Key roles of the superficial zone in articular cartilage physiology, pathology, and regeneration

Abstract

The superficial zone (SFZ) of articular cartilage is an important interface that isolates deeper zones from the microenvironment of the articular cavity and is directly exposed to various biological and mechanical stimuli. The SFZ is not only a crucial structure for maintaining the normal physiological function of articular cartilage but also the earliest site of osteoarthritis (OA) cartilage degeneration and a major site of cartilage progenitor cells, suggesting that the SFZ might represent a key target for the early diagnosis and treatment of OA. However, to date, SFZ research has not received sufficient attention, accounting for only about 0.58% of cartilage tissue research. The structure, biological composition, function, and related mechanisms of the SFZ in the physiological and pathological processes of articular cartilage remain unclear. This article reviews the key role of the SFZ in articular cartilage physiology and pathology and focuses on the characteristics of SFZ in articular cartilage degeneration and regeneration in OA, aiming to provide researchers with a systematic understanding of the current research status of the SFZ of articular cartilage, hoping that scholars will give more attention to the SFZ of articular cartilage in the future.

Introduction

Articular cartilage is a resilient shock-absorbing connective tissue that covers the ends of bones in synovial joints to disperse loads, reduce friction, and help joints move smoothly and painlessly.^[1] This tissue exhibits heterogeneity, and mature articular cartilage can be classified into four distinct zones: the superficial zone (SFZ), the middle zone, the deep zone, and the calcified zone.^[2] The SFZ is characterized by flattened cells and a tangential collagen fibril arrangement. Although the SFZ accounts for only 5–10% of the total thickness of articular cartilage, it is responsible for maintaining the normal structure and function of articular cartilage^[1]; additionally, pathological changes in cartilage are also observed in the SFZ during the onset of osteoarthritis (OA).^[3] Recently, different research groups reported that cartilage progenitor cell populations were detected mostly in the SFZ and were closely related to articular cartilage renewal and regeneration.^[4,5] Therefore, the SFZ is indispensable in the physiological, pathological, and regenerative processes of articular cartilage. However, by May 2024, only 892 articles could be found on PubMed from a search using the keywords “cartilage” and “superficial layer.” A search using the keyword “cartilage” alone returned 154,898 articles. The proportion of SFZ-related studies is only 0.58%, indicating that more attention should be given to the SFZ of articular cartilage. In this review, we summarized the recent progress in the structure, composition, mechanical function, and biological function. Additionaly, we reviewed the roles of the SFZ in cartilage degeneration and regeneration with a specific focus on OA. The aim is to increase the knowledge of the SFZ of articular cartilage with the hope that this important area receives more attention in the future.

Structure of the SFZ

Tangential collagen and flattened chondrocytes are generally regarded as unique features of the SFZ; so the SFZ is also referred to as the superficial tangential layer. However, with the development of detection technologies, researchers have discovered that the SFZ may comprise elements beyond the tangential layer and flattened chondrocytes. In 1996, Jurvelin et al^[6] used atomic force microscopy (AFM) to image the fresh bovine humeral head articular cartilage and observed an acellular non-fibrous layer that covered the collagen fibril network oriented parallel to the surface. The layer was typically 200–500 nm thick and exhibited local discontinuities, providing a very low friction lubrication surface in articular joints. The authors called this layer as the uppermost superficial layer or amorphous coat.^[6]

In addition to this amorphous coat, in 1990, Clark^[7] showed that the tangential fibers were covered by a dense, separate layer of small fibrils in the articular cartilage of the tibial plateaus, patellae, femoral heads, and medial femoral condyles of dogs, rabbits, and humans, and the fibrous network was believed to be an acellular stratum. In 2019, Boyanich et al^[8] physically peeled a 20.0 ± 4.7 μm thick transparent membrane from the femoral condyles of sheep, called it the most superficial layer/“skin” of articular cartilage, and discovered that the layer was not an acellular stratum, as reported previously. Rather, it is a relatively loose surface membrane with fewer chondrocytes and dense elastic and collagen fibers. The elastic fibers form a web-like network, whereas the collagen fibers are approximately aligned with the joint gliding direction. The diameters of various fibers were also detected, and the results showed that the diameters of the coarse elastic fibers were approximately 341 ± 89 nm; both the collagen fibrils and elastin microfibrils had similar diameters of 26.1 ± 7.1 nm and 34.8 ± 11.0 nm, respectively.^[8] This study further confirmed the existence of a fibrous network layer/most superficial layer, and an abundance of elastic fibers is believed to be the structural characteristic of this layer.

