing lymphatic vessel contraction and increasing fluid stasis (Yuan et al., 2019). The adaptive immune response is also dysregulated in lymphedema, with a predominant CD4⁺ T cell-mediated inflammatory reaction. Persistent T helper 2 (Th2) response promotes fibrosis by increasing expression of profibrotic growth factors such as IL4, IL-13, and TGF-β1, along with reducing lymphangiogenesis, and impairing lymphatic transport (Kataru et al., 2019). Regulatory T cells (Tregs) also accumulate in lymphedematous tissue, additionally suppressing innate and adaptive immune responses and contributing to local immunosuppression, thus increasing susceptibility to infections (García Nores et al., 2018).

Lymphedema can have different etiologies. It is broadly classified into primary and secondary forms. Primary lymphedema is a rare disease, occurring in about 1 in 100,000 individuals (Sleigh et al., 2024). It is characterized by a genetic abnormality that impairs the development of the lymphatic system and weakens its drainage capacity. In contrast, secondary lymphedema is more common and typically results from external damage to the lymphatic system (Drobot et al., 2024). It affects approximately 1 in 1,000 Americans (Sleigh et al., 2024) and about 250 million people worldwide (Schulze et al., 2018). Globally, secondary lymphedema is most commonly caused by infections such as filariasis, while in Western countries, it is frequently attributed to cancer treatments, including lymph node dissection and radiation therapy. This condition is particularly common among breast cancer patients who have undergone mastectomy (Drobot et al., 2024). Regardless of its cause, lymphedema leads to progressive fluid accumulation, which can result in discomfort, restricted movement, loss of function, increased risk of infection, and a significant impact on a patient’s quality of life (Brown et al., 2022).

Despite the fact that secondary lymphedema is common and morbid, there is no effective treatment. Current therapies often focus on minimizing symptoms such as pain and swelling yet do not offer a definitive cure. The gold standard therapy for lymphedema is complete decongestive therapy: a combination of manual lymphatic drainage, daily bandaging, skin care, exercising, and compression garments (Shaitelman et al., 2015). However, the effect of this treatment has been doubted in recent years (Drobot et al., 2024). In severe or refractory cases, further pharmacologic or surgical intervention is needed. In experimental models, pharmacologic therapies focus on stimulating lymphangiogenic factors, such as vascular endothelial growth factor-C (VEGF-C) and fibroblast growth factor 2 (FGF2). Other therapies include anti-inflammatory agents, such as tacrolimus, which works to inhibit CD4⁺ cell proliferation, and anti-fibrotic agents, such as pirfenidone, which works against transforming growth factor beta-1 (TGF-β1), a key regulatory of fibrosis (Brown et al., 2023). It should be noted, that to date, there is no approved pharmacologic therapy for lymphedema. Surgical intervention aims to restore normal lymphatic function through the development of alternative drainage methods such as lymphaticovenular anastomosis (LVA) or vascularized autologous lymph node transfer (VLNT), which have been depicted in Figure 2, however these procedures do not correct the fibroadipose accumulation (Drobot et al., 2024). The addition of liposuction to these procedures has shown to help with the removal of fibroadipose tissue (Brazio and Nguyen, 2021). While surgical intervention can provide symptomatic relief, procedures are often lengthy and complex, with an additional risk of inadvertently inducing lymphedema at the donor lymph node harvest site (Schaverien et al., 2018). The emerging field of tissue engineering holds potential for treating lymphedema by using lymphatic grafting to reconstruct damaged vasculature (Kanapathy et al., 2014). This new strategy aims to promote lymphangiogenesis and create functional lymphatic tissue in vitro for transplantation (Jia et al., 2021).

This review centers on identifying the processes involved in the development of artificial lymphatic tissue. By examining the architecture and physiological functions of natural lymphatic tissue, we aim to deepen our understanding of the foundational principles. Additionally, we assess the current biomaterials available for tissue generation and explore the challenges, recent advancements, and future implications of this emerging technology.

