A facile strategy for tuning the density of surface-grafted biomolecules for melt extrusion-based additive manufacturing applications

Melt extrusion-based additive manufacturing(ME-AM)is a promising technique to fabricate porous scaffolds for tissue engi-neering applications.However,most synthetic semicrystalline polymers do not possess the intrinsic biological activity required to control cell fate.Grafting of biomolecules on polymeric surfaces of AM scaffolds enhances the bioactivity of a construct;however,there are limited strategies available to control the surface density.Here,we report a strategy to tune the surface density of bioactive groups by blending a low molecular weight poly(ε-caprolactone)5k(PCL5k)containing orthogonally reactive azide groups with an unfunctionalized high molecular weight PCL75k at different ratios.Stable porous three-dimensional(3D)scaf-folds were then fabricated using a high weight percentage(75 wt.%)of the low molecular weight PCL 5k.As a proof-of-concept test,we prepared films of three different mass ratios of low and high molecular weight polymers with a thermopress and reacted with an alkynated fluorescent model compound on the surface,yielding a density of 201-561 pmol/cm^(2).Subsequently,a bone morphogenetic protein 2(BMP-2)-derived peptide was grafted onto the films comprising different blend compositions,and the effect of peptide surface density on the osteogenic differentiation of human mesenchymal stromal cells(hMSCs)was assessed.After two weeks of culturing in a basic medium,cells expressed higher levels of BMP receptor II(BMPRII)on films with the conjugated peptide.In addition,we found that alkaline phosphatase activity was only significantly enhanced on films contain-ing the highest peptide density(i.e.,561 pmol/cm^(2)),indicating the importance of the surface density.Taken together,these results emphasize that the density of surface peptides on cell differentiation must be considered at the cell-material interface.Moreover,we have presented a viable strategy for ME-AM community that desires to tune the bulk and surface functionality via blending of(modified)polymers.Furthermore,the use of alkyne-azide“click”chemistry enables spatial control over bioconjugation of many tissue-specific moieties,making this approach a versatile strategy for tissue engineering applications.

Highly sensitive ratiometric fluorescent fiber matrices for oxygen sensing with micrometer spatial resolution

Oxygen(O_(2))-sensing matrices are promising tools for the live monitoring of extracellular O_(2) consumption levels in long-term cell cultures.In this study,ratiometric O_(2)-sensing membranes were prepared by electrospinning,an easy,low-cost,scalable,and robust method for fabricating nanofibers.Poly(ε-caprolactone)and poly(dimethyl)siloxane polymers were blended with tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)dichloride,which was used as the O_(2)-sensing probe,and rhodamine B isothiocyanate,which was used as the reference dye.The functionalized scaffolds were morphologically characterized by scanning electron microscopy,and their physicochemical profiles were obtained by Fourier transform infrared spectroscopy,thermogravimetric analysis,and water contact angle measurement.The sensing capabilities were investigated by confocal laser scanning microscopy,performing photobleaching,reversibility,and calibration curve studies toward different dissolved O_(2)(DO)concentrations.Electrospun sensing nanofibers showed a high response to changes in DO concentrations in the physiological-pathological range from 0.5%to 20%and good stability under ratiometric imaging.In addition,the sensing systems were highly biocompatible for cell growth promoting adhesiveness and growth of three cancer cell lines,namely metastatic melanoma cell line SK-MEL2,breast cancer cell line MCF-7,and pancreatic ductal adenocarcinoma cell line Panc-1,thus recreating a suitable biological environment in vitro.These O_(2)-sensing biomaterials can potentially measure alterations in cell metabolism caused by changes in ambient O_(2)content during drug testing/validation and tissue regeneration processes.