As the core components, tangential collagen and flattened chondrocytes have been studied extensively, and the results demonstrated that compared with those in the middle and deep zones, tangential collagen fibers in SFZ are finer, smaller, and more densely packed.^[9] In the majority of studies, histological analyses of tissue were performed in the vertical plane, explaining why superficial cells were described as small, flattened cells that appeared individually and arranged parallel to the articular cartilage surface. In 2008, Rolauffs et al^[10] analyzed the arrangement of superficial cells in non-degenerated human joints (shoulder, elbow, knee, and ankle) and discovered that superficial cells were present in four distinct patterns, namely, strings, clusters, pairs, or single chondrocytes.^[10] These findings not only provide a new understanding of the spatial arrangement of superficial chondrocytes but are also beneficial for current strategies in tissue engineering that involve the use of a layered design for the repair of cartilage lesions.

The above-mentioned studies demonstrated that the SFZ might consist of multiple layers, not just a tangential layer. In 2013, Fujioka et al^[11] observed the structure of the SFZ of pig cartilage, and the results showed that the SFZ is subdivided into four layers from the outside to the inside: an electron-dense amorphous layer, a low-electron density layer, a collagen bundle and chondrocyte layer, and a tangential layer. In 2021, Raspanti et al^[12] also revealed that the SFZ of bovine cartilage might be divided into two layers: a very thin, delicate superficial layer rich in glycoconjugates and a thick coat of thin, highly uniform, slightly wavy collagen fibrils lying parallel to the surface below the superficial sheet. These results further confirmed that defining the SFZ as comprising only tangential collagen and flattened cells might not be accurate. However, there is no definite conclusion that the SFZ consists of several layers. Regarding the unique structural characteristics of each sublayer in the SFZ, we propose the three-layer structure hypothesis, which states that the SFZ of cartilage includes three layers: an amorphous layer, a fibrous network layer, and a tangential layer [Figure 1]. In fact, the three sublayers not only have unique structures but also have their own compositional and functional characteristics, all of which are important bases for the three-layer structure hypothesis.

Figure 1.

The three-layer structure of the SFZ. Amorphous layer is an acellular non-fibrous layer with local discontinuities that cover the articular cartilage surface. Fibrous network layer has fewer chondrocytes and dense elastic and collagen fibers, and the elastic fibers form a web-like network. Tangential layer contains tangential collagen and flattened chondrocytes, which are the crucial structural features of the SFZ. SFZ: Superficial zone.

Composition of the SFZ

Similar to the different zones of cartilage having diverse compositions,^[13] the different sublayers within the SFZ have various biological compositions. Some studies have suggested that the amorphous layer might be a mixture of extraneous material (e.g., precipitated synovial fluid) and/or material extruded from the cartilage (e.g., lipid debris and degraded metabolites discharged into the joint), but the exact biochemical composition of the layer has been debated.^[3,14] Recently, studies showed that the main components of the lubrication layer (also called the amorphous layer in this study) of human joints include hyaluronic acid (HA),^[15] cartilage oligomeric matrix protein (COMP),^[16] lubricin (proteoglycan 4 [Prg4]),^[17] phospholipids (PLs),^[18] and glycosaminoglycans (GAGs).^[19] All these substances form an electron-dense particulate film that covers the articular cartilage surface and cooperates to achieve an almost frictionless coefficient.^[14] Other substances, such as albumin,^[20] globulin,^[21] phosphatidylcholine, and chondroitin sulfate,^[22] were also present in the layer, but their roles and mechanisms of action are unclear. Compared with amorphous layers, minimal research has been focused on fibrous network layers. Boyanich et al^[8] showed that the fibrous network layer might consist of fewer chondrocytes and dense elastic and collagen fibers. Fujioka et al^[11] discovered that the most superficial layer, extending beyond the tangential layer and facing the joint cavity directly, can be divided into three sublayers. The first layer comprises an amorphous substance. The second layer is rich in small fibrils. The third layer contains extracellular materials, in which collagen fibrils are arranged parallel to the articular surface, and collagen subtypes I, II, and III are present in this layer. However, it remains unclear if the second and third layers detected by Fujioka et al^[11] are the fiber network layers mentioned in this review.

The tangential layer is mainly composed of collagen, proteoglycans, water, and cells.^[23] Collagen is the indispensable structural component of the layer, and its special arrangement parallel to the articular surface determines not only the direction of mechanical anisotropic conduction^[24] and fluid flow^[25] but also the crack morphology.^[26] However, research on the content and the types of collagen in the SFZ remains inconclusive. Study of human cartilage has shown that the collagen and water contents are the highest in the SFZ and decrease with depth.^[13] Study of human and bovine cartilage samples has reported that the collagen content increases toward the cartilage–bone interface.^[27] In addition, the majority of previous studies assumed that superficial collagen proteins were mainly composed of type II collagen, and Wu et al^[28] reported that type IX collagen was present in type II collagen in bovine cartilage and was cross-linked to its surface in an antiparallel fashion. However, in 2004, Teshima et al^[29] suggested that the superficial tangential layer of normal adult human articular cartilage consisted not of type II collagen but of type I and III collagen.