Two theories have dominated much of the discourse regarding the embryonic development of the lymphatic system. One theory suggests that LECs originate from veins and spread outwards to form the lymphatic system (Sabin, 1902). Another theory suggests that LECs develop from mesenchymal cells in tissue and eventually connect to the venous system (Null et al., 2024). However, recent studies have indicated that lymphatic vessel formation is complex, and can have both venous and non-venous origin, depending on the organ and the species (Semo et al., 2016). During embryonic development, lymphatic vessels develop after circulatory vasculature, at approximately 6 weeks after fertilization. The precursory endothelial cells that line this system are derived from the embryonic cardinal veins. Lymphatic vessels form in a similar fashion to blood vessels–creating lymphovenous and intra-lymphatic anastomoses. Approximately 8 weeks post-fertilization, six primary lymph sacs form: the cisterna chyli, located in the abdomen; the retroperitoneal lymph sac, located behind the abdomen; two iliac lymph sacs, located in the pelvic region; and two jugular lymph sacs, located in the neck region. Lymphatic vessels then form from these lymph sacs in a process of self-proliferation and sprouting to help connect the sacs to one another and to other parts of the body. Migrating mesenchymal cells enter these lymph sacs and contribute to the formation of lymphatic networks, connective tissue, and other layers of the lymph sacs. During the fetal period, these lymph sacs develop into clusters of lymph nodes (Null et al., 2024). Nearby proximal nerve fibers release retinoic acid (RA) to signal the mesenchymal cells of the lymph sacs to produce CXCL13 chemokine. CXCL13 plays a crucial role in lymph node development by attracting lymphoid tissue inducer cells (LTiCs), which are essential for the formation of the lymph node. In the absence of CXCL13 or its receptor CXCR5, peripheral lymph nodes fail to develop, (van de Pavert et al., 2009). The LTiCs also express lymphotoxin-β to interact with lymphoid tissue organizer (LTo) cells, aiding in lymph node development (Hampton and Chtanova, 2019). As the lymph node matures, distinct T and B cell zones emerge. T cell development begins with hemopoietic stem cells (HSCs) in the fetal liver and bone marrow. A subset of HSCs produces recombination activating gene 1 and 2 (RAG1 and RAG2) to eventually become common lymphoid progenitor (CLP) cells. A subset of these cells then migrates to the thymus to differentiate into early thymic progenitors (ETP) cells, which can be used to generate B cells, T cells, Natural Killer cells, myeloid cells, and dendritic cells. Specifically, B and T cells are attracted and matured through the expression of chemokines CCL19, CCL21, and CXCL13 produced by follicular dendritic cells (FDCs). These chemokines facilitate the movement of B cells into the marginal zone and T cells into the cortex for the formation of the respective B and T zones (Cano et al., 2013).

Lymph nodes are small, encapsulated organs, found all throughout the body. The structure of a lymph node facilitates its functions as a filter and a site for immune responses. Lymph nodes are found along lymphatic vessels, receive fluid from an afferent lymphatic vessel, and expel fluid out through an efferent lymphatic vessel (Null et al., 2024). Lymph nodes are composed of layers, including the capsule, the subcapsular sinus, the cortex, the paracortex, and the medulla. The capsule is composed of dense connective tissue and collagenous fibers. The subcapsular sinus allows for the transportation of lymphatic fluid from the capsule to the cortex. The cortex houses B-cell lymphoid follicles. When presented with an antigen, immature B-cells form a germinal center. Dendritic cells and resting B-cells together surround the germinal center to create a mantle zone. Between the B-cell lymphoid follicles lies the paracortex, which serves as a site for lymphocyte trafficking and houses T-lymphocytes. Dendritic cells also interact with T-cells in this layer by presenting antigens. The innermost portion of the lymph node is the medulla, which contains blood vessels, medullary sinuses, and medullary cords. Medullary sinuses act as a channel for lymphatic flow and drain fluid out of an efferent vessel, while medullary cords house macrophages and plasma cells that contribute to later stages of the immune response (Jia et al., 2021; Bujoreanu and Gupta, 2024). A conduit system in the lymph node helps to quickly deliver soluble antigens, immune complexes, and dendritic cells from the subcapsular sinus to phagocytic cells and post-capillary venules (Witte et al., 2006). Figure 3 visualizes the anatomy of the lymph node, lymphatic vessel, and lymphatic junctions.

