Right ventricular (RV) failure following surgical repair of congenital heart disease affects survival. Human induced pluripotent stem cell-derived cardiomyocyte (hiPS-CM) sheet transplantation ameliorated left ventricular dysfunction in preclinical studies, indicating its efficacy in RV failure in congenital heart disease. This study aimed to evaluate whether hiPS-CMs could improve RV function in rats with pressure-overloaded RV failure. F344/NJcl-rnu/rnu rats underwent pulmonary artery banding (PAB) via left thoracotomy. Four weeks after PAB, hiPS-CM patch transplantation to the RV was performed in the hiPS-CM group (n = 33), and a sham operation was performed in the sham group (n = 18). We evaluated cardiac catheterization, positron emission tomography data, and pathological results 8 weeks following PAB. RV end-diastolic pressure, the time constant of isovolumic relaxation, and end-diastolic pressure-volume relation were significantly ameliorated in the hiPS-CM group compared with in the sham group. Picrosirius red staining revealed that anti-fibrotic effects were significantly higher in the hiPS-CM group than in the sham group. Von Willebrand factor staining revealed significantly higher myocardial capillary vascular density in the hiPS-CM group than in the sham group. hiPS-CMs were detected in the epicardium 4 weeks after hiPS-CM sheet transplantation. The angiogenic gene expression in the myocardium was significantly higher in the hiPS-CM group than in the sham group. Overall, in rats with pressure-overloaded RV failure, hiPS-CM patch transplantation could improve diastolic function, suppress ventricular fibrosis, and increase capillary density, suggesting that it is a promising treatment for RV failure.

Culturing living cells outside the body is a complex process involving various techniques. Despite advances, harvesting cells remains challenging, especially in light of new emerging and scaled-up cell culture technologies. Enzymatic adherent cell harvesting is the most used and robust technology but can harm cells. Non-enzymatic detachment methods offer advantages but also present challenges. Thermo-responsive polymers require precise control of the molecular characteristics and thickness of the thermoresponsive films, which makes this method less robust and more expensive. This review highlights the importance of controlling harvested cell quality and its relationship to cell binding and detachment mechanisms. Many alternative methods have not been extensively analyzed, and their impact on cell quality beyond standard viability assays is not yet known. Developing robust cell harvesting methods for bioreactor microcarriers is a rapidly growing challenge as the cell manufacturing industry expands. Microcarriers with stimuli-responsive coatings face challenges similar to those observed for laboratory-scale cell dishes and bring an additional aspect of the need for microbead recycling consideration. All that together underlines the importance of the research in biomaterials and biotechnology for cell manufacturing.

Freestanding cell-sheets are valuable bio-materials for use in regenerative medicine and tissue engineering. Because cell-sheets experience various mechanical stimulations during handling, it is important to understand the responses of cells to these stimulations. Here, we demonstrate changes in the localization of various proteins during the stretching of fibroblast cell-sheets. These proteins are known to be involved in mechano-sensing. Upon stretching, actin filaments appear parallel to the stretching direction. At cell-cell junctions, β-catenin forms clusters that co-localize with accumulated vinculin and zyxin as well as the actin filaments. The p130 Crk-associated substrate, known to be present in focal adhesions, is also recruited to these clusters and phosphorylated. Our results suggest that mechano-sensing machinery is formed at cell-cell junctions when the cell-sheets are stretched.

Various cell sheets have been used as effective and useful cellular tissues in tissue engineering and regenerative therapy. Poly(N-isopropylacrylamide) (PNIPAAm)-modified surfaces have been investigated for effective cell sheet preparation. In this study, the effective PNIPAAm graft density and chain length of PNIPAAm brushes for various cell types were investigated. The PNIPAAm brush-grafted glass was prepared via silanization and subsequent atom transfer radical polymerization (ATRP). The density of the PNIPAAm brushes was modulated by changing the ATRP initiator and co-adsorber composition, while the PNIPAAm brush length was modulated by changing the monomer concentration in the ATRP. The hydrophilicity of the PNIPAAm brushes increased with increasing PNIPAAm brush length because long PNIPAAm brushes tended to hydrate. Fibronectin adsorption increased with decreasing PNIPAAm brush concentration because the exposed hydrophobic co-adsorber in the dilute PNIPAAm brush enhanced the adsorption of fibronectin. The cell-sheet fabrication ability was investigated using six types of PNIPAAm brushes. An endothelial cell sheet was fabricated using a dense, short PNIPAAm brush. NIH/3T3 sheets can be fabricated using three types of PNIPAAm brushes: dense-long PNIPAAm brushes, moderately dense-short PNIPAAm brushes, and dilute-long PNIPAAm brushes. MDCK cell sheets could not be prepared using the PNIPAAm brushes. A549 cell sheets were prepared using a dense-short PNIPAAm brush and moderately dense-short PNIPAAm brushes. These results indicate that the optimal PNIPAAm brush conditions for cell sheet preparation vary depending on cell type. Thus, modulation of PNIPAAm brush density and length is an effective approach for preparing target cell sheets.

The chondrocyte sheet is a sheet-like cell structure obtained by separating in vitro expanded and fused autologous chondrocytes from the bottom of the culture dish by physical means. The cell sheet contains autologous chondrocytes, extracellular matrix secreted by chondrocytes, and connective structures established between cells and matrix, and between cells and cells. In cartilage tissue engineering, chondrocyte sheets technology has great potential for the treatment of cartilage defects. Chondrocyte sheets have a low immunogenicity because they avoid the immune reaction caused by scaffolding materials. However, chondrocyte sheets can still cause severe local tissue swelling in the short term after implantation, resulting in a poor patient experience. In individual cases, an inflammatory reaction may even occur, leading to resorption of the chondrocyte sheet. This may be immunogenetically related to chondrocyte membrane surface-associated antigens, components of the extracellular matrix secreted by chondrocytes, and various bioactive components in the culture medium used during in vitro chondrocyte culture. Therefore, in order to investigate the causes of local tissue swelling and immune-inflammatory reactions induced by the implantation of chondrocyte sheets, this article reviews the immunogenicity of chondrocyte-associated antigens, components of the extracellular matrix of cartilage, and the active components of the cell culture medium.