Proteoglycan, composed of a core protein with covalently attached glycosaminoglycans (GAG) chains, is the second most abundant macromolecule in articular cartilage after collagen.^[23] Commonly, the predominant proteoglycan found in articular cartilage, with the highest content and largest size, is aggrecan; however, aggrecan is mainly distributed in the deeper zones of articular cartilage, not in the SFZ.^[30] Prg4, also known as the SFZ protein and lubricin, is expressed in the lining layer of the synovium, tendons, and ligaments, as well as in the SFZ of articular cartilage.^[3] Studies have shown that the SFZ disappears at eight weeks in Prg4-null mice,^[31] which indicated that Prg4 plays an essential role in maintaining the SFZ of articular joints. In addition to Prg4, decorin and biglycan were found to be most concentrated in the SFZ,^[32] but studies on these compounds are limited.

Water contributes up to 80% of the wet weight of articular cartilage.^[33] The relative water concentration decreases from approximately 80% in the SFZ to 65% in the deep zone.^[13] As cartilage is loaded, water is forced to flow through the tissue and across the articular surface; thus, it plays a crucial role in shock absorption, nutrient transportation, and lubrication.^[34] Furthermore, water is not only a key factor in maintaining the viability of superficial chondrocytes,^[35] but various studies have also shown that in OA, there is an increase in water content from an average normal concentration of 60–85% to greater than 90%^[33,34]; thus, scholars have tried to quantitatively determine the water content in articular cartilage utilizing magnetic resonance imaging (MRI) to facilitate the early detection of degenerative tissue changes in cartilage.^[33,34] However, MRI has relatively low spatial resolution, which greatly limits its application in the diagnosis of early articular cartilage degeneration.

Superficial chondrocytes are considered the only resident cells in the tangential layer, and these cells secrete pericellular matrix and extracellular matrix to regulate the mechanical and biological properties of the layer for adaptation to environmental changes during development and adulthood. In 2004, Dowthwaite et al^[5] successfully isolated articular cartilage progenitor cells from the surface zone of the articular cartilage of 7-day-old calves and discovered that the cell population exhibited a high affinity for fibronectin, possessed a high colony-forming efficiency, and expressed the cell fate selector gene Notch 1. To date, these cells have been observed in human, equine, bovine, rat, and mouse articular cartilage and are called cartilage-derived stem/progenitor cells (CSPCs).^[4] These findings suggested the presence of at least two different cell types in the SFZ. The discovery of the CSPCs may mark the end of the era when articular cartilage was considered to have no self-renewal or reparative capacity.^[36]

In summary, with improvements in technology, an increasing number of biological factors are being found in the SFZ, and each layer of the SFZ has a special biological composition [Figure 2]. However, the biological composition of the fiber network layer remains unclear, and the biological composition of the amorphous layer and tangential layer is also controversial. Currently, the methods of enzyme digestion,^[37] sandpaper grinding,^[38] and physical peeling^[8] are used to extract SFZ tissues. However, these methods are difficult to perform under aseptic conditions, and the biological activity of the extracted tissues is often disrupted. Moreover, the techniques are imprecise. Laser-capture microdissection can be used to accurately obtain the SFZ^[39]; nevertheless, the amount of tissue obtained is small, and maintaining the activity and function of the isolated SFZ tissue is difficult, which limits its application in SFZ studies. Therefore, it is necessary to develop more accurate techniques to extract multiple layers of the SFZ to study its biological composition and function.

Figure 2.

The biological composition of the SFZ. The amorphous layer is mainly composed of HA, Prg4, PLs, and GAGs, which cooperate to achieve an almost frictionless coefficient. Other substances, such as COMP and albumin, have also been reported in the layer, but their roles are unclear. The fibrous network layer might consist of fewer chondrocytes and dense elastic and collagen fiber networks, but the results need further validation. The tangential layer is mainly composed of collagen, GAGs, chondrocytes, and CSPCs. Among these layers, CSPCs are a new cell type with self-renewal and multidirectional differentiation abilities and are mainly present in the SFZ of articular cartilage. COMP: Cartilage oligomeric matrix protein; SFZ: Superficial zone.

Mechanical Function of the SFZ

Unique location and mechanical distribution of the SFZ

The SFZ is the only portion of the articular cartilage that directly bears the mechanical stimuli in the microenvironment of the joint cavity.^[40] Therefore, unlike the middle and deep layers, which mainly bear compressive stress, the SFZ of the articular cartilage simultaneously bears various mechanical stimuli, such as friction, shear, tensile, and compressive stress [Figure 3A].^[40] In 2014, Hosseini et al^[24] detected various stress distributions in intact cartilage-on-bone samples using a channel-indentation experiment. Their results showed that compressive stress, shear stress, and tensile stress were greatest in the SFZ and gradually decreased in the transitional zone.^[24] It may be closely related to the unique spatial location of the SFZ. Furthermore, the unique spatial position and mechanical distribution might be the key factors resulting in the unique mechanical functions of the SFZ.