Various signaling pathways and molecules are necessary in guiding proper lymphatic vessel formation, or lymphangiogenesis. While relevant for embryonic development, this process can also be pathologic in causing tumor metastasis and cancer growth. In the early stages of development, many factors play a role in ensuring lymphatic cell specification. Before differentiating into lymphatic-specific cells, markers such as lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) are induced in a subpopulation of venous endothelial cells to signify lymphatic fate (Butler et al., 2009). Yet, while this marker has early expression in lymphatic progenitor cells, studies have shown that the absence of Lyve1 in mice results in an apparently normal phenotype, with no obvious differences in lymphatic vessel structure or function. This finding suggests that while Lyve1 is an early marker for LECs, it is not essential for baseline lymphatic function, possibly due to the compensatory mechanism of various other factors involved in lymphangiogenesis (Gale et al., 2007). Prox1 is a key transcription factor that directs lymphatic cell fate (Semo et al., 2016). Studies have shown that Prox1 knock-out (KO) mice lack lymph sacs and lymphatic vessels (Wigle and Oliver, 1999). Prox1 regulation, and therefore LEC specification, is also affected by other transcription factors, including Sox18 and Coup-TFII, while secreted factors such as Wnt5b induce and Bmp2b inhibit the process (Semo et al., 2016). After specification, lymphangiogenic growth factors such as VEGF-C and VEGF-D induce lymphatic vessel proliferation. While both factors bind to the specific lymphatic endothelial cell receptor VEGFR-3, VEGF-C binds at a higher affinity (Witte et al., 2006). Studies showed that mice who lacked VEGFR-3 died early in utero, prior to lymphatic development, while mice who lacked VEGF-C did not develop lymph sacs (Karkkainen et al., 2004). Activation of VEGFR-3 leads to phosphorylation of serine kinases Jun N-terminal kinase (JNK), AKT, and extracellular signal-regulated kinase (ERK) to promote the proliferation, migration, and survival of LECs (Jia et al., 2021; Coso et al., 2012). FGF1 and FGF2 come from a family of fibroblast growth factors and are involved in angiogenesis and lymphangiogenesis. FGF2 has been shown to bind at low- and high-affinity receptors on LECs to promote the proliferation, migration, and survival of these LECs. An important receptor from this family, FGFR-3, also serves as a target gene for Prox1, and therefore, lymphatic development (Shin et al., 2006). Another family of growth factors, known as the platelet-derived growth factors (PDGF) are involved in this process. PDGF-BB is a growth factor that has directly been linked to lymphangiogenesis. Its receptors PDGFRα and PDGFRβ are expressed in LECs and are capable of inducing lymphangiogenesis as well (Jitariu et al., 2015). Though many of these factors work together to promote lymphangiogenesis, some have shown undesirable effects, specifically in the setting of cancer cell proliferation. Understanding the complex relationship between compounds and molecules in lymphangiogenesis can help promote vessel formation in engineered tissue.

A stable, structural scaffold creates the foundation for an environment conducive for cell growth, differentiation, and spatial organization. In normal embryonic development, stromal cells create this foundation out of polymeric collagen fibers. In artificial tissue engineering, various biomaterials have been identified to create a structural framework on which cells can grow (Nosenko et al., 2016). The primary function of this scaffold is to provide a surface for cell proliferation while delivering essential nutrients to the cells. In some instances, scaffolds have been shown to maintain their structure for up to 1 year after transplantation. When selecting a scaffold, it is important that the material is biocompatible, and therefore is non-toxic, and does not cause an immune response in the host body. Ideally, a scaffold should also be biodegradable at a rate that matches tissue formation, in order to leave behind functional tissue. Scaffolding should have the strength and flexibility to withstand physical and mechanical forces (Sung et al., 2022; Kanapathy et al., 2014; Alderfer et al., 2018). These materials can be broadly categorized into two groups: natural materials and synthetic materials (Kwon et al., 2018). In this review, we will highlight the many different biomaterials that have been used as scaffolding in artificial tissue engineering, which have been summarized in Table 1.

