Use of Cryopreserved Osteogenic Matrix Cell Sheets for Bone Reconstruction

Skeletal diseases, such as nonunion and osteonecrosis, are now treatable with tissue engineering techniques. Single cell sheets called osteogenic matrix cell sheets (OMCSs) grown from cultured bone marrow-derived mesenchymal stem cells show high osteogenic potential;however, long preparation times currently limit their clinical application. Here, we report a cryopreservation OMCS transplantation method that shortens OMCS preparation time. Cryopreserved rat OMCSs were prepared using slow- and rapid-freezing methods, thawed, and subsequently injected scaffold-free into subcutaneous sites. Rapid- and slow-frozen OMCSs were also transplanted directly to the femur bone at sites of injury. Slow-freezing resulted in higher cell viability than rapid freezing, yet all two cryopreservation methods yielded OMCSs that survived and formed bone tissue. In the rapid- and slow-freezing groups, cortical gaps were repaired and bone continuity was observed within 6 weeks of OMCS transplantation. Moreover, while no significant difference was found in osteocalcin expression between the three experimental groups, the biomechanical strength of femurs treated with slow-frozen OMCSs was significantly greater than those of non-transplant at 6 weeks post-injury. Collectively, these data suggest that slow-frozen OMCSs have superior osteogenic potential and are better suited to produce a mineralized matrix and repair sites of bone injury.

Cardiac patches made of brown adipose-derived stem cell sheets and conductive electrospun nanofibers restore infarcted heart for ischemic myocardial infarction

Cell sheet engineering has been proven to be a promising strategy for cardiac remodeling post-myocardial infarction. However, insufficient mechanical strength and low cell retention lead to limited therapeutic efficiency. The thickness and area of artificial cardiac patches also affect their therapeutic efficiency. Cardiac patches prepared by combining cell sheets with electrospun nanofibers, which can be transplanted and sutured to the surface of the infarcted heart, promise to solve this problem. Here, we fabricated a novel cardiac patch by stacking brown adipose-derived stem cells (BADSCs) sheet layer by layer, and then they were combined with multi-walled carbon nanotubes (CNTs)-containing electrospun polycaprolactone/silk fibroin nanofibers (CPSN). The results demonstrated that BADSCs tended to generate myocardium-like structures seeded on CPSN. Compared with BADSCs suspension-containing electrospun nanofibers, the transplantation of the CPSN-BADSCs sheets (CNBS) cardiac patches exhibited accelerated angiogenesis and decreased inflammation in a rat myocardial infarction model. In addition, the CNBS cardiac patches could regulate macrophage polarization and promote gap junction remodeling, thus restoring cardiac functions. Overall, the hybrid cardiac patches made of electrospun nanofibers and cell sheets provide a novel solution to cardiac remodeling after ischemic myocardial infarction.

Mechanically conditioned cell sheets cultured on thermo-responsive surfaces promote bone regeneration

Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades.However,efficient harvest and handling of cell sheets remain challenging,including insufficient extracellular matrix content and poor mechanical strength.Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types.However,currently,there are no effective ways to apply mechanical loading to cell sheets.In this study,we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide)(PNIPAAm)to poly(dimethylsiloxane)(PDMS)surfaces.The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting.Subsequently,MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates.Upon maturation,the cell sheets were harvested by lowering the temperature.We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning.Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated.After implantation into the critical-sized calvarial defects of mice,the mechanically conditioned cell sheets significantly promoted new bone formation.Findings from this study reveal that thermo-responsive elastomer,together with mechanical conditioning,can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.

Therapeutic potential of dental pulp stem cells and their derivatives:Insights from basic research toward clinical applications

For more than 20 years,researchers have isolated and identified postnatal dental pulp stem cells(DPSCs)from different teeth,including natal teeth,exfoliated deciduous teeth,healthy teeth,and diseased teeth.Their mesenchymal stem cell(MSC)-like immunophenotypic characteristics,high proliferation rate,potential for multidirectional differentiation and biological features were demonstrated to be superior to those of bone marrow MSCs.In addition,several main application forms of DPSCs and their derivatives have been investigated,including stem cell injections,modified stem cells,stem cell sheets and stem cell spheroids.In vitro and in vivo administration of DPSCs and their derivatives exhibited beneficial effects in various disease models of different tissues and organs.Therefore,DPSCs and their derivatives are regarded as excellent candidates for stem cell-based tissue regeneration.In this review,we aim to provide an overview of the potential application of DPSCs and their derivatives in the field of regenerative medicine.We describe the similarities and differences of DPSCs isolated from donors of different ages and health conditions.The methodologies for therapeutic administration of DPSCs and their derivatives are introduced,including single injections and the transplantation of the cells with a support,as cell sheets,or as cell spheroids.We also summarize the underlying mechanisms of the regenerative potential of DPSCs.

