Mechanical harvesting of cell sheets: an efficient approach for bone and cartilage tissue engineering

Abstract

Cell sheet engineering (CSE) has demonstrated significant promise for the advancement and application of tissue-engineered constructs in the fields of tissue engineering and regenerative medicine. In this technology, cells are cultured to form a monolayer, which is then detached from the culture surface as a complete sheet. This process preserves cell interactions, maintains cellular phenotypes and functions, and retains the integrity of the cell–extracellular matrix (ECM). A main characteristic of the cell sheet is its ability to retain the native ECM components secreted by cells. When the ECM is preserved in cell sheets, cells are surrounded by a much more biologically appropriate environment to increase their regenerative potential, thereby offering more native conditions for cell growth and differentiation. CSE has shown promising results in a wide range of applications, including bone and cartilage. The cell sheets can be directly transplanted to the target site, where they integrate with the host tissue and enhance regeneration. The main challenge in CSE is how to detach an intact cell sheet without disturbing the ECM and cell‒cell connections. There are various methods for removing cell sheets that lead to the harvesting of intact cell sheets. Among the various methods for harvesting cell sheets, temperature-responsive systems and mechanical peeling are the most common. Mechanical harvesting, in particular, is a simple, cost-effective, accessible method that is widely used in research, especially in the scope of bone and cartilage tissue engineering. This article aims to review the application of cell sheets in bone and cartilage tissue engineering, with a focus on practical and cost-effective mechanical harvesting methods.

Introduction

Cell sheet engineering (CSE) is based on the development of a confluent monolayer of cells with tight adhesion to the culture dish surface. It is a scaffold-free method that relies on cell-to-cell junctions and the secretion of extracellular matrix (ECM) proteins for maintenance. CSE overcomes the limitations associated with scaffold degradation and offers a composition similar to that of natural tissues. Cell sheets are collected without the use of proteolytic enzymes and preserve cell‒cell interactions, resulting in a higher cell count and more uniform cell dispersion. Compared with cell injection, this technology improves the regenerative capacity of the cells and results in a higher cell survival rate and fewer apoptotic cells [1]. Cell sheets have been successfully used to treat damaged tissues such as articular cartilage, bone, cornea, and blood vessels [2]. Multilayer cell sheets were fabricated to mimic the complicated structures of native tissues. The entire strategy of cell sheet technology includes selecting an appropriate cell source, preparing and harvesting cell sheets and their translation into therapeutic applications.

For the fabrication of intact cell sheets, it is crucial to establish effective methods that detach the monolayer cell sheet with minimum damage to cell surface proteins, promote cell‒cell interactions and maintain cell viability. Enzymes such as trypsin, which are commonly used to harvest cells, are not appropriate because they destroy adhesion proteins and lead to the dispersal of single cells. Therefore, a selective detachment method is needed to remove the adhesive molecules between the cells and the surface of the culture dish and create an impeccable cell sheet. Thus far, various techniques have been reported for harvesting cell sheets, including temperature-responsive methods, surface-modified methods, and methods without surface modification. Each method has its own advantages and disadvantages. Thermoresponsive culture dishes coated with poly(N-isopropylacrylamide) (PIPAAm) have been developed to facilitate cell sheet engineering; cells are allowed to adhere and proliferate at relatively high temperatures and detach by lowering the temperature. Temperature-responsive culture dishes (TRCDs) are commercially available, but they are expensive because of the state-of-the-art technology that is used to manufacture them. Other harvesting methods require surface modification or condition modification, pH changes, magnetic particles, or electric fields that may have adverse effects on cells. Among these methods, mechanical peeling is the simplest and most affordable method, as overconfluent cell layers can be detached via the use of cell scrapers or pipette tips.

This review provides detailed information on the cell sources, harvesting methods, applications, limitations, and future research directions of cell sheet engineering. We discuss the evolution of 3D cell sheets by means of mechanical harvesting with a particular focus on bone and cartilage tissue engineering. We sought to address these challenges and further expand the clinical applications of CSE.

Cell sources

Before discussing harvesting strategies, it is important to consider the diverse cell sources used in cell sheet engineering, as these sources influence detachment efficiency and therapeutic potential. Cells with unique characteristics play crucial roles in cell sheet tissue engineering (Fig. 1). The cells used in tissue engineering should be easily obtainable, have intense propagation and continuous passage capabilities, and have the lowest chance of rejection by the immune system [3]. When a homogenous cell line is needed, somatic cells may be considered a convenient cell source. For example, mesenchymal stem cells (MSCs), which are defined by their multipotency and derived from various tissues, are the most common stem cells utilized in tissue engineering. Pluripotent stem cells (PSCs), which include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the strongest capacity for self-renewal and differentiation into various specialized cell lines for the regeneration of damaged tissues [4]. The potential of various cell sources for CSE application in the regeneration of various tissues is discussed below.

Fig. 1.

Schematic diagram of utilizing different cell sources, such as various kinds of stem cells and somatic cells, for cell sheet fabrication, cell sheets can easily form multilayer constructs.

Somatic cells

Somatic cells from homogenous tissues are a cell source that is capable of forming cell sheets. Several cell lines that can form transplantable cell sheets in culture dishes include keratinocytes, retina pigment epithelium, cornea epithelium, cornea endothelium, Occulomaxillofacia epithelium, urothelium epithelium, gumline ligament, aortic endothelial cells, cardiomyocytes, and renal epithelial cells [3]. Autologous oral mucosal cell sheets have been used as a successful novel treatment for esophageal mucosal cancer after superficial epithelial neoplasms are removed via esophageal endoscopic submucosal dissection (ESD). Sutureless and endoscopic transplantation of cell sheets promoted re-epithelialization of the esophagus after ESD and prevented inflammation and stenosis after transplantation [3]. Yoshiki Sawa et al. developed multilayered skeletal myoblast sheets and subsequently transplanted them into the heart with dilated cardiomyopathy. They reported a significant improvement in cardiovascular function [4]. Autologous skeletal myoblasts can also be prepared for transplantation, as they are ischemia resistant and differentiable into nonmyocyte lineage cells. A research group showed that skeletal myoblast cell sheet transplantation might be a suitable therapeutic strategy for cardiovascular disease [5]. Bilayered fibroblast sheets have been utilized as novel sealants for preventing lung leakage [6]. Recently, middle ear cholesteatoma or adhesive otitis patients were favorably treated with a unique strategy for transplantation via a combination of tympanoplasty and autologous nasal mucosal epithelial cell sheets [7]. Matsukura et al. established a rat defect model via treatment with human juvenile cartilage-derived chondrocyte (JCC) sheets. JCCs were isolated from the polydactylous digits of various donors, and passage 2 JCC sheets were transplanted into the chondral defects of nude rats for 4 weeks. The samples were stained with safranin O, collagen 1 and collagen 2, and each group was evaluated via modified O’Driscoll scoring. Their results revealed that histological score variations between juvenile cartilage-derived chondrocyte donors were correlated with cartilage hyaline structure and subchondral bone regeneration quality in a nude rat defect model [8]. Despite promising outcomes, cell donor conditions, such as age, health status, and the physiology of the organ from which somatic cells are derived, may alter cell sheet properties, such as cell proliferation time, cell sheet thickness, and integration.

MSCs are multipotent cells isolated from various stromal tissues, such as adipose tissue, dental pulp, bone marrow, amniotic fluid, and the umbilical cord. Mesenchymal stromal and stem cells are two distinguishable cell types. There is current controversy regarding the interchangeable use of mesenchymal stem cells and mesenchymal stromal cells. The minimum criteria for their identification were first proposed by the International Society for Cellular Therapy (ISCT) in 2006 and were updated again in 2019. The desirable identification of these two cell types could lead to the minimization of an inadvertent mixture of stem cells and stromal cells in clinical application, which could control the safety and effectiveness of the therapeutic properties of MSCs [9]. MSCs are characterized by their self-renewal capacity and multilineage differentiation potential. MSCs are fibroblast-like cells that grow as a uniform population of adhered cells after subculture. They have amazing potential for differentiating into three main lineages: osteocytes, adipocytes and chondrocytes. They express markers such as CD73, CD90 and CD105 and do not express CD11b, CD14, CD19, CD34, CD45, CD79a or HLA-DR. A more specific combination of markers for identifying MSCs on the basis of tissue of origin was published by Ullah et al. (2015) [10].

CSE utilizes various types of MSCs to fabricate scaffold-free constructs. Bone marrow-derived MSCs (BM-MSCs) are commonly used because of their ability to differentiate into various cell types and secrete paracrine factors that promote tissue healing. BM-MSC sheets have demonstrated effectiveness in cardiac repair and systemic inflammatory diseases [11]. Adipose-derived MSCs (ADSCs), owing to their high proliferation rates and accessibility, have been utilized in engineered cell sheets for islet support and have demonstrated superior viability and recovery rates in comparison with those of other cell types [12]. Periodontal ligament stem cells (PDLSCs) are vital for periodontal tissue regeneration. When PDLSCs combine with BM-MSCs in composite cell sheets, they increase the expression of bone-related gene markers, which facilitate the regeneration of complex periodontal structures [13].