Stem cells play a crucial role in regenerative medicine, which aims to repair or replace damaged tissues and organs. Understanding the different types of stem cells and their role in tissue regeneration is essential for bioengineering lymphatic tissue. Stem cells are undifferentiated cells that possess the potential to specialize into various cell types and self-renew. While stem cell therapy alone faces considerable limitations due to poor cell survival after transplantation, tissue engineering has overcome these challenges by creating supportive environments that encourage stem cell viability, growth, and differentiation (Kwon et al., 2018). The most fundamental stem cell is the embryonic stem cell (ESC). ESCs are pluripotent cells derived from blastocysts and are capable of differentiating into any cell type in the body (Sung et al., 2022). These cells hold immense potential for tissue regeneration due to their pluripotency but are faced with many obstacles. Ethical issues have delayed the use of ESCs in clinical practice because they originate from human embryos. It is also difficult to induce 100% of ESCs into differentiated cells, and thus the cells that remain undifferentiated face the risk of forming a teratoma (Kwon et al., 2018). Additionally, these cells are allogenic and could potentially subject the patient to an immune reaction following transplantation (Boyd et al., 2012). Induced pluripotent stem cells (iPSCs) address the ethical concerns of ESCs, as they are adult somatic cells that are reprogrammed to a pluripotency similar to ESCs. Another advantage of using iPSCs in research is that they are patient-derived, reducing potential immune responses; however, the potential of generating tumors still remains a risk (Li et al., 2017). Hematopoietic stem cells (HSCs) are oligopotent, meaning they can differentiate into a few specific cell types, in this case, blood and immune system cells. In mice, these cells have been shown to give rise to LECs that eventually integrate into lymphatic vessels of tissues such as the kidney, liver, stomach, and intestines. Given their ability to form LECs and lymphatic vessels, HSCs can serve as a valuable tool for regenerating damaged or deficient lymphatic tissue. In addition to aiding in normal lymphatic endothelium, it has also been shown that HSCs can be incorporated into tumor-associated lymphatic vessels. Therefore, HSCs could also serve as a potential target for attenuating lymphatic system mediated tumor metastasis (Jiang et al., 2008). Multipotent adult progenitor cells (MAPCs) have the ability to differentiate into cell types of a specific organ, and when cultured with VEGF-A, they even separate into a mixture of arterial, venous, and lymphatic endothelial cells (Sung et al., 2022). Adipose-derived stem cells (ADSCs) are a group of mesenchymal stem cells that are specifically found in adipose tissue and are a promising source of lymphangiogenesis. Two mechanisms have been proposed for promoting lymphangiogenesis: paracrine secretion of growth factors and direct differentiation into LECs (Forte et al., 2021). While ADSCs have a strong potential for use in lymphatic tissue engineering, further studies are still needed to understand the long-term effects of their uses (Sung et al., 2022). While challenges remain for the use of stem cells in tissue engineering, the field is rapidly evolving. With an understanding of the fundamental cells needed in bioengineering artificial tissue, advancements can be made in biomaterials and the construction of lymphatic vessels to help revolutionize treatment for lymphedema and other lymphatic diseases.