Dynamically thermoresponsive biomaterials, particularly those utilizing poly(N-isopropylacrylamide) (PNIPAAm), have attracted much attention in biomedical applications due to their reversible phase transition near body temperature. These biomaterials provide innovations across drug delivery system, chromatography, and tissue engineering. Molecular designs, such as the incorporation of hydrophilic comonomers or graft copolymers in PNIPAAm hydrogels, enhance rapid kinetics of the gels when jumping the temperature across the phase transition temperature, because of avoiding 'skin layer' formation on the surface of the gels. Nanocarriers possessing PNIPAAm coronas facilitate spatial drug delivery and temporally on-demand drug release to targeted cancers in combination with hyperthermic therapy. Downsizing of PNIPAAm hydrogels accelerates the kinetics of shrinkage/swelling, leading to applications as thermoresponsive chromatographic matrices and cell cultureware. PNIPAAm-modified surfaces support thermoresponsive cell culture systems for the non-invasive recovery of intact cell sheets, enabling advanced regenerative therapies and layered 3D tissue formation. Recent developments also integrate growth factor delivery for sustained cell stimulation on culturewares. Newly developed biomaterials, including dynamically thermoresponsive PNIPAAm, are expected to expand the opportunity for novel treatment technologies such as targeted therapies and regenerative medicine.

Tissue engineering plays a pivotal role in the advancement of regenerative medicine. Thermoresponsive culture dishes, coated with specialized polymers that control cell adhesion through temperature fluctuations, enable the processing of cells into sheets for medical applications while maintaining their intact state. Cell sheets prepared using these culture dishes have been incorporated into several commercial pharmaceutical products. However, controlling the detachability of cell sheets using conventional thermoresponsive culture dishes remains a challenge, and often leads to unexpected detachment during cultivation. In this study, we developed a thermoresponsive culture dish with tunable cell detachability using a thermoresponsive block copolymer, poly(butyl methacrylate)-b-poly(N-isopropylacrylamide) (PBMA-PIPAAm), which is a specialized polymer that allows precise control of the amount of surface-immobilized polymer and polymer layer thickness. Culturing periodontal ligament-derived mesenchymal stem cells on these dishes demonstrated fully tunable detachability without compromising cell properties compared to conventional thermoresponsive dishes (UpCell®). Thermoresponsive PBMA-PIPAAm-coated culture dishes enable the complete on-demand detachment of transplantable cell sheets, thereby avoiding unexpected detachment that may increase production costs and reduce technical hurdles in the manufacturing process. The PBMA-PIPAAm coating method has the potential to contribute to biomedical and clinical applications of mesenchymal stem cell sheets.

Hemophilia B is an inherited disorder caused by a mutation in the FIX gene, which results in insufficient blood clotting factor IX (FIX) production from hepatocytes. Currently, there are no treatments for hemophilia B patients. The patients should be continuously administrated with clotting factor concentrates 2-3 times a month to prevent bleeding. Therefore, this study aimed to develop an engineered FIX-secreting hepatocyte sheet that can release FIX for an extended period. Within this study, the engineered FIX-secreting hepatocyte sheet was developed by integrating two core technologies, including a gene editing platform to generate FIX-secreting cells and cell sheet technology to improve cell delivery efficacy.

The human FIX gene was inserted into the APOC3 site of iPSCs by CRISPR/Cas9, which secretes the target protein after differentiation into hepatocytes. FIX-secreting hepatocyte sheets were obtained by temperature-responsive polymer grafted cell culture dishes (TRCD). Immunohistochemical and functional tests were performed for hepatocyte-like cells differentiated from FIX KI-iPSCs and wild-type iPSCs (WT-iPSCs). After validating the functional activity and secretion of FIX protein, the engineered hepatocyte-like cell sheets were transplanted to NOD/SCID mice for the in vivo experiments.

The insertion of the human FIX gene into the APOC3 site demonstrated a significant increase in FIX secretion in hepatocyte-like cells differentiated from FIX KI-iPSCs compared with those obtained from WT-iPSCs. Among the iPSCs to hepatocyte differentiation stages, the hepatic endoderm stage was most suitable for seeding the cells on TRCD and generating cell sheets by temperature changes from 37oC to room temperature when the hepatocyte-like cells have reached maturity. The engineered FIX-secreting cell sheets showed intensive expression of the FIX proteins without losing hepatocyte morphology for 20 days. Furthermore, an in vivo study showed that engineered FIX-secreting cell sheets retained their FIX secretion functions for two weeks, whereas single-cell injected traditionally were barely detected in the experimental animals.

The engineered FIX-secreting cell sheets fabricated from functionally improved iPSCs with practical cell delivery tools could be a promising tool for clinically treating Hemophilia B.

The application of cell sheet technology for wound healing preserves dense cell tissue and the natural extracellular matrix (ECM), contributing to disease prevention. Despite the effectiveness of autologous and allograft cell sheets for wound healing, conventional cell sheets, although stable, may experience necrosis in their middle layers due to a lack of nutrients or oxygen. To address these issues, a novel approach is proposed to create cell sheets using mechanical and electrical stimulation. This method not only facilitates the transfer of cell sheets but also enhances cell bioactivity. The performance of the proposed membrane, with a mechanically controlled microstructure under electrical stimulation, is validated in both in vitro and in vivo models. The micro-structured membrane allows for diverse electrical stimulation compared to flat membranes, which accelerates the detachment of cell sheets and promotes angiogenesis and re-epithelialization. These findings indicate that the innovative cell sheet technology could significantly enhance rapid wound healing in regenerative medicine.

Recent advances in scaffold-free three-dimensional (3D) culture methods have significantly enhanced the potential of stem cell-based therapies in regenerative medicine. This cutting-edge technology circumvents the use of exogenous biomaterial and prevents its associated complications. The 3D culture system preserves crucial intercellular interactions and extracellular matrix support, closely mimicking natural biological niches. Therefore, stem cells cultured in 3D formats exhibit distinct characteristics, showcasing their capabilities in promoting angiogenesis and immunomodulation. This review aims to elucidate foundational technologies and recent breakthroughs in 3D scaffold-free stem cell engineering, offering comprehensive guidance for researchers to advance this technology across various clinical applications. We first introduce the various sources of stem cells and provide a comparative analysis of two-dimensional (2D) and 3D culture systems. Given the advantages of 3D culture systems, we delve into the specific fabrication and harvesting techniques for cell sheets and spheroids. Furthermore, we explore their applications in pre-clinical studies, particularly in large animal models and clinical trials. We also discuss multidisciplinary strategies to overcome existing limitations such as insufficient efficacy, hostile microenvironments, and the need for scalability and standardization of stem cell-based products.