Engineered tissue constructs created from MSC sheets have shown promising outcomes, specifically with respect to host tissue-transplanted cell sheet integration and their anti-inflammatory efficiency when they are utilized for treatment. MSC sheets have significant potential for treating diseases and for tissue regeneration. They have been used for the treatment of cornea, skin, cartilage, bone, meniscus, tendons, oral ulcers, cardiac disease, and spine cord defects [14]. For example, engineered MSC sheets could prevent myocardial ischemia and improve left ventricle remodeling, indicating the potential of MSCs in cardiac tissue engineering [11]. The immunomodulatory properties of cell sheets derived from clonal BM-MSCs have been demonstrated to be enhanced, which is vital for reducing inflammation in therapeutic applications [15]. Transplanting multilayer cell sheets fabricated from patient-derived periodontal ligament MSCs expedited bone regeneration, cementogenesis, and the development of complete collagen fibers and reduced the incidence of periodontal diseases[16]. The transplantation of monolayer MSC sheets enhanced bone ossification in a nonunion rat model [17, 18]. Similarly, MSC sheets made of magnetite nanoparticles stimulated ossification in rats. Chen et al. reported that prevascularized MSC sheets repaired full-thickness wounds in the skin in contrast with nonprevascularized cell sheets [19]. Kawamura et al. integrated MSCs and endothelial progenitor cells (EPCs) derived from rat bone marrow. MSCs seeded on a fibronectin-coated culture dish differentiated into smooth muscle cells (SMCs). These SMC-EPC cocultured cell sheets prevent cardiac dysfunction and microvascular disease associated with diabetes mellitus-induced cardiomyopathy [20]. Furthermore, MSC sheets may promote angiogenesis in ischemic and wounded tissues. The application of MSC-derived cell sheet technology is not limited to these examples. When appropriate media and growth supplements are used, MSCs can be committed to the mesoderm, endoderm, or ectoderm lineages.

MSCs offer remarkable advantages in regenerative medicine, including their vast in vitro differentiation ability, lack of teratoma formation, and are easily accessible and expandable with exceptional genomic stability and few ethical issues. Nevertheless, several limitations are allocated to MSCs, such as their inability to spontaneously differentiate, their source tissue, cell behavior at the target site, and the donor's age and health status. The engraftment and retention of target tissues are other issues that must be addressed for preclinical and clinical applications. Further research may be needed to optimize the clinical application of cell sheets and ensure the consistency of therapeutic outcomes.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)

ESCs and iPSCs, known as pluripotent stem cells, possess significant features, such as unlimited proliferation ability and efficient differentiation potential. Although they have not been investigated as extensively as MSCs are, cell sheet engineering based on ESCs and iPSCs has shown promising results. A research group reported that ESC-derived cardiac cell sheets, which include endothelial cells, mural cells, and cardiomyocytes, induce angiogenesis in myocardial infarcted tissue and promote cardiovascular function [21]. Kawamura et al. confirmed the feasibility, safety and therapeutic effects of hiPSC-derived cardiomyocyte sheets in ischemic cardiomyopathy in a porcine model [22]. The primary mechanisms that improved cardiovascular function were achieved after ESC transplantation and iPSC sheets in heart infarction models. Transplantation of ESC-derived cardiac cell sheets induced angiogenesis in the marginal area and increased the expression level of angiogenic markers and neovascularization in cardiomyocytes [7]. Suzuki et al. demonstrated the potential of iPSC-derived cell sheets in restoring structural integrity and improving muscle function in hemiplegic mice [23]. These findings underscore the therapeutic value of iPSCs in treating neurological diseases.

ESCs may cause teratoma formation and are associated with many ethical concerns. Unlike ESCs, iPSCs are reprogrammed somatic cells that bypass ethical limitations. On the other hand, the risk of mutation during iPSC reprogramming can lead to tumor formation and reduced differentiation capacity, and different iPSC lines may show considerable phenotypic variation, which could hamper data integration and the use of iPSCs for CSE. Despite these advantages and disadvantages, these cell types are widely used as cell sources in CSE.

Cell sheet formation

In addition to harvesting methods to detach the cell sheet, cell sheet fabrication, owing to its cell-based nature, is artful and bothersome. Culturing ultraconfluent adherent 2D monolayer cells and converting them into 3D monolayer cell sheets is a sophisticated procedure that requires expert hands in cell culture techniques. Various culture vessels, such as common polystyrene culture dishes and multiwell plates, have been used for creating cell sheets [24]. On the basis of postdetachment shrinkage and a reduction in the diameter of the cell sheet, the culture dish diameter should be considered a vital factor in determining the final size of the cell sheet [25, 26].

Determining adequate cell density on the basis of culture dish diameter is another crucial factor. An appropriate initial seeding density leads to fewer culture incubation days and rapid confluent monolayer formation. CSE researchers reported initial seeding cell densities ranging from 6 × 10⁵ cells/cm² culture dish to 2.2 × 10⁴ cells/cm² [27, 28]. The use of natural supplements such as high concentrations of FBS, which are more than common at 10–15%, and the addition of the optimum concentration of L-ascorbic acid, which is 50 µg/ml, to culture media could lead to more rapid cell proliferation and cell sheet formation and a thicker cell sheet with robust ECM [29].

Cell sheet stacking is an easy method for creating multilayer cell sheet constructs with different layer counts and various thicknesses [18]. 3D construct fabrication with stacked cell sheets is a significant approach for tissue repair and provides vital in vitro tissue/organ models for diseases [30]. Thorp et al. utilized multilayer cell sheets to fabricate 3D hyaline-like constructs in vitro. These results confirmed that the MSC multilayer construct could increase the construct thickness and 3D cellular interactions due to in vitro chondrogenesis. The thickness increased from 14 µm in the 1-layer construct to 25 µm in the 2-layer construct, which improved cellular interactions and increased in vitro MSC chondrogenesis [31]. Cell sheet fabrication can reveal various aspects of differentiation procedures and cell behavior. Wongin-Sangphet et al. assessed the effect of chondrogenic differentiation medium (CDM) on the behavior of chondrocyte sheets. Tissue qualities were evaluated via structural analysis, mechanical testing and proteomics. They utilized time-lapse observations and bioinformatic analysis to confirm the relationship between CDM and cell migration proteins. After 48 h, CDM altered chondrocyte behavior by reducing cell migration. Compared with the basal medium, the contraction of monolayered chondrocyte sheets is affected by CDM. In response to cell sheet contraction, tissue thickness and tissue stiffness improved after 7 days. Bioinformatic analysis revealed that TGFb1 is associated with cell migration and cartilage functions. The involvement of MAPK signaling in cell migration was confirmed in both types of chondrocyte sheet cultures. CDM supplemented with TGFb1 might trigger cartilage protein production via the TGFb pathway and control cell migration via the MAPK signaling pathway. Cell behavior and protein expression are vital for the development of engineered cartilage [32]. Scientific attempts to create engineered tissues with the greatest resemblance to the structure and morphology of native tissue are still ongoing. Although the cell sheet serves as a tissue-like structure and shows significant cellular and molecular features, a desirable cell morphology after cell sheet detachment has not yet been reached.

Cell sheet harvesting strategies

Cell sheet harvesting allows the generation of viable and transplanted cells for various tissue engineering applications. Thermoresponsive systems have been widely utilized for harvesting cell sheets. Other effective methods have been described to reduce the duration of cell sheet production. When they reach a proper confluency level, the cells adhere together and form a confluent monolayer on the culture dish surface, creating a tight bond between them. Since adhesion proteins are destroyed by enzymes such as trypsin, resulting in single-cell formation, this approach is not appropriate for cell detachment. Therefore, the selective loss of adhesion between the cells and the culture dish surface led to detachment of the cells as a cell sheet. Even though polymers with temperature-responsive technology have led to the creation and expansion of cell sheet technology, additional techniques have also been described to produce cell sheets. In this review, cell sheet harvesting methods are classified into the following categories: responsive methods, surface-modified methods, and strategies that do not use surface modifications, such as enzymatic and mechanical harvesting (Fig. 2). Each one has its own particular advantages and may also have suitable applications.

Fig. 2.