3D bioprinting offers a promising approach to create multi-layered structures that mimic the complexity of natural tissues. This technology has been widely used to create tissues such as blood vessels, heart, liver, and kidneys. The printing process requires stem cells to form the 3D structure, along with bioinks, such as hydrogels, which act as scaffolds for cell growth. Bioprinting technology includes inkjet printing and laser deposition (Sung et al., 2022; Kwon et al., 2018). Inkjet printer technology is similar to that of a commercial printer, with controlled amounts of liquid being delivered to a predefined location. The printer uses thermal or acoustic forces to place drops of liquid onto a substrate to form the construct. Advantages to this technology include low cost, high availability, high uniformity, high resolution, and high biocompatibility. Despite these advantages, there are limitations that still remain. Printing exposes cells to thermal and mechanical stress, and some cells may not be able to withstand these conditions. Additionally, the biological material would need to be in a liquid state for printing, and then solidify into a 3D structure with proper organization and functionality, which could be unpredictable. Inkjet bioprinting has successfully been used to create functional skin and cartilage (Murphy and Atala, 2014; Skardal et al., 2012; Cui et al., 2012). Laser printing, in contrast, does not exert physical stress on the cells and does not require the biological material to begin in liquid form. As a result, the viability of cells is over 95% after printing, and other cellular functions, such as apoptosis and proliferation, are not affected (Kwon et al., 2018). Laser printing can also be employed for its higher resolution for building microvascular structures, such as lymphatic and blood capillaries (Hwang et al., 2021).

In lymphatic tissue engineering, 3D bioprinting has significantly advanced lymphatic system research. The integration of bioprinted lymphatic vessels has enabled the development of sophisticated tumor models, such as the tumor-on-a-chip system, for studying the tumor microenvironment. For instance, Cao et al. created a tumor-mimetic hydrogel embedded with bioprinted blood and lymphatic vessels. By optimizing vessel permeability through tailored bioink compositions, the researchers tested various combinations of bioprinted blood and lymphatic vessels with different permeabilities to examine drug diffusion through tumor tissue and its subsequent drainage via the lymphatic system. Their findings suggest that this model effectively simulates the tumor microenvironment and complex transport mechanisms, potentially enhancing the accuracy of future cancer drug screening (Cao et al., 2019).

Beyond oncology, bioprinting has also been used to study lymphangiogenesis. Sacrificial bioprinting—a technique where a printed template dissolves or is removed, leaving behind an endothelialized microchannel (Zhang et al., 2018)—has been employed to create a volumetric breast tumor model with embedded multiscale lymphatic vessels. This model facilitated the investigation of tumor-lymphatic interactions, including lymphatic sprouting and breast tumor cell migration. Notably, findings revealed that VEGF-C induced lymphatic endothelial cell proliferation and migration within the system (Liu et al., 2021).

While research on bioprinted lymphatic vessels for lymphedema treatment remains in its early stages, existing studies demonstrate the potential for fabricating functional lymphatic networks and inducing lymphangiogenesis. These advancements suggest that bioprinted lymphatic tissue could 1 day be applied to restore lymphatic drainage and improve outcomes for lymphedema patients.

ESWT has been used for decades across various medical fields to either break down tissue or stimulate tissue repair and regeneration. In urology, it is commonly employed to disintegrate kidney stones, while in orthopedic surgery, it is utilized for treating tendinopathies and non-union fractures (Wang, 2012). More recently, it has emerged as a promising, non-invasive treatment option for lymphedema. It aids in the regeneration of lymphatic valves by increasing cell permeability and facilitating the transport of molecules essential for tissue repair (Alderfer et al., 2018). The therapy delivers pressure waves as mechanical stimuli, exerting physical force on edematous tissue. This physical stress upregulates the expression of VEGF-C and FGF, which are crucial in promoting lymphangiogenesis and enhancing lymphatic drainage (Jia et al., 2021). Numerous preclinical studies have demonstrated the benefits of ESWT. In one study using a rabbit ear lymphedema model, ESWT significantly increased the expression of VEGF-C and VEGFR-3, leading to improved lymphedema outcomes (Kubo et al., 2010). Another study in a rat tail model showed that ESWT induced lymphangiogenesis by upregulating VEGF-C and bFGF, which helped alleviate lymphedema (Serizawa et al., 2011). Additionally, combining ESWT with a VEGF-C hydrogel demonstrated a synergistic effect in a mouse lymphedema model (Kim et al., 2013). Clinically, ESWT has also been applied in patients with breast cancer-related lymphedema (BCRL). Recent studies have shown that ESWT effectively reduces limb volume and enhances patients’ quality of life, with effects lasting up to 6 months post-therapy, making it a long-lasting and potentially cost-effective alternative to conventional lymphedema therapies (Lee et al., 2022; Joos et al., 2021; Cebicci et al., 2016). With its therapeutic benefits and proven effectiveness in both animal and human studies of lymphedema, ESWT holds promise as a valuable addition to lymphatic tissue engineering therapies.