Figure 3.

The function of the SFZ. (A) The mechanical function of the SFZ, including maintaining the ultralow friction on the cartilage surface, mechanical dispersion, mechanical buffering, and anti-tension. (B) The biological function of the SFZ, including being a molecular barrier to molecules with different particle sizes and a fixed charged barrier to molecules with different charges. (C) The function of the various components of the sublayers of the SFZ: various macromolecules in the amorphous layer providing an ultralow friction surface; the elastic fibers and the small fibrous network in the fibrous network layer providing resilience to the shear forces; the collagen network playing a key role in molecular barrier, mechanical dispersion, mechanical buffering, anti-tension, and the arrangement of the collagen influencing the direction of the fluid flow; GAG providing charges barrier; chondrocytes playing matrix secretion function; and the CSPCs performing cartilage repair function. SFZ: Superficial zone.

Role of the SFZ in maintaining low friction on the cartilage surface

Healthy articular cartilage is the most efficiently lubricated surface known in nature, with friction coefficients as low as 0.001 in response to physiologically high pressures.^[13] Such low friction is indeed essential for its well-being. Researchers are exploring natural lubrication mechanisms, and previous studies have concluded that synovial fluid plays a key role in maintaining the very low friction between cartilage surfaces.^[41] A recent theory showed that during joint movement, a hydrophilic gel layer is formed on the articular cartilage surface and causes an increase in the apparent surface viscosity, which serves to promote the separation of cartilage surfaces and results in low friction.^[42] The study suggested that for soft, permeable surfaces such as articular cartilage, synovial fluid is absorbed on to the surface to form a hydrophilic gel layer, and biomacromolecules, such as HA, Prg4, and PLs, are responsible for lubricating synovial fluid.^[42] In addition, most scholars believe that macromolecular substances in the layer interact with each other to ensure the nearly frictionless motion of knee joints, but the mechanism of various macromolecular interactions is currently unclear.^[14] Based on its function and composition, the hydrophilic gel layer is potentially the “amorphous layer” described in this article.

Role of the SFZ in mechanical buffering and dispersion

Similar to its structure and composition, the mechanical characteristics of articular cartilage have been shown to exhibit strong spatial variations.^[43] The equilibrium and compressive and shear moduli of articular cartilage exhibit an increasing trend as the depth from the SFZ toward the deep zone of the tissue increases,^[43] which makes the SFZ more deformable, acting as an energy-dissipation mechanism. Furthermore, during the process of deformation, to adapt to various mechanical stimuli, tensile resistance can be generated in cartilage tissue, and a thin SFZ has the greatest tensile strength in articular cartilage. Briefly, compared with the other layers, the SFZ has the lowest compressive and equilibrium modulus and shear modulus and the highest tensile strength, and it plays important roles in the load-bearing, low-friction, and wear-resistant properties of articular cartilage in vivo.^[44] Currently, many studies have confirmed this conclusion. For example, Gannon et al^[45] reported that removal of the SFZ causes a decrease in the dynamic modulus of mature cartilage. Grenfier et al^[46] also reported that using collagenase to degrade collagen at the surface of cartilage explants decreased both the instantaneous and equilibrium-confined compression moduli of the cartilage SFZ and middle zone. Komeili et al^[47] created macrocracks on the surface of intact articular cartilage, and the force–time responses of intact cartilage and cartilage with macrocracks were compared based on multiple nominal axial compressive strain levels and strain rates. The results showed a significant reduction in the transient and steady-state load-bearing capacity of the cartilage samples following the introduction of macrocracks.^[47]

With SFZ studies gradually increasing, researchers found that the SFZ not only exhibits different mechanical properties from the middle and deep layers of articular cartilage, but also exhibits some mechanical properties unique to the SFZ. In 2021, Yuh et al^[48] used a bioreactor to simulate articular loading of cartilage in the context of human gait, and stiffness was measured using microindentation immediately after bioreactor testing. The results showed that cartilage surface stiffness increases immediately after articular loading and returns to baseline values within 3 h. In addition, this stiffening response was unique to the SFZ, as articular loading on cartilage with the SFZ removed showed no changes in stiffness.^[48] In the study by Hosseini et al^[24] cartilage-on-bone blocks were subjected to creep loading under a nominal stress of 4.5 MPa using an indenter consisting of two rectangular platens separated by a narrow channel relief space to create a specific region where cartilage would not be directly loaded. The stress distribution in the intact sample and the SFZ-removed samples was detected, and the results showed that the SFZ is able to recruit a larger area of deep zone cartilage to carry compressive loads, ensuring that the cartilage with an intact SFZ has superior load-bearing properties and that these properties are unique to the SFZ.^[24]