The preparation of cells is a critical step in cell therapy. To ensure the effectiveness of cells used for clinical treatments, it is essential to harvest adherent cells from the culture media in a way that preserves their high viability and full functionality. In this study, we developed temperature-responsive poly(N-isopropylacrylamide) (PNIPAM)-grafted polystyrene (PS) microspheres using reversible addition-fragmentation chain transfer polymerization. These microspheres allow for the non-destructive harvesting of cultured cells through temperature changes. The composition and physicochemical properties of the PNIPAM-grafted PS microspheres were confirmed using infrared spectroscopy, elemental analysis, dynamic light scattering, and thermogravimetric analysis.In vitroexperiments demonstrated that these microspheres exhibit excellent biocompatibility, supporting the adhesion and proliferation of various cells. Moreover, the microspheres showed good temperature responsiveness in thermosensitive detachment experiments with GFP-HepG2cells and umbilical cord mesenchymal stem cells (UC-MSCs). Additionally, through orthogonal experiments, we identified a cell detachment aid mixture that significantly improved the dispersibility of cells detached from the microspheres, enhancing the efficiency of thermosensitive cell detachment by approximately 40%. The harvested UC-MSCs retained their capacity for re-proliferation and trilineage differentiation. Consequently, the temperature-responsive microspheres developed in this study, combined with the cell detachment aid mixtures, hold great potential for large-scale culture and harvesting of therapeutic cells in clinical applications.

Cell therapy has emerged as a viable approach for treating damaged organs or tissues, particularly with advancements in stem cell research and regenerative medicine. The innovative technique of cell sheet engineering offers the potential to create a cell-dense lamellar structure that preserves the extracellular matrix (ECM) secreted by cells, along with the cell-matrix and intercellular junctions formed during in vitro cultivation. In recent years, significant progress has been made in developing cell sheet engineering technology. A variety of novel materials and methods were utilized for enzyme-free cell detachment during the cell sheet formation process. The complexity of cell sheet structures increased to meet advanced usage demands. This review aims to provide an overview of the preparation methods and types of cell sheets, thereby enhancing the understanding of this rapidly evolving technology and offering a fresh perspective on the development and future application of cell sheet engineering.

This study aimed to establish a combined histological assessment system of neo-cartilage outcomes and to evaluate variations in an established rat defect model treated with human juvenile cartilage-derived chondrocyte (JCC) sheets fabricated from various donors.

JCCs were isolated from the polydactylous digits of eight patients. Passage 2 (P2) JCC sheets from all donors were transplanted into nude rat chondral defects for 4 weeks (27 nude rats in total). Defect-only group served as control. Histological samples were stained for safranin O, collagen 1 (COL1), and collagen 2 (COL2). (1) All samples were scored, and correlation coefficients for each score were calculated. (2) Donors were divided into "more effective" and "less effective" groups based on these scores. Then, differences between each group in each category of modified O'Driscoll scoring were evaluated.

(1) Modified O'Driscoll scores were negatively correlated with %COL1 area, and positively correlated with %COL2 area and COL2/1 ratio. (2) Four of 8 donors exhibited significantly higher modified O'Driscoll scores and %COL2 areas. JCC donors were divided into two groups by average score values. Significant differences between the two groups were observed in modified O'Driscoll categories of "Nature of predominant tissue," "Reconstruction of subchondral bone," and "Safranin O staining."

The combined histological evaluation method is useful for detailed in vivo efficacy assessments of cartilage defect regeneration models. Variations in histological scores among juvenile cartilage-derived chondrocyte donors were correlated to the quality of regenerated cartilage hyaline structure and subchondral bone remodeling observed in the nude rat defect model.

Periodontal regeneration is a challenge, and tissue engineering based on periodontal ligament stem cells (PDLSCs) has been shown to be a promising alternative to this process. However, the need for scaffolds has limited the therapeutic use of PDLSCs. In this context, scaffold-free tissue engineering using the cell sheet (CS) technique has been developed as an alternative approach to improve tissue regeneration. Previously, we showed that Protease-activated receptor-1 (PAR1) can regulate PDLSCs. Herein, we evaluate whether PAR1 influences osteogenesis in CSs produced from PDLSCs, without the use of scaffolds. PDLSCs were isolated and immunophenotyped. Then, CSs were obtained by supplementing the culture medium with ascorbic acid (50 µg/mL), and PAR1 was activated through its agonist peptide (100 nM). Scaffold-free 3D CSs were successfully produced from PDLSCs, and they showed higher proliferation potential than isolated PDLSCs. Also, PAR1 activation decreased senescence and improved osteogenic differentiation of CSs by increasing mineralized nodule deposition and alkaline phosphatase concentration; PAR1 also modulated osteogenic markers at the gene and protein levels. We further demonstrated that this effect was regulated by Wnt, TGF-βI, MEK, p38 MAPK, and FGF/VEGF signaling pathways in PDLSCs (p < 0.05%). Overall, PAR1 activation increased osteogenic activity in CSs, emerging as a promising scaffold-free therapeutic approach for periodontal regeneration.

Osteoarthritis and rheumatoid arthritis, though etiologically distinct, are both inflammatory joint diseases that cause progressive joint injury, chronic pain, and loss of function. Therefore, long-term treatment with a focus on relieving symptoms is needed. At present, the primary treatment for arthritis is drug therapy, both oral and intravenous. Although significant progress has been achieved for these treatment methods in alleviating symptoms, certain prominent drawbacks such as the substantial side effects and limited absorption of medications call for an urgent need for improved drug delivery methods. Injected hydrogels can be used as a delivery system to deliver drugs to the joint cavity in a controlled manner and continuously release them, thereby enhancing drug retention in the joint cavity to improve therapeutic effectiveness, which is attributed to the desirable attributes of the delivery system such as low immunogenicity, good biodegradability and biocompatibility. This review summarizes the types of injectable hydrogels and analyzes their applications as delivery systems in arthritis treatment. We also explored how hydrogels counteract inflammation, bone and cartilage degradation, and oxidative stress, while promoting joint cartilage regeneration in the treatment of osteoarthritis (OA) and rheumatoid arthritis (RA). This review also highlights new approaches to developing injectable hydrogels as delivery systems for OA and RA.

