Bioorthogonal targeted cell membrane vesicles/cell-sheet composites reduce postoperative tumor recurrence and scar formation of melanoma.

While surgical resection is the predominant clinical strategy in the treatment of melanoma, postoperative recurrence and undetectable metastasis are both pernicious drawbacks to this otherwise highly successful approach. Furthermore, the deep cavities result from tumor excision can leave long lasting wounds which are slow to heal and often leave visible scars. These unmet needs are addressed in the present work through the use of a multidimensional strategy, and also promotes wound healing and scar reduction. In the first phase, cell membrane-derived nanovesicles (NVs) are engineered to show PD-1 and dibenzocyclooctyne (DBCO). These are capable of reactivating T cells by blocking the PD-1/PD-L1 pathway. In the second phase, azido (N3) labeled mesenchymal stem cells (MSCs) are cultured into cell sheets using tissue engineering, then apply directly to surgical wounds to enhance tissue repair. Owing to the complementary association between DBCO and N3 groups, PD-1 NVs were accumulated at the site of excision. This strategy can inhibit postoperative tumor recurrence and metastasis, whilst also promoting wound healing and reducing scar formation. The results of this study set a precedent for a new and innovative multidimensional therapeutic strategy in the postoperative treatment of melanoma.

Human platelet lysate-cultured adipose-derived stem cell sheets promote angiogenesis and accelerate wound healing via CCL5 modulation.

BACKGROUND: A rising population faces challenges with healing-impaired cutaneous wounds, often leading to physical disabilities. Adipose-derived stem cells (ASCs), specifically in the cell sheet format, have emerged as a promising remedy for impaired wound healing. Human platelet lysate (HPL) provides an attractive alternative to fetal bovine serum (FBS) for culturing clinical-grade ASCs. However, the potential of HPL sheets in promoting wound healing has not been fully investigated. This study aimed to explore the anti-fibrotic and pro-angiogenic capabilities of HPL-cultured ASC sheets and delve into the molecular mechanism. METHODS: A rat burn model was utilized to evaluate the efficacy of HPL-cultured ASC sheets in promoting wound healing. ASC sheets were fabricated with HPL, and those with FBS were included for comparison. Various analyses were conducted to assess the impact of HPL sheets on wound healing. Histological examination of wound tissues provided insights into aspects such as wound closure, collagen deposition, and overall tissue regeneration. Immunofluorescence was employed to assess the presence and distribution of transplanted ASCs after treatment. Further in vitro studies were conducted to decipher the specific factors in HPL sheets contributing to angiogenesis. RESULTS: HPL-cultured ASC sheets significantly accelerated wound closure, fostering ample and organized collagen deposition in the neo-dermis. Significantly more retained ASCs were observed in wound tissues treated with HPL sheets compared to the FBS counterparts. Moreover, HPL sheets mitigated macrophage recruitment and decreased subsequent wound tissue fibrosis in vivo. Immunohistochemistry also indicated enhanced angiogenesis in the HPL sheet group. The in vitro analyses showed upregulation of C-C motif chemokine ligand 5 (CCL5) and angiogenin in HPL sheets, including both gene expression and protein secretion. Culturing endothelial cells in the conditioned media compared to media supplemented with CCL5 or angiogenin suggested a correlation between CCL5 and the pro-angiogenic effect of HPL sheets. Additionally, through neutralizing antibody experiments, we further validated the crucial role of CCL5 in HPL sheet-mediated angiogenesis in vitro. CONCLUSIONS: The present study underscores CCL5 as an essential factor in the pro-angiogenic effect of HPL-cultured ASC sheets during the wound healing process. These findings highlight the potential of HPL-cultured ASC sheets as a promising therapeutic option for healing-impaired cutaneous wounds in clinical settings. Furthermore, the mechanism exploration yields valuable information for optimizing regenerative strategies with ASC products. BRIEF ACKNOWLEDGMENT: This research was supported by the National Science and Technology Council, Taiwan (NSTC112-2321-B-002-018), National Taiwan University Hospital (111C-007), and E-Da Hospital-National Taiwan University Hospital Joint Research Program (111-EDN0001, 112-EDN0002).

Harvesting methods of umbilical cord-derived mesenchymal stem cells from culture modulate cell properties and functions.