Schematic diagram of various cell sheet detachment methods. (a) In mechanical harvesting systems, the cell sheet can be easily peeled off with a cell scraper. (b) In temperature–responsive systems, by altering the temperature due to changes in the hydrophobicity of PIPAAm, the cell sheet can be detached within 1 h. (c) In enzymatic treatment systems, Dispase can selectively digest the ECM, which causes cell sheet detachment. (d) In photoresponsive systems, UV radiation stimulates the detachment of a cell sheet, which is cultured in Ti2O-coated dishes. (e) In magnetic-responsive systems, cells treated with magnetic nanoparticles are cultured in ultralow-attachment culture dishes completely detached by magnetic force. (f) In electroresponsive systems, electrical stimulation of cell sheets cultured in gold- and RGD-coated culture dishes leads to cell sheet detachment. (g) In reactive oxygen species (ROS)-responsive systems, exogenous ROS products from the Hp-PK films induce cell sheet detachment and easy transfer with the fibrin gel. (h) In pH-responsive systems, pH reduction results in complete cell sheet fabrication.

Mechanical harvesting system

Mechanical peeling of cell sheets preserves the ECM and is cost-effective, making it a practical alternative harvesting method. Zhou et al. reported this straightforward method for harvesting without the need for unique materials in 2007. Owing to their potential in regenerative medicine, mechanical systems allow the preparation of cell sheets without any specific culture substrates or techniques [33]. The scraping method, which is an efficient and simple technique, uses a cell scraper or pipette tip to collect cell sheets from the bottom of a culture dish. Once the cell sheet is developed and a confluent monolayer sheet is folded on the edge, it can be easily and quickly peeled off and lifted with tweezers. In this process, there is no need to reduce the temperature [34]. Another promising approach involves the use of a cell sheet osteogenic medium containing gelatin, which leads to more proliferative cells. These cell sheets, with their robust ECMs, result in thicker sheets that are easily collected [35]. This strategy has already been successfully applied in bone and cartilage regeneration, demonstrating its potential and inspiring optimism in the field of tissue engineering. Chen et al. favorably harvested MSC sheets approximately 2 cm in diameter by gently peeling the cell layer on a glass cover for full-thickness autologous skin graft hMSC cell sheets (HCSs), and prevascularized hMSC cell sheets (PHCSs) were cultured in vitro and implanted in a rat full-thickness skin wound model. Their results revealed that HCSs and PHCSs remarkably reduce skin contraction and improve cosmetic appearance [19]. Unlike other harvesting methods, this method does not require the addition of any substances. It provides high biosafety and cost-effectiveness. Importantly, however, culture manipulation may be complex, and there may be limitations in terms of scalability and repeatability (Fig. 2a) (Table 1).

Table 1.

A comparative summary of the main harvesting methods

Harvesting system	Advantages	Disadvantages
Temperature-responsive	• Commercially available • Highly effective cell detachment • Multilayer cell sheets preparation • In vivo tested biocompatibility and feasibility • In vivo and clinical trials applications • Coculture cell sheet detachment	• Highly cost method • Complicated and time-consuming grafting method • Special equipment like electron beam is required for PIPAAm grafting • Adverse effect of temperature changes on cell cycle, metabolism, and cell viability • Commercial TRCD are much more expensive than common polystyrene dishes • Various detachment time based on cell types
Mechanical peeling	• Cost-effective • Easy and accessible • In vivo tested biocompatibility and feasibility • Suitable for various cell types	• Slight cell‒cell connection and ECM disruption • Mechanical stress may affect cell viability • Mechanical detachment may tear fragile sheet • Cell culture expert scientist required
Enzymatic detachment	• Easy and accessible • Mostly suitable for fibroblasts and keratinocytes	• Higher cell‒cell connection and ECM disruption • Dispase and collagenase may reduce cell viability • Needs more time to completely detached sheets

Temperature–responsive systems

Thermoresponsive systems have been widely used and have dramatically accelerated the development of cell sheet technology. Poly(N-isopropyl acrylamide) (PIPAAm) has yet to be the most studied temperature-sensitive polymer. The PIPAAm lower critical solution temperature (LCST) is 32 °C, which means that PIPAAm shows hydrophobic characteristics at > 32 °C, whereas it changes to a hydrophilic form at temperatures below 32 °C. Temperature-responsive PIPAAm is covalently bonded to the surface of a nanometer-thick culture dish. Owing to the accelerated hydration and swelling of the grafted PIPAAm, the PIPAAm coating allows common cell culture at an average temperature of 37 °C. However, the cultured cells detached from the surface at 32 °C and below. This expedites the spontaneous detachment of the cell sheets from the culture dish surface within a one-hour time span. In aqueous solutions, this polymer undergoes reversible and discontinuous state alteration with a lower critical solution temperature (LCST) of 32 °C. Above the LCST, intra- and intermolecular hydrophobic interactions are enhanced among the isopropyl groups of PIPAAm, releasing H₂O molecules through the hydrated coil-to-collapse transition [36, 37]. This process allows cell adherence and proliferation on a PIPAAm-immobilized surface. In contrast, below the LCST, hydrogen bonding between H₂O molecules and the hydrophilic parts (amide groups) of PIPAAm is more dominant, which leads to quick detachment of cell sheets with diverse undamaged cell‒cell junctions and cell‒secreted ECM connections [25, 38, 39]. Nakao et al. focused on the phenotypic traits of human umbilical cord MSC (hUC-MSC) sheets structurally and functionally to determine the regenerative benefits of cell sheets. They reported that cellular cleavage of proteins such as vinculin, fibronectin, laminin, integrin β-1, and connexin 43 and increased apoptotic cell death occurred under the routine protocol of trypsin detachment. However, MSC sheets fabricated without trypsin via a temperature-responsive harvesting system presented intact cellular structures. Moreover, MSCs harvested via enzymatic treatment presented higher phosphorylated Yes-associated protein (pYAP) expression than MSC sheets. They also verified that the stability of cellular structures such as the ECM, cell‒cell junctions, and cell‒ECM junctions was crucial for maintaining MSC viability after detachment from culture dish surfaces [39]. Recently, Thorp et al. reported that MSC chondrogenic differentiated cell sheets cultured in thermoresponsive culture dishes developed transplantable hyaline‑like cartilage constructs. These findings support the in vitro chondrogenic differentiation of 3D MSC sheets to hyaline-like cartilage by postcontraction cytoskeletal reorganization and structural transformations. This study also revealed that the initial thickness and density of 3D cell sheets could affect MSC‑derived chondrocyte hypertrophy in vitro. Importantly, differentiated chondrogenic cell sheets attach directly to the cartilage surface, retaining adhesion molecules while maintaining their unique characteristics [40]. The main drawback of the temperature-responsive system is the long-term detachment duration. The production of cell sheets requires at least 30 min at 20 °C for the PIPAAm-coated surface, which may affect cell function and the survival rate postengraftment. To shorten the time of detachment, some researchers have modified PIPAAm engraftment. Unlike electron beam polymerization, which is utilized on tissue culture polystyrene surfaces (TCPS) or plasma polymerization, an alternative strategy to transplant PIPAAm is ultraviolet (UV) irradiation and immobilization on polydimethylsiloxane surfaces, which shorten the detachment time from 1–2 h to 30 min [39, 41]. Yang et al. created PNIPAM copolymer films on the surfaces of glass slides or silicon wafers via a two-step film-forming method including coating and grafting. These methods improved the limited stability and reduced the reusability of conventional PNIPAM-based substrates. BM-MSCs were cultured on PNIPAM copolymer films formed with various copolymer solution concentrations. The optimal culture substrate was selected on the basis of cell growth at 37 °C and effective cell detachment through temperature reduction. The findings confirmed that the copolymer films derived from the 1 mg·ml⁻¹ solution were the optimal culture substrates for BMMSCs. This study introduces a stable and efficient approach for stem cell sheet culture and harvesting [42].

Recently, the development of novel temperature–responsive strategies to facilitate cell detachment and differentiation was reported. García-Sobrino et al. activated p(vcl-co-hema) thermosensitive hydrogels with icariin-loaded nanoparticles to create osteoblastic cell sheet harvesting platforms. They used supercritical CO₂-SAS technology for the encapsulation of icariin (a small molecule involved in osteoblastic differentiation). The thermosensitive hydrogels’ cell compatibility, transplant efficiency, and bone differentiation capacity were tested and approved. Harvested osteoblastic sheets via this technique are rich in collagen type I and ECM. These findings suggest a novel cell sheet-based therapy for bone regeneration utilizing NP-activated pVCL-based cell platforms [43] (Fig. 2b) (Table 1).