Mechanical stimuli and the mechanical environment play a crucial role in lymphatic tissue engineering (Alderfer et al., 2018). Fluid shear stress (FSS), the force exerted by fluid flow on vessel walls, varies depending on the location or type of lymphatic vessel. Initial lymphatics experience lower FSS due to their exposure to interstitial fluid flow, whereas collecting lymphatic vessels encounter greater FSS because of their smooth muscle coverage and pumping capacity. The pumping function of collecting lymphatics generates low levels of nitric oxide (NO), which increases contraction amplitude. Conversely, higher NO concentrations, such as those observed in inflammatory conditions, inhibit both contraction amplitude and frequency. This indicates that higher concentrations of NO, produced by LECs in response to FSS, may contribute to shear stress and dysfunctional lymphatic pumping. This pumping, regulated by smooth muscle coverage surrounding collecting vessels, may play a role in preventing edematous conditions (Angeli and Lim, 2023).

FSS also functions as a key lymphangiogenic mediator, facilitating LEC migration, VEGF-C expression, and lymphatic organization (Swartz and Boardman, 2002). In both lymphatic endothelial cells (LECs) and collecting lymphatic vessels, VEGF-C signals through VEGFR-3 pathways to regulate distinct functions. While its role in lymphangiogenesis has been previously discussed, VEGF-C plays another role in collecting lymphatic vessels. In rat mesenteric collecting lymphatics, VEGF-C treatment has increased contraction frequency, end-diastolic diameter, stroke volume index, and pump flow (Breslin et al., 2007). Moreover, FSS influences LEC shape and alignment. Laminar shear stress (LSS) has been shown to elongate LECs and align fibers in the direction of fluid flow, whereas oscillatory shear stress (OSS) results in cuboidal LECs that are less dependent on fluid flow (Sabine et al., 2012). In addition to FSS, transmural flow significantly impacts the physiological functions of lymphatic vessels. Miteva et al. investigated the effects of transmural flow on lymphatic vessels using both in vivo and in vitro models. Their findings revealed that transmural flow increases lymphatic permeability to specific macromolecules and promotes dendritic cell transmigration through the vessels. These results suggest that transmural flow may act as an early inflammatory signal for lymphatic vessels, aiding in the delivery of soluble antigens and dendritic cells to lymph nodes (Miteva et al., 2010).

Interstitial flow also plays a part in ECM reorganization, lymphatic cell proliferation, lymphatic capillary morphogenesis, immunity, and peripheral tolerance (Wiig and Swartz, 2012). Lymphatic cells also migrate in the direction of interstitial flow, and do not form into functional capillaries if interstitial flow is severely affected (Rutkowski and Swartz, 2007). Studies have also shown that VEGF-C and interstitial flow work together to promote lymphangiogenesis, with VEGF-C working to induce cell proliferation and migration, while interstitial flow works to promote the organization and functionality of the lymphatic vessels (Goldman et al., 2007). When engineering lymphatic tissue, it is important to take into consideration the scaffolding that encourages interstitial flow, in order to aid in lymphangiogenesis. Helm et al. investigated the effects of slow interstitial flow in hydrogels with varying fibrin and collagen compositions and found that a fibrin-only matrix with slow interstitial flow promotes LEC organization (Helm et al., 2007). This study emphasizes the importance of scaffolding that supports proper interstitial flow in promoting lymphangiogenesis and engineering functional lymphatic tissue.