Introduction: Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are promising candidates for stem cell therapy. Various methods such as enzymatic treatment, cell scraping, and temperature reduction using temperature-responsive cell culture dishes have been employed to culture and harvest UC-MSCs. However, the effects of different harvesting methods on cell properties and functions in vitro remain unclear. In this study, we investigated the properties and functions of UC-MSC using various cell-harvesting methods. Methods: UC-MSC suspensions were prepared using treatments with various enzymes, cell scraping, and temperature reduction in temperature-responsive cell culture dishes. UC-MSC sheets were prepared in a temperature-responsive cell culture dish. The properties and functions of the UC-MSC suspensions and sheets were assessed according to Annexin V staining, lactate dehydrogenase (LDH) assay, re-adhesion behavior, and cytokine secretion analysis via enzyme-linked immunosorbent assay. Results: Annexin V staining revealed that accutase induced elevated UC-MSC apoptosis. Physical scraping using a cell scraper induced a relatively high LDH release due to damaged cell membranes. Dispase exhibited relatively low adhesion from initial incubation until 3 h. UC-MSC sheets exhibited rapid re-adhesion at 15 min and cell migration at 6 h. UC-MSC sheets expressed higher levels of cytokines such as HGF, TGF-ß1, IL-10, and IL-6 than did UC-MSCs in suspension. Conclusions: The choice of enzyme and physical scraping methods for harvesting UC-MSCs significantly influenced their activity and function. Thus, selecting appropriate cell-harvesting methods is important for successful stem cell therapy.

Transplantation of Neural Progenitor Cells Derived from Stem Cells from Apical Papilla Through Small-Molecule Induction in a Rat Model of Sciatic Nerve Injury.

BACKGROUND: Stem cell-based transplantation therapy holds promise for peripheral nerve injury treatment, but adult availability is limited. A cell culture protocol utilizing a small-molecule cocktail effectively reprogrammed stem cells from apical papilla (SCAPs) into neural progenitor cells, subsequently differentiating into neuron-like cells. This study aims to evaluate neural-induced SCAPs, with and without small-molecule cocktail, for sciatic nerve repair potential. METHODS: A scaffold-free cell sheet technique was used to construct a three-dimensional cell sheet. Subsequently, this cell sheet was carefully rolled into a tube and seamlessly inserted into a collagen conduit, which was then transplanted into a 5 mm sciatic nerve injury rat model. Functional sciatic nerve regeneration was evaluated via toe spread test, walking track analysis and gastrocnemius muscle weight. Additionally, degree of sciatic nerve regeneration was determined based on total amount of myelinated fibers. RESULTS: Small-molecule cocktail induced SCAPs enhanced motor function recovery, evident in improved sciatic function index and gastrocnemius muscle retention. We also observed better host myelinated fiber retention than undifferentiated SCAPs or neural-induced SCAPs without small-molecule cocktail. However, clusters of neuron-like cell bodies (surrounded by sparse myelinated fibers) were found in all cell sheet-implanted groups in the implantation region. This suggests that while the implanted cells likely survived transplantation, integration was poor and would likely hinder long-term recovery by occupying the space needed for host nerve fibers to project through. CONCLUSION: Neural-induced SCAPs with small-molecule cocktail demonstrated promising benefits for nerve repair; further research is needed to improve its integration and optimize its potential for long-term recovery.

Dual-color FISH analyses of xenogeneic human fibroblast sheets transplanted to repair lung pleural defects in an immunocompromised rat model.

BACKGROUND: Pulmonary air leaks (PALs) due to visceral pleura injury during surgery is frequently observed after pulmonary resections and the complication is difficult to avoid in thoracic surgery. The development of postoperative PALs is the most common cause of prolonged hospitalization. Previously, we reported that PALs sealants using autologous dermal fibroblast sheets (DFSs) harvested from temperature-responsive culture dishes successfully closed intraoperative PALs during lung resection. OBJECTIVE: In this study, we investigated the fate of human DFSs xenogenetically transplanted onto lung surfaces to seal PALs of immunocompromised rat. Dual-color FISH analyses of human fibroblast was employed to detect transplantation human cells on the lung surface. RESULTS: One month after transplantation, FISH analyses revealed that transplanted human fibroblasts still composed a sheet-structure, and histology also showed that beneath the sheet's angiogenesis migrating into the sheets was observed from the recipient tissues. FISH analyses revealed that even at 3 months after transplantation, the transplanted human fibroblasts still remained in the sheet. Dual-color FISH analyses of the transplanted human fibroblasts were sparsely present as a result of the cells reaching the end of their lifespan, the cells producing extracellular matrix, and remained inside the cell sheet and did not invade the lungs of the host. CONCLUSIONS: DFS-transplanted human fibroblasts showed that they are retained within cell sheets and do not invade the lungs of the host.