Enzymatic treatment systems

The first strategy utilized to manufacture cell sheets was enzymatic treatment. Autologous human epidermal cells are used to form epithelial skin grafts in vitro and prevent skin injuries such as burns. When the seeded cells reached confluence, the cell sheet was detached from the culture dish surface by incubating the cells with 1% dispase for 15 min at 37 °C, and a cell scraper was used to lift the cell sheet [44]. The viability ratio of dispase-treated cells after transplantation is significantly lower than that of cell sheets obtained from temperature-responsive culture dishes because the ECM is lost following digestion. A dispase-based strategy is used to manufacture epithelial cell sheets, particularly for making epithelium sheets. Fundamental studies on dispase-treated cell sheets have been conducted [45]. This method does not involve any other physiological stimulation process, allowing the cell sheet to be detached. There are also reports on other enzymes, such as collagenase, as another method involving the use of enzymes. For example, dispase and collagenase-treated oral mucosal epithelial cell sheets have been reported previously to be efficient ex vivo [46] (Fig. 2c) (Table 1).

Photo-responsive systems

The potential of light as a stimulus for responsive surfaces is a fascinating area of research. It can uniquely alter the wettability of hydrophilic and hydrophobic surfaces, making it an ideal candidate for controlling cell adhesion. The main photolabile substances for this method are metal oxides, particularly zinc oxide and titanium dioxide [47]. Zinc oxide (ZnO) is an economically and chemically available material that possesses high redox potential, whereas titanium dioxide (TiO₂) has been widely studied for its stable structure, nontoxic nature, anticorrosion characteristics, and excellent photocatalytic performance. In fact, the combination of TiO₂ with ZnO nanoparticles results in photocatalytic activity due to a favorable band position that produces holes and electrons when stimulated by UV light, ultimately leading to the formation of hydroxyl radicals and reactive oxygen species [48].

Several innovative methods for cell sheet detachment via light-responsive systems have been reported. Some methods regulate cell adhesion directly, whereas others use photothermal effects to separate the adhesion protein layer from the culture surface. Direct methods use photoresponsive materials, such as wettability or electrical charge, which can be managed by light stimuli, to harvest cell sheets covering the surface of the culture dish [49, 50]. Metal oxides such as zinc oxide and titanium dioxide have been examined for their ability to affect wettability as a result of UV radiation exposure. UV or visible light exposure can control the surface electrical charge, and cell sheets can be detached as a result of changes in the electrical charge. This is because electrostatic repulsion among the cell sheet and the surface of the culture occurs when the electrical charge switches from negative to positive. Therefore, UV exposure may influence cell functions, a potential concern that should be carefully considered in the development of these systems (Fig. 2d).

Magnetic-responsive systems

Magnetic nanoparticles have also been used for cell sheet detachment. Cells labeled with magnetic nanoparticles are seeded on an ultralow-attachment dish culture surface. After reaching confluence, a magnet was placed beneath the culture dish to detach the cell sheets. After some time, the magnet was removed, and the cell sheet was harvested from the ultralow-attachment culture surface. For the first time, Ito et al. introduced a magnetic-responsive system for fabricating cell sheets [51]. They detached keratinocytes with magnetic force, where magnetite-labeled keratinocytes were cultured on ultralow attachment plates with covalent hydrophilic bonds and harvested by magnetic force. Magnetic nanoparticles have been used to fabricate and handle cell sheets of keratinocytes, cardiomyocytes, hepatocytes, endothelial cells, MSCs, and retinal pigment epithelial cells without causing cell toxicity [51]. Goncalves et al. proposed the development of magnetically responsive tenogenic patches on the basis of magnetically harvested cell sheets that allow remote control of tendon-mimicking constructs. They bioengineered tendon patches via magnetic cell sheet construction with magnetic responsiveness, good mechanoelastic properties, and a tenogenic-prone stem cell population [52]. Therefore, functional cell sheet therapies represent a possible strategy for tendon regeneration. This method has great potential for tissue engineering, as it offers a noninvasive and effective way to engineer and control the growth of cell sheets. With this external force, it is easy to regulate the thickness of the cell sheets fabricated on the culture dish surface by controlling the number of seeded cells. In other approaches, cell sheets are stacked to fabricate 3D tissues; therefore, the ability to manage the thickness of the fabricated cell sheets should benefit from this method. Although magnetic nanoparticles have been added to culture media and show good biocompatibility, they may have adverse effects on cell viability and adhesion. In addition, in vivo studies conducted on several cell types have confirmed the ability of this approach to be used for regenerative medicine [47] (Fig. 2e).

Electroresponsive systems

Another common strategy for cell sheet engineering is an electroresponsive system that allows the attachment and release of cells when triggered by an electrical impulse. For example, Mrksich and colleagues tethered electroactive molecules to electroactive self-assembled monolayers of gold (Au) for oxidation by applying an electrical potential. By determining a peptide ligand that intercedes cell attachment, the system may be electrically operated to allow adhesion or detachment of the cells [47]. The main element of this system is a self-assembled monolayer (SAM) of alkanethiols connected to gold to immobilize peptide ligands consisting of Arg-Gly-Asp (RGD) as a binding site for cell adhesive molecules. The immobilized ligands are constructed to adapt to different cell types to adhere. The monomer is oxidized, rapidly releasing the immobilized ligands when a negative electrical potential is applied to the gold film. The detachment of the cell sheets from these surfaces was achieved by applying − 1.0 V, and the cells became entirely detached within 10 min, which was faster than that of temperature-responsive surfaces [53, 54]. Another example is the polyelectrolyte-modified surface. As a result of electrostatic interactions, polyelectrolytes are adsorbed to oppositely charged surfaces and desiccated from conductive substrates when subjected to electrochemical polarization [53]. Although an electroresponsive system can detach cell sheets faster than the temperature–responsive method can, the electric potential may affect cell function and viability, and designing immobilizing ligands for different cell types may limit their extensive use (Fig. 2f).

Reactive oxygen species (ROS)-responsive systems

As natural products of cellular oxidative metabolism, reactive oxygen species (ROS) play a significant role in regulating cell survival, cell death, differentiation, cell signaling, and inflammation-related factor production. ROS not only regulate cell adhesion but also have the potential to cause cell detachment at relatively high intracellular levels, opening intriguing possibilities for their application [55]. The Möhwald group used light to release fibroblasts cultured on gold nanoparticle-based surfaces. The surface was irradiated with a green laser, producing extracellular ROS. ROS damage the cell membrane at the surface–cell interface and lead to detachment [56]. The cells disintegrated completely within 24 h, but there was a possibility to recover the surface within 72 h and allow them to reintegrate into irradiated areas. Min-Ah Koo et al. reported that ROS have the potential to directly deliver intact cell sheets to desirable sites. ROS responsiveness is based on the ability of hematoporphyrin-incorporated polyketone films (Hp-PK films) to transfer cell sheets directly to the target site. After green light-emitting diode (LED) (510 nm) irradiation, exogenous ROS products from the Hp-PK films induce cell sheet detachment and are easily carried with the fibrin gel. This study indicated that the ROS-induced detachment of cell sheets cultured on the Hp-PK film was closely related to conformational alterations in the ECM proteins [57]. This method involves different cell types and even in vivo transplantation. The advantage of using ROS is the ability to easily manage the extracellular spatiotemporal detachment of cells. These techniques hold promise for effective cell transplantation for tissue regeneration and reconstruction [55, 56] (Fig. 2g).

pH-responsive systems

pH-responsive systems are challenging to use in cell-based applications because of the limited pH range (6.8–7.4) for normal cell function. Ehrbar et al. demonstrated the feasibility of controlling cell sheet detachment via local or global pH drops [58]. The pH-responsive substrates are constructed by alternate layering of cationic poly(allylamine hydrochloride) (PAH) layers and anionic poly(styrene sulfonate) (PSS) layers. An electrical activator with a current density of 30 µA/cm² can detach the cells from their intact ECM within 10–20 min. Ehrbar and colleagues suggested that a reduction in localized pH might lead to cell sheet detachment at the cell–substrate interface, contrary to the application of an electrical trigger. Therefore, cells can be detached by decreasing the culture media pH to remove the cell sheets. Cell adhesion did not change between pH 5.0 and 7.4, whereas pH 4.0 resulted in thorough cell separation in 2–3 min [47, 55]. Notably, the pH-responsive system cannot avoid damaging cells, which are sensitive to pH changes (Fig. 2h).

Comparison of the cell sheet harvesting systems

Harvesting strategies such as electroresponsive, photoresponsive, and pH responsive methods are inexpensive procedures, but they are not frequently utilized after their establishment. Few studies have been conducted using these strategies both in vitro and in vivo. Electroresponsive systems require electrochemical dissolution of polyelectrolyte coatings, leading to local pH changes, which may be harmful to sensitive cells, and properly immobilized ligands should be designed for different cell types. The potential of photoresponsive systems for the detachment of various cell types should be evaluated carefully, and the adverse effects of UV radiation on cells must be considered. A pH-responsive system may result in incomplete detachment in some types of cells. The pH range is limited to 6.8–7.4 because the normal function of reducing the cell pH can alter cell viability and normal cellular function. In addition to inducing pH reduction, cultured cells can cause pH changes, which may hinder cell sheet formation and detachment. These points should be considered when designing experimental studies [18, 31].