Why do the mechanical properties of the SFZ differ from those of middle and deep cartilage tissue? The mechanical properties are directly correlated with the depth-dependent composition and ultrastructure of the extracellular matrix (ECM).^[43,49] As mentioned earlier, the biological composition of the SFZ remains controversial, but the role of its ultrastructure, especially the collagen structure, in its unique mechanical properties has been confirmed in multiple studies. Gannon et al^[45] discovered that removal of the SFZ significantly decreased the dynamic modulus of mature, but not immature articular cartilage, and the temporal changes in the relative spatial biochemical composition of the tissue cannot fully explain this phenomenon. The changes in the organization of the collagen network of the SFZ with age, including changes in fibril alignment, significant increases in collagen fibril diameter, and decreases in fibril branching play key roles in determining the dynamic properties of the tissue.^[45] Moreover, the study suggested that the tangential collagen of the SFZ acts to limit fluid flow into and out of articular cartilage. Thus, removing the SFZ results in the reduced ability to maintain fluid load support, which may explain the reduction in the dynamic moduli and the equilibrium moduli in confined compression upon removal of the SFZ in mature articular cartilage.^[45] Hosseini et al^[24] and Mansfield et al^[50] also discovered that the presence of tangential collagen fibrils in the SFZ is essential for the load-distributing function of the tissue. Compared with that of the tangential collagen fibril layer, there is less research on the function of the fiber network layer in the SFZ of articular cartilage. Boyanich et al^[8] suggested that the fiber network layer is likely very important to the non-linear properties of articular cartilage at the micro- and nanoscale, and the tangential orientation of the coarse elastic fibers at the layer is suggested to provide the articular cartilage with resilience to the shear forces experienced by the joint surface.

Taken together, not only does the SFZ have unique and complex mechanical properties, but the relationships among the ultrastructures of the three layers of the SFZ and their mechanical functions were also determined. The studies showed that the presence of an amorphous layer is crucial for maintaining extremely low friction on the joint surface via its lubrication function. The fibrous network layer plays a key role in the resilience to the shear forces experienced by the joint surface, and the tangential layer is an important structural foundation for buffering and dispersing various mechanical stimuli in the SFZ of articular cartilage [Figure 3]. Although this conclusion needs further verification, it still provides a new direction for researchers to study the mechanical function of articular cartilage tissue.

Biological Function of the SFZ

Articular cartilage is an avascular, aneural, and alymphatic tissue, so nutrient intake and metabolite excretion in articular cartilage largely depend on fluid exchange between the articular cartilage and synovial fluid in the joint. For this reason, the SFZ, as the only gateway for fluid to move into and/or out of articular cartilage, has been the focus of attention. In 1968, Maroudas et al^[51] reported that the permeability of human femoral condyle cartilage increased from the SFZ to the middle zone by approximately 35% and decreased from the middle zone to the deep zone by approximately 200%. These results suggested that, compared with the middle zone, the SFZ acts as a biological barrier in the transport of soluble molecules into/out articular cartilage.^[51] Factors that affect the permeability of cartilage include “pore” size and fixed charge density, which are determined by the composition of the cartilage.^[51] As shown previously, in the SFZ, collagen is woven together to form a fibrous network in which large proteoglycan aggregates are trapped. Together, these aggregates form a molecular barrier for fluid flow in the articular cartilage. An examination of a substantial cohort of data demonstrated that the spacing between GAG molecules in cartilage is 4–6 nm, the spacing between collagen fibrils in the SFZ is approximately 60 nm, and the effective “pore” size of cartilage is only approximately 6 nm.^[51,52] On the other hand, GAGs contain high concentrations of fixed negatively charged groups, which serve as an electric barrier.^[52] Additionally, the collagen fiber orientation influences the fluid flow in articular cartilage; indeed, fluid flows more easily in the direction parallel to the fibers than perpendicular to them.^[25,53] Thus, there is greater permeability in the direction parallel to the articular surface in the superficial layers, not in the axial direction (through the thickness).^[25,53] These permeability characteristics at least partially enhance the biological barrier function of the SFZ [Figure 3]. However, in 2022, Sun et al^[54] divided porcine articular cartilage into three zones (the SFZ, middle zone, and deep zone) in the horizontal direction, and the permeability of the cartilage tissue in each zone was tested. The results showed that the permeability decreased gradually from the SFZ to the deep zone. Leddy et al^[53] also reported that the diffusivity ratio of 500 kDa dextran was significantly greater than that of 3 kDa dextran in the SFZ. These studies indicate that whether the superficial layer of articular cartilage has a barrier effect on biological molecules needs further study. And, the role and molecular mechanism of each layer of the SFZ in the substance exchange of articular cartilage are unclear at present.