Improving the mechanical and biological functions of cell sheet constructs: The interplay of human-derived periodontal ligament stem cells, endothelial cells and plasma rich in growth factors.

OBJECTIVE: The aim of this study was to produce and characterize triple-layered cell sheet constructs with varying cell compositions combined or not with the fibrin membrane scaffold obtained by the technology of Plasma Rich in Growth Factors (mPRGF). MATERIALS AND METHODS: Human primary cultures of periodontal ligament stem cells (hPDLSCs) were isolated, and their stemness nature was evaluated. Three types of triple-layered composite constructs were generated, composed solely of hPDLSCs or combined with human umbilical vein endothelial cells (HUVECs), either as a sandwiched endothelial layer or as coculture sheets of both cell phenotypes. These three triple-layered constructs were also manufactured using mPRGF as cell sheets' support. Necrosis, glucose consumption, secretion of extracellular matrix proteins and synthesis of proangiogenic factors were determined. Histological evaluations and proteomic analyses were also performed. RESULTS: The inclusion of HUVECs did not clearly improve the properties of the multilayered constructs and yet hindered their optimal conformation. The presence of mPRGF prevented the shrinkage of cell sheets, stimulated the metabolic activity and increased the matrix synthesis. At the proteome level, mPRGF conferred a dramatic advantage to the hPDLSC constructs in their ability to provide a suitable environment for tissue regeneration by inducing the expression of proteins necessary for bone morphogenesis and cellular proliferation. CONCLUSIONS: hPDLSCs' triple-layer construct onto mPRGF emerges as the optimal structure for its use in regenerative therapeutics. CLINICAL RELEVANCE: These results suggest the suitability of mPRGF as a promising tool to support cell sheet formation by improving their handling and biological functions.

A first-in-human clinical study of laparoscopic autologous myoblast sheet transplantation to prevent delayed perforation after duodenal endoscopic mucosal dissection.

BACKGROUND: The detection rate of superficial non-ampullary duodenal epithelial tumors (SNADETs) has recently been increasing. Large tumors may contain malignant lesions and early therapeutic intervention is recommended. Endoscopic mucosal dissection (ESD) is considered a feasible treatment modality, however, the anatomical and physiological characteristics of the duodenum create a risk of postoperative perforation after ESD. METHODS: To explore whether myoblast sheet transplantation could prevent delayed perforation after ESD, a first-in-human (FIH) clinical trial of laparoscopic autologous myoblast sheet transplantation after duodenal ESD was launched. Autologous myoblast sheets fabricated from muscle tissue obtained seven weeks before ESD were transplanted laparoscopically onto the serous side of the ESD. The primary endpoints were the onset of peritonitis due to delayed perforation within three days after surgery and all adverse events during the follow-up period. RESULTS: Three patients with SNADETs ≥ 20 mm in size underwent transplantation of a myoblast sheet onto the serous side of the duodenum after ESD. In case 1, The patient's postoperative course was uneventful. Endoscopy and abdominal computed tomography revealed no signs of delayed perforation. Despite incomplete mucosal closure in case 2, and multiple micro perforations during ESD in case 3, cell sheet transplantation could prevent the postoperative massive perforation after ESD, and endoscopy on day 49 after transplantation revealed no stenosis. CONCLUSIONS: This clinical trial showed the safety, efficacy, and procedural operability of this novel regenerative medicine approach involving transplanting an autologous myoblast sheet laparoscopically onto the serosa after ESD in cases with a high risk of delayed perforation. This result indicates the potential application of cell sheet medicine in treating various abdominal organs and conditions with minimal invasiveness in the future. TRIAL REGISTRATION: jRCT, jRCT2073210094. Registered November 8 2021, https://jrct.niph.go.jp/latest-detail/jRCT2073210094 .