The ability of thermoresponsive polymers to respond to temperature with a reversible conformational change makes them promising 'smart' materials for solutions in medical and biotechnological applications. In this work, two such polymers and structural isomers were studied: poly(N-isopropyl acrylamide) (PNiPAm) and poly(2-isopropyl-2-oxazoline) (PiPOx). We compare the critical solution temperatures (CST) of these polymers in D2O and H2O in the presence of Hofmeister series salts, as results obtained under these different solvent conditions are often compared. D2O has a higher dipole moment and electronegativity than H2O, which could significantly alter the CST transition. We used two complementary methods to measure the CST, dynamic light scattering (DLS) and differential scanning calorimetry (DSC) and found that the CST decreased significantly in D2O compared to H2O. In the presence of highly concentrated kosmotropes, the CST of both polymers decreased in both solvents. The influence of the kosmotropic anions was smaller than the water isotope effect at low ionic strengths but considerably higher at physiological ionic strengths. However, the Hofmeister anion effect was quantitatively different in H2O than in D2O, with the largest relative differences observed for Cl-, where the CSTs in D2O decreased more than in H2O measured by DLS but less by DSC. PiPOx was more sensitive than PNiPAm to the presence of chaotropes. It exhibited much higher transition enthalpies and multistep transitions, especially in aqueous solutions. Our results highlight that measurements of thermoresponsive polymer properties in D2O cannot be compared directly or quantitatively to application conditions or even measurements performed in H2O.

Owing to the rapid increase in the number of people with severe heart failure, regenerative medicine is anticipated to play a role in overcoming the limitations inherent in existing surgical interventions. There are essentially two types of cardiac regenerative therapies for a failing heart. Cellular regenerative therapies using various stem cells improve the functional recovery of the heart mainly by cytokine paracrine effects. The implantation of induced pluripotent stem cell-derived cardiomyocytes can contribute not only to the inhibition of adverse heart remodeling by paracrine effects but also to the supply of newly born functional myocytes with the recipient myocardium as "mechanically working cells." Cell transplantation, including autologous myoblast transplantation, reduces heart failure exacerbations and benefits patients without the need for other treatment options. Although cellular therapy is currently the mainstream approach, it requires an in-house cell-processing center with an aseptic environment. In addition, these stem cells are usually introduced via several invasive delivery methods, including intracoronary administration, and cellular sheet implantation. Simplifying the culture methods for these cells is a crucial problem that needs to be resolved. Drug-induced regenerative therapy is another option that enhances self-endogenous regenerative systems in the human body and does not require invasive methods or cell cultures. Therefore, drug-induced regenerative therapies may overcome the disadvantages of these cellular therapies. The purpose of this report is to summarize cell transplantation therapy in the cardiovascular system and regenerative therapy for heart failure using an autologous endogenous regenerative system.

Heart failure (HF) is a life-threatening disorder and is treated by drug therapies and surgical interventions such as heart transplantation and left ventricular assist device (LVAD). However, these treatments can lack effectiveness in the long term and are associated with issues such as donor shortage in heart transplantation, and infection, stroke, or gastrointestinal bleeding in LVADs. Therefore, alternative therapeutic strategies are still needed. In this respect, stem cell therapy has been introduced for the treatment of HF and numerous preclinical and clinical studies are employing a range of stem cell varieties. These stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have been shown to improve cardiac function and attenuate left ventricular remodeling. IPSCs, which have a capacity for unlimited proliferation and differentiation into cardiomyocytes, are a promising cell source for myocardial regeneration therapy. In this review, we discuss the following topics: (1) what are iPSCs; (2) the limitations and solutions for the translation of iPSC-CMs practically; and (3) the current therapeutic clinical trials.

Recent advances in nanotechnology design and fabrication have shaped the landscape for the development of ideal cell interfaces based on biomaterials. A holistic evaluation of the requirements for a cell interface is a highly complex task. Biocompatibility is a crucial requirement which is affected by the interface's properties, including elemental composition, morphology, and surface chemistry. This review explores the current state-of-the-art on graphene coatings produced by chemical vapor deposition (CVD) and applied as neural interfaces, detailing the key properties required to design an interface capable of physiologically interacting with neural cells. The interfaces are classified into substrates and scaffolds to differentiate the planar and three-dimensional environments where the cells can adhere and proliferate. The role of specific features such as mechanical properties, porosity and wettability are investigated. We further report on the specific brain-interface applications where CVD graphene paved the way to revolutionary advances in biomedicine. Future studies on the long-term effects of graphene-based materials in vivo will unlock even more potentially disruptive neuro-applications.

Mesenchymal stem cell (MSC)-based cardiac patches are envisioned to be a promising treatment option for patients with myocardial infarction. However, their therapeutic efficacy and duration are hampered due to their limited retention on the epicardium. We engineered a scaffold-free MSC sheet with an inherent ability to migrate into the infarcted myocardium, a strategy enabled by actively establishing a sustained intracellular hypoxic environment through the endocytosis of our FDA-approved ferumoxytol. This iron oxide nanoparticle stabilized hypoxia-induced factor-1α, triggering upregulation of the CXC chemokine receptor and subsequent MSC chemotaxis. Thus, MSCs integrated into 2/3 depth of the left ventricular anterior wall in a rat model of acute myocardial infarction and persisted for at least 28 days. This led to spatiotemporal delivery of paracrine factors by MSCs, enhancing cardiac regeneration and function. Ferumoxytol also facilitated the noninvasive MRI tracking of implanted MSCs. Our approach introduces a strategy for mobilizing MSC migration, holding promise for rapid clinical translation in myocardial infarction treatment.

Type 1 diabetes-mellitus (T1DM) is characterized by damage of beta cells in pancreatic islets. Cell-sheet engineering, one of the newest therapeutic approaches, has also been used to create functional islet systems by creating islet/beta cell-sheets and transferring these systems to areas that require minimally invasive intervention, such as extrahepatic areas. Since islets, beta cells, and pancreas transplants are allogeneic, immune problems such as tissue rejection occur after treatment, and patients become insulin dependent again. In this study, we aimed to design the most suitable cell-sheet treatment method and macrocapsule-device that could provide long-term normoglycemia in rats. Firstly, mesenchymal stem cells (MSCs) and beta cells were co-cultured in a temperature-responsive culture dish to obtain a cell-sheet and then the cell-sheets macroencapsulated using different concentrations of alginate. The mechanical properties and pore sizes of the macrocapsule-device were characterized. The viability and activity of cell-sheets in the macrocapsule were evaluatedin vitroandin vivo. Fasting blood glucose levels, body weight, and serum insulin & C-peptide levels were evaluated after transplantation in diabetic-rats. After the transplantation, the blood glucose level at 225 mg dl-1on the 10th day dropped to 168 mg dl-1on the 15th day, and remained at the normoglycemic level for 210 days. In this study, an alginate macrocapsule-device was successfully developed to protect cell-sheets from immune attacks after transplantation. The results of our study provide the basis for future animal and human studies in which this method can be used to provide long-term cellular therapy in T1DM patients.