Oxygen tension modulates cell function in an in vitro three-dimensional glioblastoma tumor model

Hypoxia is a typical feature of the tumor microenvironment,one of the most critical factors affecting cell behavior and tumor progression.However,the lack of tumor models able to precisely emulate natural brain tumor tissue has impeded the study of the effects of hypoxia on the progression and growth of tumor cells.This study reports a three-dimensional(3D)brain tumor model obtained by encapsulating U87MG(U87)cells in a hydrogel containing type I collagen.It also documents the effect of various oxygen concentrations(1%,7%,and 21%)in the culture environment on U87 cell morphology,proliferation,viability,cell cycle,apoptosis rate,and migration.Finally,it compares two-dimensional(2D)and 3D cultures.For comparison purposes,cells cultured in flat culture dishes were used as the control(2D model).Cells cultured in the 3D model proliferated more slowly but had a higher apoptosis rate and proportion of cells in the resting phase(G0 phase)/gap I phase(G1 phase)than those cultured in the 2D model.Besides,the two models yielded significantly different cell morphologies.Finally,hypoxia(e.g.,1%O2)affected cell morphology,slowed cell growth,reduced cell viability,and increased the apoptosis rate in the 3D model.These results indicate that the constructed 3D model is effective for investigating the effects of biological and chemical factors on cell morphology and function,and can be more representative of the tumor microenvironment than 2D culture systems.The developed 3D glioblastoma tumor model is equally applicable to other studies in pharmacology and pathology.

Biomaterials and emerging technologies for tissue engineering and in vitro models

The latest advances in the field of biomaterials have opened new avenues for scientific breakthroughs in tissue engineer-ing which greatly contributed for the successful translation of tissue engineering products into the market/clinics.Bio-materials are easily processed to become similar to natural extracellular matrix,making them ideal temporary supports for mimicking the three-dimensional(3D)microenvironment required for maintaining the adequate cell/tissue functions both in vitro and in vivo^([1]).

Properties of Collagen/Sodium Alginate Hydrogels for Bioprinting of Skin Models

3D printing technology has great potential for the reconstruction of human skin.However,the reconstructed skin has some differences from natural skin,largely because the hydrogel used does not have the appropriate biological and physical properties to allow healing and regeneration.This study examines the swelling,degradability,microstructure and biological properties of Collagen/Sodium Alginate(Col/SA)hydrogels of differing compositions for the purposes of skin printing.Increasing the content of sodium alginate causes the hydrogel to exhibit stronger mechanical and swelling properties,a faster degradation ratio,smaller pore size,and less favorable biological properties.An optimal 1%collagen hydrogel was used to print bi-layer skin in which fibroblasts and keratinocytes showed improved spreading and proliferation as compared to other developed formulations.The Col/SA hydrogels presented suitable tunability and properties to be used as a bioink for bioprinting of skin aiming at finding applications as 3D models for wound healing research.

Replication of natural surface topographies to generate advanced cell culture substrates

Surface topographies of cell culture substrates can be used to generate in vitro cell culture environments similar to the in vivo cell niches. In vivo, the physical properties of the extracellular matrix (ECM), such as its topography, provide physical cues that play an important role in modulating cell function. Mimicking these properties remains a challenge to provide in vitro realistic environments for cells. Artificially generated substrates’ topographies were used extensively to explore this important surface cue. More recently, the replication of natural surface topographies has been enabling to exploration of characteristics such as hierarchy and size scales relevant for cells as advanced biomimetic substrates. These substrates offer more realistic and mimetic environments regarding the topographies found in vivo. This review will highlight the use of natural surface topographies as a template to generate substrates for in-vitro cell culture. This review starts with an analysis of the main cell functions that can be regulated by the substrate’s surface topography through cell-substrate interactions. Then, we will discuss research works wherein substrates for cell biology decorated with natural surface topographies were used and investigated regarding their influence on cellular performance. At the end of this review, we will highlight the advantages and challenges of the use of natural surface topographies as a template for the generation of advanced substrates for cell culture.