Hydrogels with dual sensitivity to temperature and pH in physiologically relevant ranges as supports for versatile controlled cell detachment.

Thermosensitive hydrogels based on the N-vinyl caprolactam (VCL), capable of allowing for cell adhesion and proliferation, as well as non-aggressive detachment by controlled temperature drop, were functionalized with 23 % or lower molar percentages of the cationizable hydrophobic unit 2-(diisopropylamino) ethyl methacrylate (DPAEMA), to obtain networks with dual sensitivity to temperature and pH. The swelling analysis of the systems has shown a transition pK (pKb) close to physiological values, dependent on the temperature of the medium (pKb of 6.6 and 6.9 when the temperature of the medium is above and below the transition temperature VPTT, respectively) and little dependence on the degree of functionalization of DPAEMA. In addition, at temperatures below the transition temperature (VPTT), the systems have shown large swelling variations as a function of the pH (i.e. below and above the pKb), exhibiting greater absorption capacity at pHs below pKb, where the DPAEMA units are cationized. Cytocompatibility and transplant capacity have been evaluated using the C166-GFP endothelial cell line. None of the thermosensitive hydrogels with variable DPAEMA content showed a delay with respect to the control without DPAEMA neither in terms of adhesion nor in proliferation. However, by increasing the percentage of DPAEMA functionalization -and decreasing thermosensitivity-, a correlative decrease in mitochondrial activity was obtained in the transplant, with significant differences for the hydrogels with DPAEMA molar percentage of 3 % or higher. Taking advantage of the proximity of the pKb to the physiological value, we have evaluated the cellular response and the capacity for transplantation after lowering the pH to 6.5, below pKb. A direct relationship of the DPAEMA functionalization degree on the detachment efficiency was observed, since the hydrogels with the highest molar load of DPAEMA showed higher mitochondrial metabolic activity after cell detachment.

Cell sheet formation enhances the therapeutic effects of adipose-derived stromal vascular fraction on urethral stricture.

Urethral stricture (US) is a common disease in urology, lacking effective treatment options. Although injecting a stem cells suspension into the affected area has shown therapeutic benefits, challenges such as low retention rate and limited efficacy hinder the clinical application of stem cells. This study evaluates the therapeutic impact and the mechanism of adipose-derived vascular fraction (SVF) combined with cell sheet engineering technique on urethral fibrosis in a rat model of US. The results showed that SVF-cell sheets exhibit positive expression of α-SMA, CD31, CD34, Stro-1, and eNOS. In vivo study showed less collagen deposition, low urethral fibrosis, and minimal tissue alteration in the group receiving cell sheet transplantation. Furthermore, the formation of a three-dimensional (3D) tissue-like structure by the cell sheets enhances the paracrine effect of SVF, facilitates the infiltration of M2 macrophages, and suppresses the TGF-ß/Smad2 pathway through HGF secretion, thereby exerting antifibrotic effects. Small animal in vivo imaging demonstrates improved retention of SVF cells at the damaged urethra site with cell sheet application. Our results suggest that SVF combined with cell sheet technology more efficiently inhibits the early stages of urethral fibrosis.

Rapamycin Inhibits Senescence and Improves Immunomodulatory Function of Mesenchymal Stem Cells Through IL-8 and TGF-ß Signaling.

Mesenchymal stromal cells (MSCs) grown in high-density monolayers (sheets) are promising vehicles for numerous bioengineering applications. When MSC sheets are maintained in prolonged cultures, they undergo rapid senescence, limiting their downstream efficacy. Although rapamycin is a potential agent that can inhibit senescence in cell cultures, no study has investigated rapamycin's effect on MSCs grown in high-density culture and its effect on downstream target gene expression. In this study, placental-derived MSCs (PMSCs) were seeded at high density to generate PMSC sheets in 24 hours and were then treated with rapamycin or vehicle for up to 7 days. Autophagy activity, cell senescence and apoptosis, cell size and granularity, and senescence-associated cytokines (IL-6 and IL-8) were analyzed. Differential response in gene expression were assessed via microarray analysis. Rapamycin significantly increased PMSC sheet autophagy activity, inhibited cellular senescence, decreased cell size and granularity at all timepoints. Rapamycin also significantly decreased the number of cells in late apoptosis at day 7 of sheet culture, as well as caspase 3/7 activity at all timepoints. Notably, while rapamycin decreased IL-6 secretion, increased IL-8 levels were observed at all timepoints. Microarray analysis further confirmed the upregulation of IL-8 transcription, as well as provided a list of 396 genes with 2-fold differential expression, where transforming growth factor-ß (TGF-ß) signaling were identified as important upregulated pathways. Rapamycin both decreased senescence and has an immunomodulatory action of PMSCs grown in sheet culture, which will likely improve the chemotaxis of pro-healing cells to sites of tissue repair in future bioengineering applications.