In summary, as the first line of defense for cartilage tissue against various mechanical and biological stimuli, the SFZ plays a key role in low friction, mechanical buffering, mechanical dispersion, and material exchange between articular cartilage tissue and the articular cavity microenvironment [Figure 3]. Further studies on the function-related influencing factors and the mechanisms of SFZ sublayers will provide new perspectives and strategies for the construction of tissue-engineered cartilage,^[55] cartilage tissue organoids,^[56] and drug delivery systems.^[57]

SFZ in OA

SFZ is the initial site of OA-related articular cartilage degeneration

OA is the most common joint disorder and remains the leading cause of disability in elderly individuals. Despite decades of research, the mechanisms underlying OA initiation remain poorly understood.^[57] In the late 20th century, studies showed that the earliest visible changes in arthritic articular cartilage involved SFZ roughening and fibrillation. Panula et al^[58] induced OA models in beagles at age 3 months and suggested that superficial collagen network disorganization seems to be one of the first structural changes before the appearance of overt OA lesions. In addition, chondrocyte properties in the SFZ were also observed.^[58] The results showed that in early OA, the volume of superficial chondrocytes increased,^[59] the chondrocyte spatial organization changed from strings to double strings and then formed small or even large clusters, and the cell number decreased significantly.^[8,60] In 2022, Tschaikowsky et al^[61] isolated articular cartilage explants from the femoral condylar cartilage of OA patients and categorized them into four stages of early OA (strings, double strings, small clusters, and large clusters) as well as advanced OA using superficial chondrocyte spatial organization (SCSO) as a biomarker. The study suggested that in early OA, a loss of thick type II collagen fibers and the formation of type I collagen on the articular cartilage surface result in a macroscopically intact yet mechanically inferior tissue phenotype that lacks durability and the ability to withstand high tensile forces and, therefore, is prone to further damage.^[61] As a result, studies believed that the first changes at the onset of OA occur at the SFZ of the articular cartilage.^[62] However, OA is a whole-joint disease involving structural alterations in the hyaline articular cartilage, subchondral bone, ligaments, capsule, meniscus, synovial membrane, infrapatellar fat pad, and periarticular muscles.^[63] The above studies indicated that only the earliest OA-related cartilage degeneration occurred in the SFZ, and the relationship between SFZ degeneration and OA development needs further study.

SFZ injury might be a key event that accelerates degeneration of the deeper layer of cartilage

In 2013, Waller et al^[64] discovered that decreased lubricin secretion resulted in increased friction on the articular cartilage surface and cellular apoptosis in the deeper layers of cartilage using Prg4 knockout mice and an in vitro bovine explant cartilage-on-cartilage bearing system. In 2015, Bartell et al^[65] detected chondrocyte death in neonatal calf cartilage samples following rapid, localized impact, and the results showed that when the superficial layer was removed, strain causes the subsequent chondrocyte death penetrated deeper into the samples.^[65] Others have also reported that SFZ degeneration directly exposes the middle and deep layers of the cartilage to various mechanical stimuli, subsequently leading to the degeneration of deeper zones of articular cartilage.^[8] These studies suggest that decreased mechanical resistance or changes in the mechanical properties of the SFZ result in injury to the middle and deep cartilage. Moreover, SFZ extracellular matrix degradation, especially reduced collagen and GAG content, can destroy the particle size selection and charge selection functions of collagen and GAG, and then the deleterious bioactive molecules subsequently break through the SFZ and enter the deeper layers of cartilage tissue.^[66] So the loss of cartilage tissue matrix molecules^[66] ultimately accelerates the pathological process of OA. Taken together, considering the important mechanical and biological functions of the SFZ in maintaining the physiological characteristics of articular cartilage, this review concluded that SFZ injury is a key event that initiates and accelerates degeneration of the deeper layer of cartilage. Therefore, if superficial injuries can be detected, OA might be diagnosed and treated in a timely manner in the initial stage of cartilage degeneration [Figure 4]. However, there are currently no effective diagnostic methods for detecting SFZ injury. Histological staining, MRI detection,^[33] and arthroscopy detection^[67] can be used to detect cartilage damage, but these methods are only useful for evaluating full-thickness cartilage damage and lack sensitivity and specificity in detecting SFZ injuries; moreover, there is no specific grading system for SFZ injury. Although high-precision scanning electron microscope (SEM), transmission electron microscope (TEM), and AFM can be used to analyze the ultrastructure of SFZ,^[8] the equipment is expensive and clinical translation is difficult. The development of novel technologies to evaluate SFZ injury has thus become an urgent issue.