Calculated and Experimental Research of Sheet Resistances of Laser-Doped Silicon Solar Cells

The calculated and experimental research of sheet resistances of crystalline silicon solar cells by dry laser doping is investigated. The nonlinear numerical model on laser melting of crystalline silicon and liquid-phase diffusion of phosphorus atoms by dry laser doping is analyzed by the finite difference method implemented in MATLAB. The melting period and melting depth of crystalline silicon as a function of laser energy density is achieved. The effective liquid-phase diffusion of phosphorus atoms in melting silicon by dry laser doping is confirmed by the rapid decrease of sheet resistances in experimental measurement. The plateau of sheet resistances is reached at around 15 Ω/. The calculated sheet resistances as a function of laser energy density is obtained and the calculated results are in good agreement with the corresponding experimental measurement. Due to the successful verification by comparison between experimental measurement and calculated results, the simulation results could be used to optimize the virtual laser doping parameters.

AB012.Formation of scaffold-free cell sheet with eye-related cells for ophthalmic application

The translation of current tissue engineering approaches to clinical application is somehow limited by the use of scaffolding materials.Recently a number of in vitro scaffold-free three-dimensional culture techniques have been developed.These techniques realize the assembly of tissue-like structures including but not limited to spheroids,blood vessels and cartilage.In particular,cells can now self-assemble to form planar tissue-like structures at the interface of an aqueous-two-phase system(ATPS).The unique advantage of this technique is that without a solid substrate,planar tissue-like structures can now be assembled rapidly with very simple procedures.This technique can potentially be very useful for tissue engineering in eye because of its ability to direct cells to form monolayer.In this talk,we will introduce what ATPS is and its current applications in biomedical research.We will then present an approach to assemble cell sheets in ATPS using both primary cells isolated from porcine eyes and other cell lines.The physiological relevance of these eye-related cell sheets as well as their potentials in ophthalmic research and applications will be discussed.

One-step fabrication of cell sheet-laden hydrogel for accelerated wound healing

Full-thickness skin wounds are have continued to be reconstructive challenges in dermal and skin appendage regeneration, and skin substitutes are promising tools for addressing these reconstructive procedures. Herein, the one-step fabrication of a cell sheet integrated with a biomimetic hydrogel as a tissue engineered skin for skin wound healing generated in one step is introduced. Briefly, cell sheets with rich extracellular matrix, high cell density, and good cell connections were integrated with biomimetic hydrogel to fabricate gel + human skin fibroblasts (HSFs) sheets and gel + human umbilical vein endothelial cells (HUVECs) sheets in one step for assembly as a cell sheet-laden hydrogel (CSH). The designed biomimetic hydrogel formed with UV crosslinking and ionic crosslinking exhibited unique properties due to the photo-generated aldehyde groups, which were suitable for integrating into the cell sheet, and ionic crosslinking reduced the adhesive force toward the substrate. These properties allowed the gel + cell sheet film to be easily released from the substrate. The cells in the harvested cell sheet maintained excellent viability, proliferation, and definite migration abilities inside the hydrogel. Moreover, the CSH was implanted into a full-thickness skin defects to construct a required dermal matrix and cell microenvironment. The wound closure rate reached 60.00 ± 6.26% on the 2nd day, accelerating mature granulation and dermis formation with skin appendages after 14 days. This project can provide distinct guidance and strategies for the complete repair and regeneration of full-thickness skin defects, and provides a material with great potential for tissue regeneration in clinical applications.