Translational application of human keratinocyte-fibroblast cell sheets for accelerated wound healing in a clinically relevant type 2 diabetic rat model.

BACKGROUND AIMS: Despite advancements in wound care, wound healing remains a challenge, especially in individuals with type 2 diabetes. Cell sheet technology has emerged as an efficient and promising therapy for tissue regeneration and wound repair. Among these, bilayered human keratinocyte-fibroblast cell sheets constructed using temperature-responsive culture surfaces have been shown to mimic a normal tissue-like structure and secrete essential cytokines and growth factors that regulate the wound healing process. METHODS: This study aimed to evaluate the safety and therapeutic potential of human skin cell sheets to treat full-thickness skin defects in a rat model of type 2 diabetes. RESULTS: Our findings demonstrate that diabetic wounds transplanted with bilayered cell sheets resulted in accelerated re-epithelialization, increased angiogenesis, enhanced macrophage polarization and regeneration of tissue that closely resembled healthy skin. In contrast, the control group that did not receive cell sheet transplantation presented characteristic symptoms of impaired and delayed wound healing associated with type 2 diabetes. CONCLUSIONS: The secretory cytokines and the upregulation of Nrf2 expression in response to cell sheet transplantation are believed to have played a key role in the improved wound healing observed in diabetic rats. Our study suggests that human keratinocyte-fibroblast cell sheets hold great potential as a therapeutic alternative for diabetic ulcers.

Production of alginate macrocapsule device for long-term normoglycaemia in the treatment of type 1 diabetes mellitus with pancreatic cell sheet engineering.

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.

Fibronectin Connecting Cell Sheet Based on Click Chemistry for Wound Repair.

As a living repair material, cell sheet exhibits significant potential in wound repair. Nonetheless, wound healing is a complicated and protracted process that necessitates specific repair functions at each stage, including hemostasis and antibacterial activity. In this work, on the basis of harvesting the cell sheet via a photothermal response strategy, a fibronectin attached cell sheet (FACS) is prepared to enhance its wound repair capability. For this purpose, the azide group (N3 ) is initially tagged onto the cell surface through metabolic glycoengineering of unnatural sugars, and then the conjugate (DBCO-fibronectin) comprises of the dibenzocyclooctyne (DBCO) and fibronectin with multiple wound repair functions is linked to N3 using click chemistry. Biological evaluations following this demonstrates that the FACS preparation exhibits excellent biocompatibility, and the fibronectin modification enhances the capacity for cell proliferation and migration. Moreover, in vivo wound healing experiment confirms the reparative efficacy of FACS. It not only has a wound closure rate 1.46 times that of a conventional cell sheet but also reduces inflammatory cell infiltration, promotes hair follicle and blood vessel regeneration, and encourages collagen deposition. This strategy holds enormous clinical potential and paves the way for advanced functional modifications of cell sheets.

Recent Advances in Cell Sheet-Based Tissue Engineering for Bone Regeneration.

In conventional bone tissue engineering, cells are seeded onto scaffolds to create three-dimensional (3D) tissues, but the cells on the scaffolds are unable to effectively perform their physiological functions due to their low density and viability. Cell sheet (CS) engineering is expected to be free from this limitation. CS engineering uses the principles of self-assembly and self-organization of endothelial and mesenchymal stem cells to prepare CSs as building blocks for engineering bone grafts. This process recapitulates the native tissue development, thus attracting significant attention in the field of bone regeneration. However, the method is still in the prebasic experimental stage in bone defect repair. To make the method clinically applicable and valuable in personalized and precision medicine, current research is focused on the preparation of multifunctionalized building blocks using CS technologies, such as 3D layered CSs containing microvascular structures. Considering the great potential of CS engineering in repairing bone defects, in this review, the types of cell technologies are first outlined. We then summarize the various types of CSs as building blocks for engineering bone grafts. Furthermore, the specific applications of CSs in bone repair are discussed. Finally, we present specific suggestions for accelerating the application of CS engineering in the clinical treatment of bone defects.