Current regenerative medicine tactics focus on regenerating tissue structures pathologically modified by cell transplantation in combination with supporting scaffolds and biomolecules. Natural and synthetic polymers, bioresorbable inorganic and hybrid materials, and tissue decellularized were deemed biomaterials scaffolding because of their improved structural, mechanical, and biological abilities.Various biomaterials, existing treatment methodologies and emerging technologies in the field of Three-dimensional (3D) and hydrogel processing, and the unique fabric concerns for tissue engineering. A scaffold that acts as a transient matrix for cell proliferation and extracellular matrix deposition, with subsequent expansion, is needed to restore or regenerate the tissue. Diverse technologies are combined to produce porous tissue regenerative and tailored release of bioactive substances in applications of tissue engineering. Tissue engineering scaffolds are crucial ingredients. This paper discusses an overview of the various scaffold kinds and their material features and applications. Tabulation of the manufacturing technologies for fabric engineering and equipment, encompassing the latest fundamental and standard procedures.

Articular cartilage is a common cartilage type found in a multitude of joints throughout the human body. However, cartilage is limited in its regenerative capacity. A range of methods have been employed to aid adults under the age of 45 with cartilage defects, but other cartilage pathologies such as osteoarthritis are limited to non-steroidal anti-inflammatory drugs and total joint arthroplasty. Cell therapies and synthetic biology can be utilized to assist not only cartilage defects but have the potential as a therapeutic approach for osteoarthritis as well. In this review, we will cover current cell therapy approaches for cartilage defect regeneration with a focus on autologous chondrocyte implantation and matrix autologous chondrocyte implantation. We will then discuss the potential of stem cells for cartilage repair in osteoarthritis and the use of synthetic biology to genetically engineer cells to promote cartilage regeneration and potentially reverse osteoarthritis.

Surface modification of polydimethylsiloxane (PDMS) with an extracellular matrix (ECM) is useful for enhancing stable cell attachment. However, few studies have investigated the correlation between the stability of deposited ECM and cell behavior on the PDMS surfaces in external stretched cell culture systems. Herein, covalent collagen type I (Col)-immobilized PDMS surfaces were fabricated using 3-aminopropyl-trimethoxysilane, glutaraldehyde, and Col molecules. The immobilized collagen molecules on the PDMS surface were more stable and uniform than the physisorbed collagen. The cells stably adhered to the Col-immobilized surface and proliferated even under uniaxial cyclic mechanical stretching stress (UnCyMSt), whereas the cells gradually detached from the Col-physisorbed PDMS surface, accompanied by a decrease in the number of deposited collagen molecules. Moreover, the immobilization of collagen molecules enhanced cell alignment under the UnCyMSt. This study reveals that cell adhesion, proliferation, and alignment under the UnCyMSt can be attributed to the retention of collagen molecules on the PDMS surface.

Bioprinting is an additive manufacturing technique that combines living cells, biomaterials, and biological molecules to develop biologically functional constructs. Three-dimensional (3D) bioprinting is commonly used as anin vitromodeling system and is a more accurate representation ofin vivoconditions in comparison to two-dimensional cell culture. Although 3D bioprinting has been utilized in various tissue engineering and clinical applications, it only takes into consideration the initial state of the printed scaffold or object. Four-dimensional (4D) bioprinting has emerged in recent years to incorporate the additional dimension of time within the printed 3D scaffolds. During the 4D bioprinting process, an external stimulus is exposed to the printed construct, which ultimately changes its shape or functionality. By studying how the structures and the embedded cells respond to various stimuli, researchers can gain a deeper understanding of the functionality of native tissues. This review paper will focus on the biomaterial breakthroughs in the newly advancing field of 4D bioprinting and their applications in tissue engineering and regeneration. In addition, the use of smart biomaterials and 4D printing mechanisms for tissue engineering applications is discussed to demonstrate potential insights for novel 4D bioprinting applications. To address the current challenges with this technology, we will conclude with future perspectives involving the incorporation of biological scaffolds and self-assembling nanomaterials in bioprinted tissue constructs.

Organ transplantation is a definitive treatment for endocrine disorders, but donor shortages limit the use of this technique. The development of regenerative therapies would revolutionize the treatment of endocrine disorders. As is the case for harvested organs, the ideal bioengineered graft would comprise vascularized endocrine tissue, contain blood vessels that could be anastomosed to host vessels, have stable blood flow, and be suitable for transplantation into various sites. Here, we describe a transplantable endocrine tissue graft that was fabricated byex vivoperfusion of tricultured cell sheets (isletβ-cells, vascular endothelial cells (vECs), and mesenchymal stem cells (MSCs)) on a vascularized tissue flap ofin vivoorigin. The present study has three key findings. First, mild hypothermic conditions enhanced the success ofex vivoperfusion culture. Specifically, graft construction failed at 37 °C but succeeded at 32 °C (mild hypothermia), and endocrine tissue fabricated under mild hypothermia contained aggregations of isletβ-cells surrounded by dense vascular networks. Second, the construction of transplantable endocrine tissue byex vivoperfusion culture was better achieved using a vascular flap (VF) than a muscle flap. Third, the endocrine tissue construct generated using a VF could be transplanted into the rat by anastomosis of the graft artery and vein to host blood vessels, and the graft secreted insulin into the host's circulatory system for at least two weeks after transplantation. Endocrine tissues bioengineered using these techniques potentially could be used as novel endocrine therapies.

No effective therapies have yet been established for liver regeneration in liver failure. Autologous skeletal myoblast cell sheet transplantation has been proven to improve cardiac function in patients with heart failure, and one of the mechanisms has been reported to be a paracrine effect by various growth factors associated with liver regeneration. Therefore, the present study focused on the effect of myoblast cells on liver regeneration in vitro and in vivo.

We assessed the effect of myoblast cells on the cells comprising the liver in vitro in association with liver regeneration. In addition, we examined in vivo effect of skeletal myoblast cell sheet transplantation in C57/BL/6 mouse models of liver failure, such as liver fibrosis induced by thioacetamide and hepatectomy.

In vitro, the myoblast cells exhibited a capacity to promote the proliferation of hepatic epithelial cells and the angiogenesis of liver sinusoidal endothelial cells, and suppress the activation of hepatic stellate cells. In vivo, sheet transplantation significantly suppressed liver fibrosis in the induced liver fibrosis model and accelerated liver regeneration in the hepatectomy model.