Figure 4.

SFZ injury is a key event that can lead to early diagnosis and treatment of OA. The cartilage tissue specimen in the picture was obtained from a human. MRI: Magnetic resonance imaging; OA: Osteoarthritis; SFZ: Superficial zone.

SFZ and cartilage injury repair in OA

Delaying cartilage degeneration by reducing surface friction

Impaired articular cartilage surface lubrication can cause cartilage wear, chondroblast injury, and cartilage matrix degradation and accelerate the process of OA. Therefore, for decades, scholars have been interested in effectively reducing the friction coefficient of the cartilage surface and delaying OA. As a result, scientists have tried to synthesize different lubricin mimics, including a chondroitin sulfate backbone with type II collagen and HA,^[68] HA and Prg4,^[69] and both HA and collagen type II, to help Prg4^[20] provide a lower coefficient of friction. Early studies have also used single macromolecules, such as lubricin, HA, and Prg4, which have shown excellent performance in maintaining lubrication, to reduce friction and treat OA.^[70] Moreover, the choice of fully synthetic yet biocompatible macromolecules^[71] and multifunctional systems that both lubricate and release specific molecules, such as anti-inflammatory drugs,^[72] has been shown to reduce the friction coefficient of the cartilage surface. Although additional research is needed before synthetic replacements of natural biolubricants can be used clinically, the establishment of effective cartilage surface lubrication has become an important method for the early treatment of OA.

Promotion of cartilage repair using CSPCs in the SFZ

Over the past two decades, increasing evidence has confirmed the existence of CSPCs in the SFZ of articular cartilage and demonstrated that CSPCs exhibit strong proliferation, migration, and multiple differentiation potentials.^[73] These cells can be isolated and cultured from animals and humans and are found in articular cartilage throughout the body, including the knee joint and patellofemoral joint. These cells potentially play significant roles in cartilage regeneration and repairment in OA.^[36] In 2019, Yin et al^[74] intra-articularly injected CSPCs into a rat model of temporomandibular OA and found that CSPCs significantly delayed the degradation of articular cartilage, and lineage tracing revealed that CSPCs could directly repair articular cartilage.^[74] In another study, CSPCs were used as seed cells to repair full-layer bovine femoral cartilage defects, and the regenerated cartilage had sufficient physiological characteristics and mechanical properties.^[75] Therefore, CSPC therapy may directly initiate in situ self-repair of articular cartilage via certain means. Tong et al^[76] used an nuclear factor kappa B (NF-κB) inhibitor to protect the cartilage tissue and induce cartilage regeneration by inhibiting the NF-κB pathway and activating CSPCs during OA, suggesting that increasing the CSPC differentiation and proliferation is also an important method for delaying or repairing OA cartilage injury. The Prg4 gene, which encodes lubricin, is highly expressed in CSPCs; thus, CSPC aggregation can produce a large amount of lubricin, decrease joint surface friction, and treat OA.^[77]

However, to date, there are no precise or specific biomarkers for cartilage-resident progenitor cells. Some of the markers currently used for isolating and identifying CSPCs, such as CD73, CD105, and STRO-1, overlap with those of bone marrow stromal cells (BMSCs).^[78] New markers are constantly being explored. In 2023, using long-term cell fate tracing of adult Grem1-lineage cells in vivo, Ng et al^[79] discovered that these cells contributed to progenitor populations in the SFZ of the articular cartilage. Similarly, Gli1⁺ cells were found in the SFZ of articular cartilage and costal cartilage and the topmost layer of the growth plate underneath the cartilage endplate in vertebrae at 1 month of age.^[80] In several studies, Prg4 has been used as a key marker for CSPCs to investigate its role in the growth, development, and injury repair of articular cartilage tissue. The results indicated that Prg4 is expressed by superficial chondrocytes in young mice but expands into deeper regions of the articular cartilage as the animals age.^[81] However, progenitor tracing at prenatal or juvenile stages revealed that joint injury provoked a massive and rapid increase in synovial Prg4⁺ and CD44⁺/P75⁺ cells, some of which filled the injury site, whereas neighboring chondrocytes appeared unresponsive.^[82] Massengale et al^[83] also showed that chondrocytes in adult SFZ do not reconstitute cartilage wounds, adult Prg4-lineage progenitors predominate in wounds and do not originate in pre- or post-injury bone marrow, and Prg4⁺ articular chondrocytes, including those in the SFZ, are unlikely contributors to wound repair. The data described above suggest that CSPCs labeled with different markers play different roles in cartilage injury repair. Therefore, exploring the specific markers of CSPCs in the SFZ is highly important for studying their function and related molecular mechanisms.