A new technology for the treatment of premature ovarian insufficiency: Cell sheet.

Premature ovarian insufficiency (POI) is defined as the development of hypergonadotropic hypogonadism before the age of 40 with definitive treatment being absent. In the current study, we aim to compare the efficacy of the cell sheet method with an intravenous (IV) application of adipose-derived mesenchymal stem cells (AdMSCs) to the POI with an animal model. In the current prospective study, 6-to-8-week-old Sprague Dawley rats were generated four groups: (i) a control group in which only PBS was administered; (ii) an only-POI group generated by cyclophosphamide; (iii) a POI group treated by way of IV AdMSCs; and (iv) a POI group treated by way of the cell sheet method. Twenty-eight days after an oophorectomy was performed, intracardiac blood was taken. Follicle count, immunohistochemical examination for GDF9, BMP15, and TUNEL were conducted, gene expressions of GDF9 and BMP15 were examined, and E2 was measured in the serum samples. With hematoxylin-eosin, in the third group, multi oocytes follicles were the most remarkable finding. In the fourth group, most of the follicles presented normal morphology. GDF9 involvement was similar between the first and fourth groups. BMP-15 immunoreactivity, in contrast to fourth group, was weak in all stages in the second and third groups. The current attempt represents a pioneer study in the literature in which a cell sheet method is used for the first time in a POI model. These results suggest that the cell sheet method may be a feasible and efficient method for the stem cell treatment of models with POI and could be a new treatment approach in POI.

Cell Sheet Technology: An Emerging Approach for Tendon and Ligament Tissue Engineering.

Tendon and ligament injuries account for a substantial proportion of disorders in the musculoskeletal system. While non-operative and operative treatment strategies have advanced, the restoration of native tendon and ligament structures after injury is still challenging due to its innate limited regenerative ability. Cell sheet technology is an innovative tool for tissue fabrication and cell transplantation in regenerative medicine. In this review, we first summarize different harvesting procedures and advantages of cell sheet technology, which preserves intact cell-to-cell connections and extracellular matrix. We then describe the recent progress of cell sheet technology from preclinical studies, focusing on the application of stem cell-derived sheets in treating tendon and ligament injuries, as well as highlighting its effects on mitigating inflammation and promoting tendon/graft-bone interface healing. Finally, we discuss several prerequisites for future clinical translation including the selection of appropriate cell source, optimization of preparation process, establishment of suitable animal model, and the fabrication of vascularized complex tissue. We believe this review could potentially provoke new ideas and drive the development of more functional biomimetic tissues using cell sheet technology to meet the needs of clinical patients.

Fabricating Composite Cell Sheets for Wound Healing: Cell Sheets Based on the Communication Between BMSCs and HFSCs Facilitate Full-Thickness Cutaneous Wound Healing.

BACKGROUND: Insufficient angiogenesis and the lack of skin appendages are critical challenges in cutaneous wound healing. Stem cell-fabricated cell sheets have become a promising strategy, but cell sheets constructed by a single cell type are inadequate to provide a comprehensive proregenerative microenvironment for wound tissue. METHODS: Based on the communication between cells, in this study, bone marrow mesenchymal stem cells (BMSCs) and hair follicle stem cells (HFSCs) were cocultured to fabricate a composite cell sheet (H/M-CS) for the treatment of full-thickness skin wounds in mice. RESULTS: Experiments confirmed that there is cell-cell communication between BMSCs and HFSCs, which enhances the cell proliferation and migration abilities of both cell types. Cell-cell talk also upregulates the gene expression of pro-angiogenic-related cytokines in BMSCs and pro-hair follicle-related cytokines in HFSCs, as well as causing changes in the properties of secreted extracellular matrix components. CONCLUSIONS: Therefore, the composite cell sheet is more conducive for cutaneous wound healing and promoting the regeneration of blood vessels and hair follicles.