Autologous skeletal myoblast cell sheet transplantation significantly improved the liver failure in the in vitro and in vivo models. Sheet transplantation is expected to have the potential to be a clinically therapeutic option for liver regeneration in liver failure.

In recent years, research on human umbilical cord mesenchymal stem cells (hUCMSCs) derived from human umbilical cord tissue has accelerated and entered clinical application research. Compared with mesenchymal stem cells (MSCs) from other sources, hUCMSCs can be extracted from different parts of umbilical cord or from the whole umbilical cord. It has the characteristics of less ethical controversy, high differentiation potential, strong proliferation ability, efficient expansion in vitro, avoiding immune rejection and immune privilege, and avoids the limitations of lack of embryonic stem cells, heterogeneity, ethical and moral constraints. hUCMSCs avoid the need for embryonic stem cell sources, heterogeneity, and ethical and moral constraints. Bone defects are very common in clinical practice, but completely effective bone tissue regeneration treatment is challenging. Currently, autologous bone transplantation and allogeneic bone transplantation are main treatment approaches in clinical work, but each has different shortcomings, such as limited sources, invasiveness, immune rejection and insufficient osteogenic ability. Therefore, to solve the bottleneck of bone tissue regeneration and repair, a great amount of research has been carried out to explore the clinical advantages of hUCMSCs as seed cells to promote osteogenesis.However, the regulation of osteogenic differentiation of hUCMSCs is an extremely complex process. Although a large number of studies have demonstrated that the role of hUCMSCs in enhancing local bone regeneration and repair through osteogenic differentiation and transplantation into the body involves multiple signaling pathways, there is no relevant article that summarize the findings. This article discusses the osteogenesis-related regulatory mechanisms of hUCMSCs, summarizes the currently known related mechanisms, and speculates on the possible signals.

This study aimed to explore the effect of periostin in the osteogenic abilities of dental follicle stem cells (DFSCs) and DFSC sheets in the inflammatory microenvironment.

DFSCs were isolated from dental follicles and identified. A lentiviral vector was used to knock down periostin in DFSCs. 250 ng/ml lipopolysaccharide from Porphyromonas gingivalis (P.g-LPS) was used to construct the inflammatory microenvironment. Osteogenic differentiation was evaluated by alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot. The formation of extracellular matrix was assessed by qRT-PCR and immunofluorescence. The expressions of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) were measured by western blot.

Knockdown of periostin inhibited osteogenic differentiation and promoted adipogenic differentiation of DFSCs. In an inflammatory microenvironment, knockdown of periostin attenuated the proliferation and osteogenic differentiation of DFSCs. Knockdown of periostin inhibited the formation of extracellular matrix collagen I (COL-I), fibronectin, and laminin in DFSC sheets, but did not affect the expression of osteogenesis-related markers alkaline phosphatase (ALP) and osteocalcin (OCN). In the inflammatory microenvironment, knocking down periostin inhibited the expression of OCN and OPG in DFSC sheets, and promoted the expression of RANKL.

Periostin played a key role in maintaining the osteogenic abilities of DFSCs and DFSC sheets in the inflammatory microenvironment and might be an important molecule in the process of DFSCs coping with inflammatory microenvironment and promoting periodontal tissues regeneration.

Cell sheet fabrication for articular cartilage regenerative medicine necessitates a large number of chondrocytes of consistent quality as a cell source. Previously, we have developed human-induced pluripotent stem cell (iPSC)-derived expandable PRRX1⁺ limb-bud mesenchymal cells (ExpLBM) with stable expansion and high chondrogenic capacity, while in this study; our ExpLBM technology was combined with cell sheet engineering to assess its potential as a stable cell source for articular cartilage regeneration.

ExpLBM cells derived from human-induced pluripotent stem cells (hiPSCs), including 414C2 and Ff-KVs09 (HLA homozygous), were seeded onto a culture plate and two-dimensional chondrogenic induction (2-DCI) was initiated. After 2-DCI, ExpLBM-derived chondrocytes were stripped and transferred to temperature-responsive culture inserts and the chondrocyte sheets were histologically examined or transplanted into osteochondral knee defects of immunodeficient rats.

Immunohistochemistry revealed that ExpLBM-derived cell sheets were positive for Safranin O, COL2, and ACAN but that they were negative for COL1 and RUNX2. Furthermore, the engrafted tissues in osteochondral knee defects in immunodeficient rats were stained with SafO, human VIMENTIN, ACAN, and COL2.

The present study is the first to report the chondrocyte sheet fabrication with hiPSC-derived cell source. hiPSC-derived ExpLBM would be a promising cell source for cell sheet technology in articular cartilage regenerative medicine.

In this research, we introduced a novel strategy for fabricating cell sheets (CSs) prepared by simply adding a fibrinogen solution to growth medium without using any synthetic polymers or chemical agents. We confirmed that the fibrinogen-based CS could be modified for target tissue regardless of size, shape, and cell types. Also, fibrinogen-based CSs were versatile and could be used to form three-dimensional (3D) CSs such as multi-layered CSs and those mimicking native blood vessels. We also prepared fibrinogen-based spheroid sheets for the treatment of ischemic disease. The fibrinogen-based spheroid sheets had much higherin vitrotubule formation and released more angiogenic factors compared to other types of platform in this research. We transplanted fibrinogen-based spheroid sheets into a mouse hindlimb ischemia model and found that fibrinogen-based spheroid sheets showed significantly improved physiological function and blood perfusion rates compared to the other types of platform in this research.

Methods to induce islet β-cells from induced pluripotent stem cells or embryonic stem cells have been established. However, islet β-cells are susceptible to apoptosis under hypoxic conditions, so the technique used to transplant β-cells must maintain the viability of cells in vivo. This study describes the development of a tricultured cell sheet, which was made by coculturing islet β-cells, vascular endothelial cells, and mesenchymal stem cells for 1 day. The islet β-cells in the tricultured cell sheet self-organized into islet-like structures surrounded by a dense vascular network in vitro. Triple-layered tricultured cell sheets engrafted well after transplantation in vivo and developed into insulin-secreting tissue with abundant blood vessels and a high density of islet β-cells. We anticipate that the tricultured cell sheet could be used as an in vitro pseudo-islet model for pharmaceutical testing and may have potential for development into transplantable grafts for use in regenerative medicine.