Among these diverse harvesting strategies, mechanical harvesting and temperature-responsive methods are the two strategies most utilized by CSE researchers. Both have advantages and disadvantages. A comparison of the mechanical harvesting of cell sheets with that of the temperature-responsive method reveals several key drawbacks of the mechanical harvesting strategy, which is vital to consider. Cell sheets are very thin and fragile, so mechanical harvesting of the sheets and postdetachment handling can be very difficult and can cause tearing or other damage. Mechanical scraping may cause damage, such as disruption of cell‒cell junctions and ECM damage to thin, delicate cell sheets. Mechanical stress can impair essential cell surface proteins, which are critical for cell signaling, adhesion, interaction with surrounding tissues, posttransplant tissue integrity and cell sheet function. This damage could lead to a reduction in cell viability and may compromise the functionality of the harvested cell sheets. Mechanical harvesting may increase the risk of contamination, which is a significant concern in tissue engineering products.

Temperature-responsive methods allow detachment of intact cell sheets without the need for harsh enzymatic or mechanical treatments. This strategy preserves cell‒cell junctions, the ECM, and cell surface proteins, resulting in increased cell viability and functionality. By simply lowering the temperature, the cell sheets detach spontaneously, which minimizes physical stress and damage. The gentle nature of this method reduces contamination risk. Temperature-responsive harvesting is a precise approach that preserves the integrity of cell sheets, whereas mechanical harvesting causes more damage. Although temperature-responsive harvesting offers significant advantages, a breakdown of the disadvantages associated with this method are as follows. Creating temperature-responsive culture surfaces, particularly those using poly(N-isopropylacrylamide) (PIPAAm) polymers, involves complex and costly procedures. Procedures include polymerization and immobilization on culture surfaces and long-term stability of the polymer coating. The state-of-the-art materials and equipment required may limit the accessibility of this technique. Although temperature-responsive culture dishes are commercially available, they seem very expensive. Precise temperature control is crucial for cell sheets. Deviations from the optimal temperature range may have a negative impact on cell detachment and viability, and the temperature transition is potentially stressful for cells. Although this method is gentler than mechanical methods are used, rapid or extreme temperature changes may still lead to stress or damage to cells. Since all cell types do not respond equally to temperature-responsive detachment, some cells may require optimization of their surface properties or temperature conditions. The cost and complexity of substrate preparation are also important factors to be considered.

These two strategies are frequently used in CSE, but there is a large scientific research gap in the comparison of these methods in terms of cell viability, postdetachment adherent properties, and ECM content at the same time. The information provided by future research on this matter may be very constructive for determining which strategy is more convenient.

Overall, among these various detachment systems, mechanical harvesting stands out as one of the most conventional and widely used methods in bone and cartilage regeneration. Its high level of biosafety, economic availability, surface modification independence, and accessibility make it a reliable choice for researchers and professionals in the field.

Optimization of mechanical harvesting for the generation of functional osteogenic cell sheets

Although regeneration of bone defects through cell suspensions and the incorporation of MSCs with various materials have yielded satisfactory results, the biocompatibility and immunogenicity of biomaterials, low cell density, and survival rate are significant challenges for biomaterials. Therefore, cell sheet engineering with remarkably efficient cell delivery represents a compelling approach for innovation and a revolution in bone defect regeneration. The ability of MSCs to differentiate into osteogenic and chondrogenic cells makes them an ideal cell source for engineering cell sheets to regenerate bone and cartilage. Numerous studies have employed MSC sheets to assess in vitro osteogenesis and in vivo bone defect regeneration. Having introduced mechanical harvesting as a general approach, we now discuss its specific optimization and applications in bone and cartilage tissue engineering. Modification of culture media with supplementary materials such as L-ascorbic acid and gelatin significantly affects cell sheet formation and lineage differentiation, further increasing the potential of this technique. Adding gelatin to osteogenic media can increase osteogenic cell sheet formation, sheet thickness, the expression of osteogenic gene markers, and the number of calcium deposits [35]. Guo et al. made a significant contribution to the field by modifying conventional culture media with different concentrations of vitamin C (0 µg/mL, 10 µg/mL, 20 µg/mL, and 50 µg/mL). They suggested that 50 µg/mL vitamin C was the optimal concentration and resulted in the secretion of large amounts of collagen in MSCs. Vitamin C shortens the formation time of the cell-sheet layer, providing valuable insights into the potential of vitamin C in cell sheet engineering [29]. Nakamura et al. demonstrated enhanced bone union resulting from mesenchymal cell sheet transplantation. They cultured rat bone marrow cells to fabricate a cell sheet with the ability to be scraped off as a monolayer. These cell sheets were then grafted onto fractured femurs without using a scaffold. Radiological and histological analysis revealed callus formation near the fracture site in the cell sheet-grafted group, indicating a positive impact on bone union. In contrast, the control group (without a cell sheet graft) demonstrated nonuniformity in terms of femur fracture. These results strongly suggested that the femur fracture in the rat model was completely regenerated by the cell sheet graft fabricated by mechanical harvesting of the cell sheets, demonstrating the potential of this technique for bone regeneration in preclinical settings [17]. Tatsuhiro et al. developed a scaffold-free tissue using human dental pulp stem cells (hDPSCs). After mechanical scraping, the hDPSCs were cultured for four weeks to prepare basal sheets, followed by a 1-week culture to obtain a 3D construct, with or without osteogenic induction. Compared with those in the control group, the expression levels of osteogenic genes in the hDPSC constructs were substantially upregulated. Moreover, the hDPSC constructs with osteogenic induction resulted in greater calcified matrix formation and higher expression levels of osteogenic genes than did the hDPSC constructs without osteogenic induction. These results suggest that hDPSC constructs with osteogenic induction hold promise as scaffold-free constructs for bone regeneration, suggesting optimism for the future of tissue engineering in this field [59]. In conclusion, the mechanical peeling of cell sheets as a harvesting method is an excellent and accessible way to regenerate bone via cell sheet engineering (Table 2).

Table 2.

Summary of cell sheet engineering research using mechanical harvesting in the bone and cartilage fields

Cell source	Fabrication method	Strategy	Stage of study	Application	Outcomes	Year, References
MSCs	Mechanical	Culture media modified with L-Ascorbic acid	In vitro	MSCs undifferentiated sheet	L-Ascorbic acid promote collagen secretion	2015,[29]
Chondrocytes	Mechanical	Wrapped around a silicon tube	In vitro	Cartilage	Type II collagen and glycosaminoglycan content increased	2010,[60]
Chondrocytes	Mechanical	BMSC engineered cartilagea (BEC) composed of polyglycolic acid b(PGA) polylactic acidc (PLA) wrapped with chondrocyte sheets and acellular small intestinal submucosa	In vivo	Cartilage	Chondrocyte sheet could create a chondrogenic niche to retain chondrogenic phenotype	2017,[61]
Chondrocytes	Mechanical	Released cell sheets were cultured in vitro 12 days with chondrogenic media to form gelatinous chondroid mass	In vivo	Cartilage	ECM formation and high expression of type II collagen was observed	2015,[62]
Chondrocytes	Mechanical	Decellularized cell sheet	In vivo	Cartilage	Preserved the integrity and bioactivity, appropriate for osteochondral regeneration	2018,[63]
MSCs	Mechanical	MSC sheets wrapped around MSC-loaded bilayer poly- (lactic-co-glycolic acid) (PLGA)d scaffolds	In vivo	Cartilage	The optimal integration between the repaired cartilage and surrounding normal cartilage and subchondral bone	2014,[73]
MSCs	Mechanical	Decellularized cell sheet	In vivo	Osteochondral	Promoted the osteogenic and chondrogenic differentiation potential of BMSCs under differentiative conditions	2020,[64]
MSCs	Mechanical	Sheets were wrapped on demineralized bone or frozen tendon grafts	In vitro	Bone	MSCs sheets can maintain their differentiation potential within particular scaffolds	2006,[65]
MSCs	Mechanical	Assembled with tubular coral scaffolds	In vivo	Bone	The new formed bone has a woven bone matrix and fully mineralized compact bone and it was similar to endochondral osteogenesis	2009,[66]
MSCs	Mechanical	The MSC sheets were wrapped around preseeded polycaprolactone–calcium phosphate e(mPCL–CaP) scaffolds	In vivo	Bone	Large bone tissues are similar to native bone, can be regenerated utilizing BMSC sheets with scaffolds	2007,[33]
MSCs	Mechanical	MSC wrapped around allograft structures	In vivo	Bone	The MSC sheet increased the repopulation of the bone graft after implantation and thicker cortical bone formation	2009,[67]
MSCs	Mechanical	(rhBMP-2)f -loaded calcium sulfateg (CS) combined with MSCs sheets	In vivo	Bone	Showed higher scores by X‑ray analysis and more bone formation	2012,[68]
MSCs	Mechanical	Simvastatin-loaded calcium sulfate CS with MSC sheet	In vivo	Bone	Complete bone union	2013,[69]
MSCs	Mechanical	Demineralized bone matrix wrapped with cell sheet	In vivo	Bone	Promote bone bonding at the bone-implant interface by providing cells and ECM	2010,[70]
MSCs	Mechanical	Multilayered MSC sheets wrapped around two types of implant surface‑modified titanium and zirconia	In vivo	Bone	New bone tissue formed around the implants by endochondral pathway	2010,[71]
MSCs	Mechanical	Titanium implants wrapped with MSC sheets	In vivo	Bone	Development of MSC implants with osteogenic and vascularization properties	2011,[72]
MSCs	Mechanical	Culture media modified with gelatin	In vitro	Bone	Gelatin enhances osteogenic differentiation	2017,[35]
MSCs	Mechanical	Cell sheet transplantation	In vivo	Bone	Enhanced bone union	2010,[17]
hDPSCsh	Mechanical	Obtain a three-dimensional construct from the cell sheet	In vitro	Bone	Enhanced calcified matrix formation and higher bone-related gene expression levels	2018,[59]