In recent years, advancements in the profiling of cartilage tissues at single-cell resolution have provided unique insights into the organization and function of these tissues in health and disease, demonstrating the biomarkers, functions, and molecular mechanisms of CSPCs in cartilage. In 2021, single-cell RNA-seq (scRNA-seq) was performed on knee cartilage of 10-week-old normal and OA adult mice, and chondrocytes were divided into 9 cell subpopulations.^[84] The data of this study showed that dividing chondrocytes, Ucma^high chondrocytes, Mef2c^high chondrocytes, and Krt16^high chondrocytes likely constitute the SFZ of articular cartilage. However, it was not clear whether dividing chondrocytes or any of the other nearby clusters, such as Ucma^high chondrocytes, represented a progenitor-like population.^[84] In addition to scRNA-seq, the single-cell proteomic technique (Cytometry by time of flight, CyTOF)) has played an important role in the advancement of single-cell proteomics. In a study of 20 samples obtained from patients with OA undergoing total knee joint arthroplasty and five normal cartilage samples obtained from non-OA donors, chondrocytes were isolated from the samples and cultured for CyTOF profiling.^[85] And the CSPCs were divided into three subpopulations: CSPC I, CSPC II, and CSPC III. Among the subpopulations, CSPC I was CD105^low and CD54^high, with the highest percentage of cycling cells and active extracellular regulated protein kinases (ERK)1/2 signaling, and the percentage of these subpopulations decreased in OA. In contrast, CD105^high CPC III subpopulations were enriched in OA.^[85] Single-cell level studies can provide a clearer description of the population characteristics and some cellular biomarkers of CSPCs. However, it is still difficult to isolate MSCs, CSPCs, and chondrocytes.

Conclusions

The SFZ of articular cartilage potentially includes at least three layers: the amorphous layer, the fibrous network layer, and the tangential layer. Each sublayer has its own unique structural characteristics, biological composition, and function [Table 1]. Importantly, the SFZ not only plays a crucial role in maintaining the normal mechanical and biological functions of articular cartilage, but is also the location where pathological changes are observed during OA initiation. Therefore, SFZ injury might be a novel target for the early diagnosis and treatment of OA. However, to date, we know very little about the SFZ. One reason might be that researchers do not pay much attention to the SFZ. But, the most important reason is that the SFZ is too thin to extract and detect. As the improtant roles of SFZ in articular cartilage, in the future, more attention should be given to the SFZ, and novel techniques targeting SFZ should be developed to reveal its role and molecular mechanism in articular cartilage physiology, pathology, and regeneration, laying the foundation for the early diagnosis, prevention, and treatment of OA.

Table 1.

The structure, composition, mechanical function, biological function, and role of the SFZ in OA (including key references).

Structure	Main composition	Mechanical function	Biological function	Role in OA
Amorphous layer[6] (The uppermost superficial layer, the amorphous coat, the lubrication layer or the outer surface of cartilage)	HA Lubricin (Prg4) PLs Aggrecan[14,20–22]	Provides a frictionless surface for articular cartilage The mechanism is unclear[14]	Unknown	Supplementing the main components in this layer may delay the OA process, but there is still controversy
Fibrous network layer[8] (The most superficial layer or “skin” of articular cartilage)	Fewer chondrocytes Dense elastic fibers Collagen fibers[8]	Provides articular cartilage resilience to various forces The mechanism is unclear[8]	Unknown	Unknown
Tangential layer[8] (The superficial tangential layer)	Chondrocytes CSPCs Types I, II, III, IX collagen Prg4[4,5,28,29]	Disperses loads Resists compression, lesion and shearing forces The mechanism is unclear[24,43,50]	Unknown	Unknown
The SFZ (The layer may be composed of the above three layers )	All of the above	All of the above	The biological barrier between the mid-deep zone of articular cartilage and synovial fluid The mechanism is unclear[25,51–54]	Protects articular cartilage from OA[65] Pathological changes upon OA onset are observed here first[10,24,58–62] Responsible for OA cartilage regeneration[24,65,66] The mechanism is unclear

CSPCs: Cartilage-derived stem/progenitor cells; HA: Hyaluronic acid; OA: Osteoarthritis; PLs: Phospholipids; Prg4: Proteoglycan 4; SFZ: Superficial zone.

Funding

This manuscript was supported by funds from the Regional Innovation Joint Fund of the National Natural Science Foundation of China (Integrated Project) (No. U23A6009), Regional Innovation Joint Fund of the National Natural Science Foundation of China (Key Project) (No. U21A20353), Natural Science Foundation of Shanxi Province (No. 20210302123285), Key R&D Program Projects of Shanxi Province (No. 202202040201012), and Hainan Provincial Medical and Health Research Program (No. 21A200349).

Conflicts of interest

None.