In regenerative medicine, cell sheet engineering has various advantages, including the retention of cells at the transplantation site for a longer period and the local delivery of growth factors and cytokines. Adipose-derived stem cell (ASC) is widely used owing to their various functions such as wound healing, immunomodulation, and nerve regeneration, in addition to their ability to differentiate into adipocytes, chondrocytes, and osteoblasts. ASC sheet generated using cell sheet engineering is considered effective in preventing anastomotic leakage, a serious postoperative complication in gastrointestinal surgery. However, the ASC sheet is too soft, thin, and brittle to handle with laparoscopic forceps during the operation. Therefore, we considered using the peritoneum, which is stiff and easy to collect while operating, as an alternative support. In this study, we explored the feasibility of using the peritoneum as a support for the precise transplantation of ASC sheets during surgery.

ASCs were isolated from the subcutaneous fat of the inguinal region of Sprague-Dawley (SD) transgenic rats expressing green fluorescent protein. ASCs were cultured until passage 3, seeded in temperature-responsive culture dishes, and the resulting ASC sheet was harvested at more than 80% confluency. Non-transgenic SD rats were used for transplant experiments. The wall peritoneum was harvested from SD rats following laparotomy, and hybrid adipose-derived stem cell (HASC) sheet was prepared by laminating the peritoneum with ASC sheet. The cell sheets were transplanted on the backs of SD rats following the incision. On post-transplantation days 3 and 7, the specimens were extracted. ASC and HASC sheets were then compared macroscopically and histopathologically.

HASC sheet transplantation was macroscopically and histopathologically more effective than ASC sheet transplantation. The peritoneum provided sufficient stiffness as a support for precise transplantation.

The newly developed HASC sheet, which combine the advantages of ASC sheet with those of the peritoneum, could be more useful for clinical application than the ASC sheet alone.

Cell sheet technique using mesenchymal stem cells is a high-level strategy in periodontal regenerative medicine. Although recent studies have shown the role of MSCSs in increased dental supporting tissues and bone, there is no systematic review focused specifically on assessing periodontal regeneration in orthotopic animal models.

To evaluate the potential of mesenchymal stem cell sheets (MSCSs) on periodontal regeneration, compared to control, in experimental animal models Methods: Pre-clinical studies in periodontal defects of animal models were considered eligible. The electronic search included the MEDLINE, Web of Science, EMBASE and LILACS databases. The review was conducted according to the Preferred Reporting Item for Systematic Reviews and Meta-Analyses statement guidelines.

A total of 17 of the 3989 studies obtained from the electronic database search were included. MSCSs included dental follicle (DF) MSCSs, periodontal ligament (PL) MSCSs, dental pulp (DP) MSCSs, bone marrow (BM) MSCSs, alveolar periosteal (AP) MSCSs and gingival (G) MSCSs. Regarding cell sheet inducing protocol, most of the studies used ascorbic acid (52.94%). Others used culture dishes grafted with a temperature-responsive polymer (47.06%). Adverse effects were not identified in the majority of studies. Meta-analysis was not considered because of methodological heterogeneities. PDL-MSCSs were superior for periodontal regeneration enhancement compared to the control, but in an induced inflammatory microenvironment, DF-MSCSs were better. Moreover, DF-MSCSs, DP-MSCSs, and BM-MSCSs showed improved results compared to the control.

MSCSs can improve periodontal regeneration in animal periodontal defect models.

A direct transfer of a cell sheet from a culture surface to a target tissue is introduced. Commercially available, flexible parylene is used as the culture surface, and it is proposed that the UV-treated parylene offers adequate and intermediate levels of cell adhesiveness for both the stable cell attachment during culture and for the efficient cell transfer to a target surface. The versatility of this cell-transfer process is demonstrated with various cell types, including MRC-5, HDFn, HULEC-5a, MC3T3-E1, A549, C2C12 cells, and MDCK-II cells. The novel cell-sheet engineering is based on a mechanism of interfacial cell migration between two surfaces with different adhesion preferences. Monitoring of cytoskeletal dynamics and drug treatments during the cell-transfer process reveals that the interfacial cell migration occurs by utilizing the existing transmembrane proteins on the cell surface to bind to the targeted surface. The re-establishment and reversal of cell polarity after the transfer process are also identified. Its unique capabilities of 3D multilayer stacking, freeform design, and curved surface application are demonstrated. Finally, the therapeutic potential of the cell-sheet delivery system is demonstrated by applying it to cutaneous wound healing and skin-tissue regeneration in mice models.

Allogeneic cell therapies are not fully effective in treating osteoarthritis of the knee (OAK). We recently reported that transplantation of autologous chondrocyte cell-sheets along with open-wedge high tibial osteotomy promoted hyaline cartilage repair in humans. Here we describe our regenerative therapy for OAK using polydactyly-derived allogeneic chondrocyte cell-sheets (PD sheets) and temperature-responsive culture inserts. Ten patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV received the therapy. Cartilage viscoelasticity and thickness were assessed before and after transplantation. Arthroscopic biopsies obtained 12 months after transplantation were analyzed histologically. Gene expression was analyzed to evaluate the PD sheets. In this small initial longitudinal series, PD sheet transplantation was effective in treating OAK, as indicated by changes in cartilage properties. Gene marker sets in PD sheets may predict outcomes after therapy and provide markers for the selection of donor cells. This combined surgery may be an ideal regenerative therapy with disease-modifying effects in OAK patients.

Organoids are 3D structures generated from stem cells. Their functions and physiological characteristics are similar to those of normal organs. They are used in disease mechanism research, new drug development, organ transplantation and other fields. In recent years, the application of 3D materials in plastic surgery for repairing injuries, filling, tissue reconstruction and regeneration has also been investigated. The PubMed/MEDLINE database was queried to search for animal and human studies published through July of 2022 with search terms related to Organoids, Plastic Surgery, Pluripotent Stem Cells, Bioscaffold, Skin Reconstruction, Bone and Cartilage Regeneration. This review presents stem cells, scaffold materials and methods for the construction of organoids for plastic surgery, and it summarizes their research progress in plastic surgery in recent years.Level of Evidence III This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

In recent years, tissue engineering studies have proposed several approaches to regenerate periodontium based on the use of three-dimensional (3D) tissue scaffolds alone or in association with periodontal ligament stem cells (PDLSCs). The rapid evolution of bioprinting has sped up classic regenerative medicine, making the fabrication of multilayered scaffolds-which are essential in targeting the periodontal ligament (PDL)-conceivable. Physiological mechanical loading is fundamental to generate this complex anatomical structure ex vivo. Indeed, loading induces the correct orientation of the fibers forming the PDL and maintains tissue homeostasis, whereas overloading or a failure to adapt to mechanical load can be at least in part responsible for a wrong tissue regeneration using PDLSCs. This review provides a brief overview of the most recent achievements in periodontal tissue engineering, with a particular focus on the use of PDLSCs, which are the best choice for regenerating PDL as well as alveolar bone and cementum. Different scaffolds associated with various manufacturing methods and data derived from the application of different mechanical loading protocols have been analyzed, demonstrating that periodontal tissue engineering represents a proof of concept with high potential for innovative therapies in the near future.