^aBMSC engineered cartilage (BEC)

^bpolyglycolic acid (PGA)

^cpolylactic acid (PLA)

^dpoly-lactic-co-glycolic acid (PLGA)

^epolycaprolactone–calcium phosphate (mPCL–CaP)

^fRecombinant Human Bone Morphogenetic Protein-2

^gCalcium sulfate (CS)

^hHuman Dental Pulp Stem Cell

Mechanical harvesting of chondrogenic cell sheets for cartilage regeneration

Owing to the restricted ability of cartilage to heal itself on the basis of its avascular form, injured cartilage often leads to untreated deterioration. Tissue engineering has been shown to be a promising strategy for the regeneration of cartilage defects. The fabrication of chondrogenic cell sheets is a therapeutic strategy that can regenerate cartilage defects. Cell sheets can provide an extracellular matrix, large numbers of cells, and adhesion molecules on the cell surface, as well as cell‒cell interactions, which may provide consistency between the implanted cell sheet and host cartilage, leading to integrative cartilage regeneration. Although temperature-responsive harvesting is the most conventional method for constructing a chondrogenic sheet, mechanical peeling is considered an alternative method. Unlike MSCs, the conventional cell source for cell sheet engineering, chondrocytes are a typical somatic cell source for chondrogenic cell sheet fabrication. The use of engineered cartilage tissue may be useful for treating many cartilage diseases. Combining bioscaffolds and chondrocyte sheets has yielded significant outcomes in cartilage tissue engineering. Tani et al. demonstrated a novel strategy to construct scaffold-free cartilage tissue in cylindrical form in vitro to develop functionally engineered tracheas. The auricular chondrocytes, which were carefully isolated from New Zealand white rabbits, were cultured at high density to generate a chondrocyte sheet. These sheets were peeled off, wrapped around a silicon tube, and cultured under static or dynamic conditions for six weeks. The cylindrical cartilage was meticulously assessed histologically and macroscopically. The collagen expression level, amount of glycosaminoglycans, and mechanical properties were defined precisely. The cylindrical cartilaginous matrix was stained completely with Safranin-O as a result of type II collagen expression. The amount of glycosaminoglycan increased after six weeks of culture. This novel method, developed via sheet-based tissue engineering techniques, fabricates engineered cartilage that maintains its shape, rigidity, and flexibility under in vitro conditions [60]. Li et al. investigated the ability of a chondrocyte sheet to create a chondrogenic niche that retains the chondrogenic phenotype in subcutaneous environments. Porcine bone marrow mesenchymal stem cells (BMSCs) were seeded into biodegradable scaffolds and cultured in chondrogenic media for four weeks in vitro. These were wrapped with chondrocyte sheets (Sheet group), acellular small intestinal submucosa (SIS group), or left unwrapped (Blank group). They were subsequently implanted subcutaneously into nude mice to maintain the cartilaginous phenotype. The results demonstrated that all the constructs in the Sheet group presented typical cartilage features, such as numerous lacunae and deposition of the extracellular matrix. Cell labeling confirmed the direct involvement of the implanted construct in cartilage formation in the Sheet group. In contrast, samples in both the SIS and blank groups presented bone formation, with minimal formation of fibrous cartilage in a few samples. These findings suggested that the chondrocyte sheet could effectively create a chondrogenic niche to retain the cartilaginous phenotype under subcutaneous conditions [61]. Zhou et al. employed chondrocyte cell sheets in both in vitro and in vivo studies, and the initial formation of the ECM and type II collagen expression were observed in the cell sheets during in vitro culture. Eight weeks after implantation into nude mice, mature cartilage discs were successfully harvested. These neocartilage samples exhibited robust ECM formation, and significant expression of type II collagen was observed surrounding the evenly distributed chondrocytes. The primary ECM components, glycosaminoglycans, and hydroxyproline of neocartilage are similar to those of native human costal cartilage [62]. These studies provide a novel approach for stable ectopic cartilage regeneration utilizing chondrocytes and engineered monolayer and bilayer chondrocyte cell sheets. These sheets were harvested with a cell scraper via a simple and low-cost technique instead of temperature-responsive culture dishes (Table 1).

Cell sheet engineering for osteochondral defect repair: a promising approach

Recent investigations have revealed the significant potential of decellularized cell sheets as biological scaffolds for osteochondral regeneration. These sheets have shown the ability to enhance cartilage and subchondral bone formation in critical-sized osteochondral defects, suggesting optimism for the future of tissue engineering in this field. Wang et al. utilized chondrocyte sheets to facilitate osteochondral regeneration. They isolated chondrocytes from the auricular cartilage of New Zealand rabbits for cell sheet fabrication through gentle peeling and subsequent decellularization via sodium dodecyl sulfate (SDS) at different concentrations, Triton X-100, and deoxyribonuclease enzyme solution. Implantation of decellularized chondrocyte sheets in osteochondral defects in rabbits resulted in significant host remodeling and variant regeneration of osteochondral tissues. These findings suggested that the decellularized chondrocyte sheets effectively preserved structural integrity and biological activity, which was appropriate for osteochondral regeneration in the knee joints of rabbits and offered a promising method for articular cartilage regeneration without cell transplantation [63]. Moreover, Wang et al. investigated the regenerative effects of decellularized bone marrow stromal cell (BMSC) sheets in vivo. These sheets were fabricated on the basis of a previously described methodology and implanted into osteochondral defects in rabbits. The results demonstrated that decellularized cell sheets promoted the osteogenic differentiation potential of BMSCs under osteogenic conditions, enhanced the chondrogenic differentiation potential of BMSCs under chondrogenic conditions and significantly improved osteochondral defect regeneration in rabbits [64]. Although decellularized cell sheet approaches may reconstruct osteochondral tissue by retaining natural ECM as a bioscaffold, other strategies, such as multilayer differentiated cell sheet-based structures and cell sheets combined with scaffolds, may be considered novel methods for osteochondral critical-sized defect regeneration (Table 1).

Integrated cell sheet and biomaterial approaches for enhanced bone and cartilage regeneration

Combining different approaches, such as cell sheet engineering and biomaterials, is suggested to significantly improve tissue engineering outcomes (Fig. 3). Common polystyrene culture dishes have been used to fabricate cell sheets via the mechanical harvesting method with a scraper instead of thermoresponsive culture dishes. They combined cell sheets with diverse biomaterials and scaffolds. The combination of mechanically detached cell sheets and scaffolds offers several advantages in tissue engineering. In contrast with traditional cell seeding methods, in which cells are randomly distributed within a scaffold, cell sheets provide a preorganized layer of cells with their native ECM and promote cell‒cell interactions and functional tissue formation. Scaffolds provide structural support and allow the cell sheet to maintain shape and integrate with the surrounding tissue, which is particularly important for tissues that require mechanical strength, such as cartilage and bone. Engineered scaffolds release growth factors in a controlled manner, stimulating cell proliferation, differentiation, and tissue regeneration, which may enhance the healing process and improve tissue function. Combining cell sheets and scaffolds allows the fabrication of tissue constructs that more closely mimic the architecture of native tissue and leads to enhanced biocompatibility and integration with the host tissue. Overall, this approach enables the development of biocompatible and functional tissue constructs for a wide range of applications.

Fig. 3.

The combination of diverse biomaterials, scaffolds, demineralized bone grafts and implants is an effective approach.