Adaptable hydrogel with reversible linkages for regenerative medicine:Dynamic mechanical microenvironment for cells

Hydrogels are three-dimensional platforms that serve as substitutes for native extracellular matrix.These materials are starting to play important roles in regenerative medicine because of their similarities to native matrix in water content and flexibility.It would be very advantagoues for researchers to be able to regulate cell behavior and fate with specific hydrogels that have tunable mechanical properties as biophysical cues.Recent developments in dynamic chemistry have yielded designs of adaptable hydrogels that mimic dynamic nature of extracellular matrix.The current review provides a comprehensive overview for adaptable hydrogel in regenerative medicine as follows.First,we outline strategies to design adaptable hydrogel network with reversible linkages according to previous findings in supramolecular chemistry and dynamic covalent chemistry.Next,we describe the mechanism of dynamic mechanical microenvironment influence cell behaviors and fate,including how stress relaxation influences on cell behavior and how mechanosignals regulate matrix remodeling.Finally,we highlight techniques such as bioprinting which utilize adaptable hydrogel in regenerative medicine.We conclude by discussing the limitations and challenges for adaptable hydrogel,and we present perspectives for future studies.

Translation of biomaterials from bench to clinic

Scientific research originates from curiosity and interests. Translational research of biomaterials should always focus on addressing specific needs of the targeted clinical applications. The guest editors of this special issue hope that the included articles have provided cutting-edge biomaterials research as well as insights of the translation of biomaterials from bench to clinic.

Osteogenic lithium-doped brushite cements for bone regeneration

This study investigated the osteogenic performance of new brushite cements obtained from Li+-dopedβ-tricalcium phosphate as a promising strategy for bone regeneration.Lithium(Li+)is a promising trace element to encourage the migration and proliferation of adipose-derived stem cells(hASCs)and the osteogenic differentiation-related gene expression,essential for osteogenesis.In-situ X-ray diffraction(XRD)and in-situ 1H nuclear magnetic resonance(1H NMR)measurements proved the precipitation of brushite,as main phase,and monetite,indicating that Li+favored the formation of monetite under certain conditions.Li+was detected in the remaining pore solution in significant amounts after the completion of hydration.Isothermal calorimetry results showed an accelerating effect of Li+,especially for low concentration of the setting retarder(phytic acid).A decrease of initial and final setting times with increasing amount of Li+was detected and setting times could be well adjusted by varying the setting retarder concentration.The cements presented compressive mechanical strength within the ranges reported for cancellous bone.In vitro assays using hASCs showed normal metabolic and proliferative levels.The immunodetection and gene expression profile of osteogenic-related markers highlight the incorporation of Li+for increasing the in vivo bone density.The osteogenic potential of Li-doped brushite cements may be recommended for further research on bone defect repair strategies.

Osteochondral scaffolds for early treatment of cartilage defects in osteoarthritic joints:from bench to clinic

Osteoarthritis is a degenerative joint disease,typified by the loss in the quality of cartilage and bone at the interface of a synovial joint,resulting in pain,stiffness and reduced mobility.The current surgical treatment for advanced stages of the disease is joint replacement,where the non-surgical therapeutic options or less invasive surgical treatments are no longer effective.These are major surgical procedures which have a substantial impact on patients’quality of life and lifetime risk of requiring revision surgery.Treatments using regenerative methods such as tissue engineering methods have been established and are promising for the early treatment of cartilage degeneration in osteoarthritis joints.In this approach,3-dimensional scaffolds(with or without cells)are employed to provide support for tissue growth.However,none of the currently available tissue engineering and regenerative medicine products promotes satisfactory durable regeneration of large cartilage defects.Herein,we discuss the current regenerative treatment options for cartilage and osteochondral(cartilage and underlying subchondral bone)defects in the articulating joints.We further identify the main hurdles in osteochondral scaffold development for achieving satisfactory and durable regeneration of osteochondral tissues.The evolution of the osteochondral scaffolds–from monophasic to multiphasic constructs–is overviewed and the osteochondral scaffolds that have progressed to clinical trials are examined with respect to their clinical performances and their potential impact on the clinical practices.Development of an osteochondral scaffold which bridges the gap between small defect treatment and joint replacement is still a grand challenge.Such scaffold could be used for early treatment of cartilage and osteochondral defects at early stage of osteoarthritis and could either negate or delay the need for joint replacements.