Mechanotransduction is the process by which cells convert mechanical stimuli into biochemical signals, typically mediated by molecules and transcription factors. Several flow-induced transcription factors have been identified as key mediators of FSS responses in lymphatic vessels. Forkhead box protein C2 (FOXC2) and PROX1 are key regulators of lymphatic vessel and valve development, as well as LEC alignment and organization under LSS and OSS. Studies show that OSS upregulates FOXC2, while the loss of PROX1 disrupts the cuboidal LEC arrangement induced by OSS and instead enhances the elongated LEC arrangement promoted by LSS (Sabine et al., 2012). FOXC2 activates FOXP2, a transcription factor that collaborates with the NFATc1 molecule to regulate collecting vessel morphogenesis and lymphatic valve development (Hernández Vásquez et al., 2021). Conversely, FOXC2-deficient LECs exhibit a disorganized cytoskeleton with disrupted cell-to-cell junctions, leading to abnormal responses to shear stress, increased cell proliferation, and apoptosis (Sabine et al., 2012).

In vitro models, LSS has been shown to enhance calcium levels in LECs via ORAI1, an early mediator of laminar flow. ORAI1 subsequently upregulates KLF2 and KLF4 in flow-activated LECs, leading to the regulation of VEGF-A, VEGF-C, FGFR3, and p57. Mouse embryos deficient in ORAI1 and its downstream molecules KLF2 and KLF4 demonstrated impaired lymphatic development and reduced lymphatic density (Choi et al., 2017). The upstream mechanosensor of ORAI1-mediated calcium entry in LECs is Piezo1. Embryos of Piezo1-knockout mice showed defects in lymphatic sprouting, while adult mice exhibited lymphatic regression. A potential therapeutic option for lymphedema involves Yoda1, a Piezo1 agonist, to stimulate lymphatic regeneration (Choi et al., 2022).

Another critical lymphatic mechanosensor is PECAM-1. Mice deficient in this sensor exhibited impaired flow sensing across LECs, leading to abnormal lymphatic vessel remodeling and valve development. Their lymphatic vessels displayed irregular, enlarged branches, while LECs appeared randomly oriented with abnormal valves. In vivo, Pecam1 and Sdc4 knockout mice developed significant edema, highlighting the necessity of both molecules for proper lymphatic vessel and valve formation (Wang et al., 2016).

Disruption in these mechanical forces contributes to the pathophysiology of lymphedema. Impaired lymphatic drainage alters the biomechanical environment, leading to endothelial dysfunction and progressive tissue changes, exacerbating lymphedema symptoms (Angeli and Lim, 2023). While conventional lymphedema diagnostic measures, such as circumferential limb measurement or water displacement, provide limited information on underlying tissue changes, recent studies suggest that biomechanical tools can offer a more precise approach for detecting lymphedema at earlier stages. For instance, the MyotonPro device, a noninvasive instrument used to measure skin stiffness, elasticity, oscillation frequency, relaxation time, and creep (the ratio of deformation to relaxation time) can assess biomechanical changes in lymphedematous tissue. Glassman et al. used this device to compare skin biochemical parameters between affected and unaffected arms in patients with breast cancer-related lymphedema. They found that on average, the affected arms exhibited decreased skin stiffness and oscillation frequency, and increased relaxation time and creep compared to the contralateral unaffected arms (Glassman et al., 2022). Similarly, Naczk et al. used MyotonPro to analyze the viscoelastic property of tissue in breast cancer-related lymphedema patients compared to controls, with similar findings to the previous study (Naczk et al., 2022). These findings suggest the potential of biomechanical assessment tools in improving early disease detection, monitoring of disease progression, and guiding personalized treatment plans for patients with lymphedema.

Beyond diagnostics, biomechanical control also holds promise for therapeutic strategies in lymphedema management. One key application is the optimization of compression therapy. By incorporating biomechanical measurements of tissue stiffness and elasticity, compression therapy can be personalized to ensure optimal pressure distribution for intermittent pneumatic compression devices (Zaleska et al., 2018). Additionally, tissue engineering offers a promising approach by leveraging the connection between the extracellular matrix (ECM) and the mechanical environment, both of which regulate lymphatic structure and function (Angeli and Lim, 2023). By developing biomechanical scaffolds that mimic the ECM, tissue engineering can enhance the biomechanical environment of the lymphatic system, ultimately promoting lymphatic vessel regeneration and stabilization.