The feasibility of producing MSC sheets for the reconstitution of frozen grafts explored by Ouyang et al. has significant implications for the field of regenerative medicine. hMSC sheets were cultured on demineralized bone grafts or frozen tendon grafts via a wrapping method for three weeks. The ability of the MSC matrix resembling the in situ periosteum to differentiate into osteochondral lineages when mixed with the demineralized bone matrix was demonstrated. When combined with a frozen tendon graft, the MSC sheet was integrated within the tissue sheath (peritenon) around the tendon and adopted the spindle-shaped morphology of tenocyte‑like cells. This study provides valuable insights into the potential for in situ‑specific differentiation of MSC sheets, paving the way for future research and clinical applications. [65]. Gao et al. assembled multilayered BMSC sheets with tubular coral scaffolds for long bone regeneration in rabbits. Cortical bone, resembling the shape and structure of native bones, was observed. New bone formation follows an endochondral ossification process, with the bone matrix subsequently developing into completely mineralized cortical bone [66]. Zhou et al. engineered structural and functional bone grafts by assembling multilayered porcine BMSC sheets with fully interconnected polycaprolactone‒calcium phosphate scaffolds. Two sets of cell-sheet scaffold/cell constructs were transplanted under the skin of nude rats in vivo. The first constructs were assembled in BMSC sheets and cultured for eight weeks before implantation. The second set of constructs was implanted immediately after assembly with the BMSC sheets. In both groups, neocortical and well-vascularized cancellous bone formed within the constructs. Bone formation predominantly follows an endochondral pathway with the primary bone matrix, eventually evolving into complete cortical BMD, showing histological features similar to those of native bone [33]. Zou et al. demonstrated that wrapping BMSC sheets onto allograft structures enhanced bone graft repopulation in nude mice and promoted greater cortical bone formation and more efficient graft-to-bone end fusion in rabbits with segmental bone defects [67]. Qi et al. investigated the effects of human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium sulfate (CS) combined with MSC sheets for repairing ulnar segmental defects in rabbits. Compared with the other groups, the defects treated with MSC sheet/rhBMP-2-loaded CS presented significantly greater bone formation, as evidenced by X-ray analysis, histology, and microcomputed tomography, than the other groups did 4 and 8 weeks later. This positive outcome underscores the significant role of the MSC sheet in bone regeneration [68]. Furthermore, Qi et al. explored the effects of simvastatin on fracture healing via the local application of CS/MSC sheets in a rat tibia model. Complete bone union was achieved in the CS/simvastatin/MSC sheet group at eight weeks. On the other hand, in the other groups, only new bone tissue exhibited incomplete gap bridging or nonunion [69]. Ma et al. investigated the capacity of BMSC sheets to repair critical-sized calvarial defects in rabbits. The bone defects treated with demineralized bone matrix and cell sheets presented the most significant bone formation at 6 and 12 weeks postimplantation. This study suggested that MSC sheets at the interface between bone and implants offer therapeutic opportunities for bone defect repair [70]. Zhou et al. assembled multilayered rabbit BMSC sheets with two types of implants (surface‑modified titanium and zirconia) to construct an MSC sheet-implant complex. After being cultured in osteogenic medium and subsequently implanted, both complexes demonstrated promising results. New bone tissue formed around the implants, following predominantly an endochondral pathway for the MSC sheet‒titanium implant complex. The MSC sheet–zirconia implant complex exhibited typical osteocytes embedded in a dense matrix accompanied by microvessels and marrow cavities, indicating intramembranous ossification on the surface of the zirconia implants [71]. Similarly, Yu et al. explored the potential of MSC sheets when attached to titanium implants. The effects of an MSC sheet-implant complex were assessed in the right tibia of a diabetic rat model. Compared with the titanium implants alone, the MSC sheet-implant complexes resulted in significantly greater bone volume ratios, greater trabecular thickness, and lower trabecular separation. The amount of new bone tissue formed around the MSC-implant constructs was notably greater than that formed around the titanium implants. These findings suggest that the combination of cell sheet engineering techniques with standard implant materials has led to the development of MSC implants with osteogenic and vascularization properties, promoting osseous healing and potentially benefiting diabetic patients [72]. Qi et al. incorporated MSC sheets into MSC-loaded bilayer poly(lactic-co-glycolic acid) (PLGA) scaffolds to enhance the repair and integration of cartilage defects in a rabbit model. Compared with the other groups, the MSC sheet-encapsulated PLGA/MCSs group presented significantly more hyaline cartilage and better histological scores. This group also demonstrated optimal integration between the repaired cartilage and surrounding normal cartilage and subchondral bone. Furthermore, Qi and Yan suggested that MSC sheet-encapsulated cartilage debris may effectively assist in cartilage regeneration in osteoarthritis (OA) patients [73]. Therefore, many attempts have been made to evaluate bone and cartilage regeneration via cell sheet engineering and scaffolds, most of which have focused on bone reconstitution, and there is an extensive opportunity in the field of cartilage regeneration to address scientific gaps.

Preclinical research and clinical trials of CSE

As discussed above, several preclinical studies have utilized CSE for regeneration of damaged tissue or organs. Corneal injury, esophageal lesions, ischemic heart diseases, bone and cartilage defects, and liver diseases are widely studied. This novel therapeutic approach has shown efficacy and safety in lesion and defect regeneration in animal models, which provides remarkable evidence for clinical applications. ECM preserved in CSE leads to tight adhesion of cell sheets to the target site without the use of biological scaffolds or suturing. Currently, CSE application in regenerative medicine has switched from fundamental research and preclinical studies to clinical trials.

CSE clinical trials have focused mostly on ophthalmic disease treatment. Other studies, such as those on esophageal diseases and cardiovascular diseases, are ranked second and third. According to the International Clinical Trials Registry Platform (ICTRP), there are at least 4 clinical trials related to cartilage-related defects (Table 3). Statistics show that more than 80% of CSE clinical trials have been registered by Japanese scientists because they are pioneers in CSE research and its laboratory products. In 2019, Sato et al. developed chondrocyte sheets for autologous transplantation; tested them under in vitro conditions and in vivo in rat, rabbit, and minipig models; and confirmed that autologous chondrocyte sheet transplantation promoted hyaline cartilage repair. Since cartilage regenerative therapies are not effective enough to treat knee osteoarthritis (OAK), they designed a clinical trial (UMIN Clinical Trials Registry, UMIN000006650), which involved a combination of surgical treatment with OAK, followed by autologous triple-layered chondrocyte sheet transplantation. Eight patients with grade III or IV OAK and cartilage defects were included in the trial. Patients were evaluated by preoperative X-ray imaging, magnetic resonance imaging (MRI), knee injury and osteoarthritis outcome score (KOOS), and Lysholm knee score (LKS). Postoperative assessments were performed at 1, 3, 6, 12, 24, and 36 months after surgery. Patient cartilage viscoelasticity and histological evaluation of arthroscopic biopsies were performed 12 months after transplantation. Autologous chondrocyte sheet gene expression was analyzed to predict the clinical and structural outcomes of therapy. These findings confirmed that combination therapy is an effective procedure for OAK treatment [74]. In 2022, Hamahashi et al. reported regenerative therapy of OAK using polydactyly derived allogeneic chondrocyte cell sheets (PD sheets) and TRCD inserts in humans. They designed a clinical trial (UMIN Clinical Trials Registry, UMIN000015205) with ten (four men and six women) grade III or IV OAK patients who received therapy. Before and after cell sheet transplantation, cartilage viscoelasticity and thickness were assessed. Arthroscopic biopsies were performed 12 months posttransplantation for histological evaluation. This combined surgery with PD cell sheet transplantation is an ideal and effective regenerative therapy for treating OAK. Gene markers in PD cell sheets could predict therapeutic outcomes and provide a marker set for donor cell selection [75].

Table 3.

Summary of clinical trials

Title	Identifier	Country	Participants No	Recruitment status	Phase objective	Disease	Cell sheet type
Clinical study on the regenerative therapy for articular cartilage using autologous cell sheets	jRCTb030190166	Japan	20	Recruiting	Safety efficacy	Cartilage defects associated with osteoarthritis of the knee	Autologous chondrocyte sheet
The clinical study for the joint treatment by cell sheet	UMIN000006650	Japan	8	Complete: follow-up complete	Efficacy	Traumatic and/or degenerative cartilage defect of the knee joint	Autologous chondrocyte sheets
The clinical study for the joint treatment by allogeneic cell sheet	UMIN000015205	Japan	10	Complete: follow-up continuing	Safety efficacy	Cartilage defects associated with osteoarthritis of the knee	Chondrocyte sheet
safety and efficacy study of cells sheet-autologous chondrocyte implantation to treat articular cartilage defects (CS-ACI)	NCT01694823	China	10	Recruiting	Safety efficacy	Osteochondritis/osteochondritis dissecans/joint diseases	Cells sheet-autologous chondrocyte (CS-AC)

Although nearly all these clinical trials use commercial TRCD for cell sheet fabrication, the use of mechanical detachment may provide a significant opportunity to expand preclinical studies and clinical trials of various organs.