基于压印细胞片组装的仿生肝小叶

Liver-tissue engineering has proven valuable in treating liver diseases,but the construction of liver tissues with high fidelity remains challenging.Here,we present a novel three-dimensional(3D)-imprinted cell-sheet strategy for the synchronous construction of biomimetic hepatic microtissues with high accuracy in terms of cell type,density,and distribution.To achieve this,the specific composition of hepatic cells in a normal human liver was determined using a spatial proteogenomics dataset.The data and biomimetic hepatic micro-tissues with hexagonal hollow cross-sections indicate that cell information was successfully generated using a homemade 3D-imprinted device for layer-by-layer imprinting and assembling the hepatic cell sheets.By infiltrating vascular endothelial cells into the hollow section of the assembly,biomimetic hepatic microtissues with vascularized channels for nutrient diffusion and drug perfusion can be obtained.We demonstrate that the resultant vascularized biomimetic hepatic micro-tissues can not only be integrated into a microfluidic drug-screening liver-on-a-chip but also assembled into an enlarged physiological structure to promote liver regeneration.We believe that our 3D-imprinted cell sheets strategy will open new avenues for biomimetic microtissue construction.

A cell retrievable strategy for harvesting extracellular matrix as active biointerface

Extracellular matrix(ECM) provides a variety of physical and chemical cues for cells. Here, a very simple and smart method is developed to glue living cells away for harvesting their ECMs. The obtained ECM coatings show less cell fragment residues comparing with those obtained by the traditional cell lysis. The glued cell sheets can even be re-cultured and reused after transferring to new environment. This moderate way well maintains the activity of the ECM proteins, which can promote cell adhesion and growth.Strikingly, the ECM coatings acquired from different functional cells can guide stem cell differentiation,which is attributed to the natural physical and biochemical cues on ECM coatings. Consequently, this method provides a substantial progress for preparing natural ECM coatings and shows promising potential in regenerative medicine and other related fields of biomedical engineering.

Cell sheet formation enhances the therapeutic effects of human umbilical cord mesenchymal stem cells on myocardial infarction as a bioactive material

Stem cell-based therapy has been used to treat ischaemic heart diseases for two decades.However,optimal cell types and transplantation methods remain unclear.This study evaluated the therapeutic effects of human umbilical cord mesenchymal stem cell(hUCMSC)sheet on myocardial infarction(MI).Methods:hUCMSCs expressing luciferase were generated by lentiviral transduction for in vivo bio-luminescent imaging tracking of cells.We applied a temperature-responsive cell culture surface-based method to form the hUCMSC sheet.Cell retention was evaluated using an in vivo bio-luminescent imaging tracking system.Unbiased transcriptional profiling of infarcted hearts and further immunohistochemical assessment of monocyte and macrophage subtypes were used to determine the mechanisms underlying the therapeutic effects of the hUCMSC sheet.Echocardiography and pathological analyses of heart sections were performed to evaluate cardiac function,angiogenesis and left ventricular remodelling.Results:When transplanted to the infarcted mouse hearts,hUCMSC sheet significantly improved the retention and survival compared with cell suspension.At the early stage of MI,hUCMSC sheet modulated inflammation by decreasing Mcp1-positive monocytes and CD68-positive macrophages and increasing Cx3cr1-positive non-classical macrophages,preserving the cardiomyocytes from acute injury.Moreover,the extracellular matrix produced by hUCMSC sheet then served as bioactive scaffold for the host cells to graft and generate new epicardial tissue,providing mechanical support and routes for revascularsation.These effects of hUCMSC sheet treatment significantly improved the cardiac function at days 7 and 28 post-MI.Conclusions:hUCMSC sheet formation dramatically improved the biological functions of hUCMSCs,mitigating adverse post-MI remodelling by modulating the inflammatory response and providing bioactive scaffold upon transplantation into the heart.Translational perspective:Due to its excellent availability as well as superior local cellular retention and survival,allogenic transplantation of hUCMSC sheets can more effectively acquire the biological functions of hUCMSCs,such as modulating inflammation and enhancing angiogenesis.Moreover,the hUCMSC sheet method allows the transfer of an intact extracellular matrix without introducing exogenous or synthetic biomaterial,further improving its clinical applicability.