The robust thermoresponsive and bioactive surfaces for tissue engineering by combining poly-N-isopropylacrylamide (PNIPAM) and cellulose sulfate (CS) remain highly in demand but not yet realized. Herein, PNIPAM-grafted cellulose sulfates (PCSs) with diverse degrees of substitution ascribed to sulfate groups (DS_S) are synthesized for the first time. Higher sulfated PCS2 generally forms larger aggregates than lower sulfated PCS1 at their cloud point temperatures (TCP) of around 33 °C, whereas PCS1 leads to larger aggregates at body temperature (37 °C). Via the layer-by-layer (LbL) technique, biocompatible polyelectrolyte multilayers (PEMs) composed of PCSs as polyanions in combination with poly-l-lysine (PLL) or quaternized chitosan (QCHI) as polycations were fabricated. The resulting surfaces contained a more intermingled structure of polyanions with both polycations, while higher sulfated cellulose derivatives (CS2 and PCS2) displayed greater stability. Studies on toxicity and biocompatibility of PEM using 3T3 mouse fibroblasts showed a lower cytotoxicity of PEM with PCS2 and CS2 than PCS1 and CS1. Furthermore, the PEM using PCS2 particularly in combination with QCHI demonstrated excellent biocompatibility that is promising for new bioactive, thermoresponsive coatings on biomaterials and substrata for culturing adhesion-dependent cells.

Many cell-based therapies are challenged by the poor localization of introduced cells and the use of biomaterial scaffolds with questionable biocompatibility or bio-functionality. Endothelial progenitor cells (EPCs), a popular cell type used in cell-based therapies due to their robust angiogenic potential, are limited in their therapeutic capacity to develop into mature vasculature. Here, we demonstrate a joint delivery of human-derived endothelial progenitor cells (EPC) and smooth muscle cells (SMC) as a scaffold-free, bi-level cell sheet platform to improve ventricular remodeling and function in an athymic rat model of myocardial infarction. The transplanted bi-level cell sheet on the ischemic heart provides a biomimetic microenvironment and improved cell-cell communication, enhancing cell engraftment and angiogenesis, thereby improving ventricular remodeling. Notably, the increased density of vessel-like structures and upregulation of biological adhesion and vasculature developmental genes, such as Cxcl12 and Notch3, particularly in the ischemic border zone myocardium, were observed following cell sheet transplantation. We provide compelling evidence that this SMC-EPC bi-level cell sheet construct can be a promising therapy to repair ischemic cardiomyopathy.

Polyrotaxanes (PRXs) containing acetylated α-cyclodextrins exhibit a temperature-dependent phase transition in aqueous solutions across their lower critical solution temperature (LCST) of approximately 26.6 °C. To gain insights into the interactions of acetylated PRXs (Ac-PRXs) with biological components, thermoresponsive supramolecular surfaces were prepared by coating tissue culture polystyrene (TCPS) surfaces with Ac-PRX triblock copolymers, and their surface properties across the LCST were evaluated. The wettability and protein adsorption of Ac-PRX-coated surfaces changed significantly between 10 and 37 °C, whereas the uncoated TCPS and unmodified PRX-coated surfaces did not alter the wettability and protein adsorption at 10 and 37 °C. The adhesion, proliferation, morphology, and adhesion strength of NIH/3T3 cells on Ac-PRX-coated surfaces were found to be similar to those of the uncoated and unmodified PRX-coated surfaces. However, the adhesion strength of NIH/3T3 cells on Ac-PRX-coated surfaces decreased drastically at 10 °C. Consequently, the cells spontaneously detached from the Ac-PRX-coated surfaces without enzymatic treatment. Additionally, when incubating confluent cells at 10 °C, the cells detached from Ac-PRX-coated surfaces as cell sheets while retaining extracellular matrix proteins. The findings of this study provide new directions for the design of thermoresponsive supramolecular biointerfaces for applications in bioseparation and cell manipulation.

The choice of bone substitutes for the treatment of infected bone defects (IBDs) has attracted the attention of surgeons for years. However, single-stage bioabsorbable materials that are used as carriers for antibiotic release, as well as scaffolds for BMSC sheets, need further exploration. Our study was designed to investigate the effect of vancomycin-loaded calcium sulfate hemihydrate/nanohydroxyapatite/carboxymethyl chitosan (CSH/n-HA/CMCS) hydrogels combined with BMSC sheets as bone substitutes for the treatment of IBDs.

BMSCs were harvested and cultured into cell sheets. After the successful establishment of an animal model with chronic osteomyelitis, 48 New Zealand white rabbits were randomly divided into 4 groups. Animals in Group A were treated with thorough debridement as a control. Group B was treated with BMSC sheets. CSH/n-HA/CMCS hydrogels were implanted in the treatment of Group C, and Group D was treated with CSH/n-HA/CMCS+BMSC sheets. Gross observation and micro-CT 3D reconstruction were performed to assess the osteogenic and infection elimination abilities of the treatment materials. Histological staining (haematoxylin and eosin and Van Gieson) was used to observe inflammatory cell infiltration and the formation of collagen fibres at 4, 8, and 12 weeks after implantation.

The bone defects of the control group were not repaired at 12 weeks, as chronic osteomyelitis was still observed. HE staining showed a large amount of inflammatory cell infiltration around the tissue, and VG staining showed no new collagen fibres formation. In the BMSC sheet group, although new bone formation was observed by gross observation and micro-CT scanning, infection was not effectively controlled due to unfilled cavities. Some neutrophils and only a small amount of collagen fibres could be observed. Both the hydrogel and hydrogel/BMSCs groups achieved satisfactory repair effects and infection control. Micro-CT 3D reconstruction at 4 weeks showed that the hydrogel/BMSC sheet group had higher reconstruction efficiency and better bone modelling with normal morphology. HE staining showed little aggregation of inflammatory cells, and VG staining showed a large number of new collagen fibres.

Our preliminary results suggested that compared to a single material, the novel antibiotic-impregnated hydrogels acted as superior scaffolds for BMSC sheets and excellent antibiotic vectors against infection, which provided a basis for applying tissue engineering technology to the treatment of chronic osteomyelitis.