Scaffold-Free Bone Marrow-Derived Mesenchymal Stem Cell Sheets Enhance Bone Formation in a Weight-Bearing Rat Critical Bone Defect Model.

Researchers have been exploring alternative methods for bone tissue engineering, as current management of critical bone defects may be a significant challenge for both patient and surgeon with conventional surgical treatments associated with several potential complications and drawbacks. Recent studies have shown mesenchymal stem cell sheets may enhance bone regeneration in different animal models. We investigated the efficacy of implanted scaffold-free bone marrow-derived mesenchymal stem cell (BMSC) sheets on bone regeneration of a critical bone defect in a weight-bearing rat model. BMSCs were isolated from the femora of male Sprague-Dawley rats 5-6 weeks of age and cell sheets were produced on temperature-responsive culture dishes. Nine male Sprague-Dawley rats 6-8 weeks of age were utilized. A bilateral femoral critical bone defect was created with a bridge plate serving as internal fixation. One side was randomly selected and BMSC sheets were implanted into the bone defect (BMSC group), with the contralateral side receiving no treatment (control). Rats were anesthetized and radiographs were performed at 2-week intervals. At the 8-week time point, rats were euthanized, femurs harvested, and microcomputed tomography and histological analysis was performed. We found a statistically significant increase in new bone formation and bone volume fraction compared with the control. Histomorphometry analysis revealed a larger percent of newly formed bone and a higher total histological score. Our results suggest that scaffold-free BMSC sheets may be used in the management of large weight-bearing bone defects to complement a different surgical technique or as a standalone approach followed by internal fixation. However, further research is still needed.

Culture of rabbit corneal stromal cells on thermo-sensitive materials by the radiation polymerization / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore the synthesis of thermo-sensitive poly N-isopropylacry-lamide(PNIPAAm)and the petri dish grafted with PNIPAAm hydrogels by the electron accelerator, as well as the growth conditions and the biological characteristics of rabbit corneal stromal cells on thermo-sensitive PNIPAAm hydrogels, and the cell sheets obtained from the PNIPAAm hydrogels.METHODS: NIPAAm monomer was dissolved in 2-propanol at concentrations of 55% with 0.5% N,N'-Methylenebisacry-lamide(MBA). Solution(70 μL)was added and spread uniformly over 35 mm petri dish. These dishes were immediately subjected to irradiation. After follow-up treatment, rabbit corneal stromal cells were cultured on thermo-sensitive petri dish in vitro.RESULTS: According to the monomer formula and radiation synthesis scheme in this experiment, PNIPAAm can be synthesized on the surface of the petri dish. Rabbit corneal stromal cells grew well in the thermo- sensitive surface and can be separated into sheets.CONCLUSION: The single and multilayer carrier-free cell sheets can be obtained from the use of thermo-sensitive petri dish.

bFGF-releasing biodegradable nanoparticles for effectively engrafting transplanted hepatocyte sheet.

Hepatic tissue engineering has been applied for the treatment of intractable liver diseases, and hepatocyte sheets are promising for this purpose. However, hepatocyte sheets have poor survival after transplantation because of their high metabolic activity. In this study, we aimed to develop basic fibroblast growth factor (bFGF)-releasing nanoparticles to prolong the survival of hepatocyte sheets after transplantation. The nanoparticles were prepared by electrospraying a bFGF-dispersed poly(D,l-lactide-co-glycolide) emulsion. bFGF-loaded PLGA nanoparticles can be developed by optimizing the applied electrospray voltage and the oil:water ratio of the emulsion. The prepared nanoparticles exhibited prompt release at the initial duration and continuous gradual release at the subsequent duration. Hepatocyte sheet engraftment was evaluated by transplanting hepatocyte sheets containing the prepared nanoparticles into rats. The hepatocyte sheets with the prepared nanoparticles exhibited longer survival than those without the bFGF nanoparticles or solution owing to the local and continuous release of bFGF from the nanoparticles and the subsequent enhanced angiogenesis at the transplantation site. These results indicated that the prepared bFGF-releasing nanoparticles can enhance the efficiency of hepatocyte sheet transplantation. The developed bFGF-releasing nanoparticles would be useful for the transplantation of cellular tissue with post-transplantation survival challenges.