Recent development of temperature-responsive surfaces and their application for cell sheet engineering

Cell sheet engineering,which fabricates sheet-like tissues without biodegradable scaffolds,has been proposed as a novel approach for tissue engineering.Cells have been cultured and proliferate to confluence on a temperature-responsive cell culture surface at 37℃.By decreasing temperature to 20℃,an intact cell sheet can be harvested from the culture surface without enzymatic treatment.This new approach enables cells to keep their cell–cell junction,cell surface proteins and extracellular matrix.Therefore,recovered cell sheet can be easily not only transplanted to host tissue,but also constructed a three-dimensional(3D)tissue by layering cell sheets.Moreover,cell sheet manipulation technology and bioreactor have been combined with the cell sheet technology to fabricate a complex and functional 3D tissue in vitro.So far,cell sheet technology has been applied in regenerative medicine for several tissues,and a number of clinical studies have been performed.In this review,recent advances in the preparation of temperature-responsive cell culture surface,the fabrication of organ-like tissue and the clinical application of cell sheet engineering are summarized and discussed.

Bioactive polydimethylsiloxane surface for optimal human mesenchymal stem cell sheet culture

Human mesenchymal stem cell(hMSC)sheets hold great potential in engineering three-dimensional(3D)completely biological tissues for diverse applications.Conventional cell sheet culturing methods employing thermoresponsive surfaces are cost ineffective,and rely heavily on available facilities.In this study,a cost-effective method of layer-by-layer grafting was utilized for covalently binding a homogenous collagen I layer on a commonly used polydimethylsiloxane(PDMS)substrate surface in order to improve its cell adhesion as well as the uniformity of the resulting hMSC cell sheet.Results showed that a homogenous collagen I layer was obtained via this grafting method,which improved hMSC adhesion and attachment through reliable collagen I binding sites.By utilizing this low-cost method,a uniform hMSC sheet was generated.This technology potentially allows for mass production of hMSC sheets to fulfill the demand of thick hMSC constructs for tissue engineering and biomanufacturing applications.

Application of Induced Pluripotent Stem Cells Derived from Human Periodontal Ligament Cells in Bone Regeneration

The purpose of this study was to primarily culture human periodontal ligament cells(hPDLCs)and to reprogram hPDLCs with exogenous genes via a lentivirus-mediated transfection system.Then induced pluripotent stem cells derived from h PDLCs(hPDLC-iPSCs)were identified.Alizarin red staining was used to observe the formation of mineralized nodules and real-time Polymerase Chain Reaction(PCR)was used to detect the expression of osteogenic genes.For the in vivo experiment,nude mouse skull defect models were established and cell sheets were made to repair the bone defect.The reprogrammed cells were positive for alkaline phosphatase(ALP)staining and embryonic stem cells(ESCs)-specific proteins,and could form teratomas.After osteogenic induction,alizarin red staining showed that the number of mineralized nodules in the h PDLC-i PSCs group was more and the osteogenic related factors ALP,osteocalcin(OCN),Col-I and Runx2 were also expressed higher in hPDLC-iPSCs.The hPDLC-iPSC cell sheets were all successfully made.Histological analysis showed that the h PDLC-i PSC cell sheet got new bone formation.These results demonstrated that hPDLC-iPSCs were successfully generated from human periodontal ligament fibroblasts and hPDLC-iPSC cell sheets provided new options for bone tissue engineering.

Novel pre-vascularized tissue-engineered dermis based on stem cell sheet technique used for dermis-defect healing

Insufficient donor dermis and the shortage of three-dimensional vascular networks are the main limitations in the tissue-engineered dermis(TED).To solve these problems,we initially constructed pre-vascularized bone marrow mesenchymal stem cell sheet(PBMCS)and pre-vascularized fibroblasts cell sheet(PFCS)by cell sheet technology,and then superimposed or folded them together to construct a pre-vascularized TED(PTED),aiming to mimic the real dermis structure.The constructed PTED was implanted in nude mice dorsal dermis-defect wound and the wound-healing effect was quantified at Days 1,7 and 14 via the methods of histochemistry and immunohistochemistry.The results showed that PTED could rapidly promote the wound closure,especially at Day 14,and the wound-healing rate of three-layer PTED could reach 97.2%(P<0.01),which was faster than the blank control group(89.1%),PBMCS(92.4%),PFCS(93.8%)and six-layer PTED(92.3%).In addition,the vessel density in the PTED group was higher than the other groups on the 14th day.Taken together,it is proved that the PTED,especially three-layer PTED,is more conducive to the fullthickness dermis-defect repair and the construction of the three-dimensional vascular networks,indicating its potential application in dermis-defect repair.