Commercial aspects of the CSE

Commercially available CSE products, such as TRCD plates provided by a cutting edge Japanese company, have led to a wide range of cell sheet research for various organs. TRCDs are known as UpCell plates. These plates are made by CellSeed and invented by Teruo Okano in Japan and are considered a key tool in cell sheet engineering. The CellSeed company offers these plates at variable prices, depending on the size, brand, and supplier. The use of cell sheets is highly recommended for autologous cell therapy, and there has also been a remarkable effort to develop allogeneic cell sheet products. The importance of cell sheet products has been identified, and many companies are attempting to establish cell sheet production. CSE products include a wide range of products, from the epidermis and corneal epithelium to chondrocyte sheets. Table 4 provides a detailed description of available commercial cell sheet products for different clinical applications. Two products are manufactured for cartilage regeneration. RevaFlex™ is an allogeneic juvenile chondrocyte sheet used in phase II clinical trials for the treatment of articular cartilage injury and was manufactured by ISTO Technologies in the USA. CellSeed Inc., which is the main TRDC company in Japan, manufactured allogenic chondrocyte sheets in clinical trials for the treatment of cartilage defects and knee osteoarthritis (Table 4). The manufacturing of cell sheet products has its own GMP and regulatory limitations. Four weeks are required to fabricate autologous cell sheets after the initial biopsy, although more typically, 2–3 weeks are needed. Cell sheets have a short shelf-life, so patients should be ready to receive the graft while the sheet is ready for transplant. The transportation of cell sheets from the laboratory to the hospital requires manufacturers close to the medical facility. The use of biological reagents such as fetal bovine serum (FBS) and L-ascorbic acid is susceptible to changes in quality, which can lead to product variability. Cell sheet manufacturing is still manual, requires a highly skilled operator to produce SCE products, and requires surgeons who are trained to handle fragile sheets and use this technology appropriately [76].

Table 4.

Commercially available cell sheet products

Company	Product	Clinical indication	Description
ISTO Technologies (USA)	RevaFlex™	Cartilage	Allogeneic juvenile Chondrocytes sheets under phase II clinical trials for the treatment of articular cartilage injury
CellSeed Inc (Japan)	–	Cartilage	Allogenic Chondrocytes sheets under clinical trials for the treatment of cartilage defects and knee osteoarthritis
Japan Tissue Engineering Corp (J-TEC) (Japan)	Autologous cultured epidermis	Skin	Cultured epidermal autograft
Japan Tissue Engineering Corp (J-TEC) (Japan)	Autologous cultured corneal epithelium	Cornea	Autologous corneal epithelial cells cultured on hydrogel
Vericel (USA)	Epicel	Skin	Cultured epidermal autograft
Chiesi Farmaceutici (Italy)	Holoclar	Cornea	Autologous epithelial corneal cell sheet licensed for the treatment of limbal stem cells deficiency
Cytograft (USA)	LifeLine	Blood vessel	Autologous fibroblasts tubular constructs licensed as shunts for hemodialysis
CellSeed Inc (Japan)	–	Esophagus	Autologous oral epithelial cell sheets licensed for the treatment of esophageal ulcers after endoscopic surgery for esophageal cancer
Terumo (Japan)	HeartSheet	Heart	Autologous skeletal myoblast sheets licensed for the treatment of severe heart failure caused by chronic ischemic heart disease

Conclusion

In the fields of biological and medical science, cell sheets, as a revolutionary concept in tissue engineering, have gained considerable attention. These two-dimensional cellular constructs offer a unique advantage over traditional cell-based therapies by preserving the extracellular matrix and cell‒cell interactions. The efficient and gentle harvesting of such fragile structures, which has been extensively studied, is one of the essential aspects of cell packaging technology. An enzymatic procedure, such as trypsinization, to remove cell sheets from the culture surface is a conventional method for harvesting cells. The integrity of the interactions between the cell and matrix may be disturbed by this approach, which could impair the functionality of a cell sheet. To overcome this challenge, researchers have explored alternative methods, with a particular focus on mechanical techniques, which allow for easy removal of the cell sheet by scrapers or tweezers without the need for enzymatic treatment or surface modification. This approach has been shown to preserve the structural and functional properties of the cell sheet, making it a promising strategy for tissue engineering applications. Particularly in bone and cartilage repair, it allows the formation of cohesive cell sheets that can be transplanted directly to the site of injury or defect. The integration and function of the graft may be enhanced by such a natural architecture and cellular organization, resulting in better tissue regeneration and a reduced risk for immunological rejection. Moreover, the need for scaffolds or biomaterials that can sometimes interfere with natural tissue formation has been eliminated through cell sheet engineering. The cell sheets can be applied directly to the target site so that they are more easily integrated with the host tissues. Cell sheet engineering has shown promising results in bone and cartilage regeneration. In studies to repair bone defects and cartilage lesions, the successful use of cell sheets derived from different types of cells, such as mesenchymal stem cells or chondrocytes, has been demonstrated. Finally, significant advances in tissue engineering can be attributed to the mechanical harvesting of cell sheets. This approach has the potential to increase the therapeutic possibilities of cell therapies and help develop more effective strategies for revitalizing medicine, as it preserves the integrity of interactions between cells and the matrix.

Cell sheet engineering is an advancing discipline with considerable promise for revolutionizing regenerative medicine. The development of more intricate tissues that mimic the functional characteristics of natural tissues and the possibility of generating patient-specific cell sheets is noteworthy. This customization can increase compatibility and minimize the possibility of transplant rejection. Furthermore, the incorporation of complex biomaterials with cell sheets can improve the mechanical properties and support better cell viability and functionality. This integration may result in superior scaffolding for tissue regeneration. Additionally, the fusion of engineered cell sheets with 3D bioprinting technology has the potential to transform tissue construction, enabling precise control over cell arrangement and tissue structure. The future of cell sheet engineering is promising, driven by technological progress, personalized strategies, and a deeper understanding of cellular behavior in the tissue formation process.

One of the most promising approaches is the automation of the harvesting process. Manual detachment of delicate cell sheets could be technically prone to variability, depending on the operator’s lab skill and expertise. There is a fundamental question here: could robotic or automated systems be developed to standardize the harvesting procedure? The answer is that by minimizing human error and improving reproducibility, automation would provide more consistent results across different laboratories and clinical settings, accelerating the pathway from bench to bedside.

The development of hybrid approaches is another exciting strategy. Although mechanical harvesting methods offer advantages, such as avoiding chemical or temperature switching, combining this method with existing chemical or thermoresponsive methods might lead to further optimized procedures. For example, the use of mild temperature changes to assist a mechanical peel could reduce stress on cells and improve cell sheet integrity. Exploring synergistic strategies might help overcome the limitations of a single technique.

There is a need to address the standardization and scalability criteria of cell sheet fabrication. What kinds of technological innovations will be necessary to make large-scale production of mechanically harvested sheets a reality? This involves not only tools and protocols but also developing significant quality control measures and adapting systems for mass production. Cell therapies are moving toward broader clinical applications; thus, logistical considerations will become just as important as biological considerations.

Abbreviations

CSE

Cell sheet engineering

ECM

Cell–extracellular matrix

PIPAAm

Poly(N-isopropylacrylamide)

TRCDs

Temperature-responsive culture dishes

MSCs

Mesenchymal stem cells

PSCs

Pluripotent stem cells

ESCs

Embryonic stem cells

iPSCs

Induced pluripotent stem cells

EPCs

Endothelial progenitor cells

SMCs

Smooth muscle cells

ESD

Endoscopic submucosal dissection

LCST

Lower critical solution temperature

hUC-MSCs

Human umbilical cord mesenchymal stem cells

pYAP

Phosphorylated Yes-associated protein

3D

Three-dimensional

TCPS

Tissue culture polystyrene surfaces

UV

Ultraviolet

SAM

Self-assembled monolayer

RGD

Arg-Gly-Asp

PAH

Poly(allylamine hydrochloride)

PSS

Poly(styrene sulfonate)

ROS

Reactive oxygen species

Hp-PK films

Hematoporphyrin-incorporated polyketone films

LED

Light-emitting diode

hDPSCs

Human dental pulp stem cells

BMSCs

Bone marrow mesenchymal stem cells

SDS

Sodium dodecyl sulfate

rhBMP-2

Human bone morphogenetic protein-2

CS

Calcium sulfate

PLGA

Poly(lactic-co-glycolic acid)

OA

Osteoarthritis

Author contributions

F.L.M Conceptualization, Writing- original draft. M.B.E. and S.H. Conceptualization and writing-review and editing. Y.A Preparing the figures. All the authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

Not applicable.

Code availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

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Competing interests

The authors declare that they have no conflicts of interest. The authors declare that they have not used artificial intelligence to generate the paper.