Translation through collaboration:practice applied in BAMOS project in in vivo testing of innovative osteochondral scaffolds

Osteoarthritis is the most common chronic degenerative joint disease,recognized by the World Health Organization as a public health problem that affects millions of people worldwide.The project Biomaterials and Additive Manufacturing:Osteochondral Scaffold(BAMOS)innovation applied to osteoarthritis,funded under the frame of the Horizon 2020 Research and Innovation Staff Exchanges(RISE)program,aims to delay or avoid the use of joint replacements by developing novel cost-effective osteochondral scaffold technology for early intervention of osteoarthritis.The multidisciplinary consortium of BAMOS,formed by international leading research centres,collaborates through research and innovation staff exchanges.The project covers all the stages of the development before the clinical trials:design of scaffolds,biomaterials development,processability under additive manufacturing,in vitro test,and in vivo test.This paper reports the translational practice adopted in the project in in vivo assessment of the osteochondral scaffolds developed.

Osteogenic differentiation of encapsulated cells in dexamethasone-loaded phospholipid-induced silk fibroin hydrogels

The tissue engineering triad comprises the combination of cells,scaffolds and biological factors.Therefore,we prepared cell-and drug-loaded hydrogels using in situ silk fibroin(SF)hydrogels induced by dimyristoyl glycerophosphoglycerol(DMPG).DMPG is reported to induce rapid hydrogel formation by SF,facilitating cell encapsulation in the hydrogel matrix while maintaining high cell viability and proliferative capacity.In addition,DMPG can be used for liposome formulations in entrapping drug molecules.Dexamethasone(Dex)was loaded into the DMPG-induced SF hydrogels together with human osteoblast-like SaOS-2 cells,then the osteogenic differentiation of the entrapped cells was evaluated in vitro and compared to cells cultured under standard conditions.Calcium production by cells cultured in DMPG/Dex-SF hydrogels with Dex-depleted osteogenic medium was equivalent to that of cells cultured in conventional osteogenic medium containing Dex.The extended-release of the entrapped Dex by the hydrogels was able to provide a sufficient drug amount for osteogenic induction.The controlled release of Dex was also advantageous for cell viability even though its dose in the hydrogels was far higher than that in osteogenic medium.The results confirmed the possibility of using DMPG-induced SF hydrogels to enable dual cell and drug encapsulation to fulfil the practical applications of tissue-engineered constructs.

Extracellular matrix-derived materials for tissue engineering and regenerative medicine:A journey from isolation to characterization and application

Biomaterial choice is an essential step during the development tissue engineering and regenerative medicine(TERM)applications.The selected biomaterial must present properties allowing the physiological-like recapitulation of several processes that lead to the reestablishment of homeostatic tissue or organ function.Biomaterials derived from the extracellular matrix(ECM)present many such properties and their use in the field has been steadily increasing.Considering this growing importance,it becomes imperative to provide a comprehensive overview of ECM biomaterials,encompassing their sourcing,processing,and integration into TERM applications.This review compiles the main strategies used to isolate and process ECM-derived biomaterials as well as different techniques used for its characterization,namely biochemical and chemical,physical,morphological,and biological.Lastly,some of their applications in the TERM field are explored and discussed.

Prevascularized spongy-like hydrogels maintain their angiogenic potential after prolonged hypothermic storage

The chronic shortage of organs and tissues for transplantation represents a dramatic burden on healthcare systems worldwide.Tissue engineering offers a potential solution to address these shortages,but several challenges remain,with prevascularization being a critical factor for in vivo survival and integration of tissue engineering products.Concurrently,a different challenge hindering the clinical implementation of such products,regards their efficient preservation from the fabrication site to the bedside.Hypothermia has emerged as a potential solution for this issue due to its milder effects on biologic systems in comparison with other cold preservation methodologies.Its impact on prevascularization,however,has not been well studied.In this work,3D prevascularized constructs were fabricated using adipose-derived stromal vascular fraction cells and preserved at 4◦C using Hypothermosol or basal culture media(α-MEM).Hypothermosol efficiently preserved the structural and cellular integrity of prevascular networks as compared to constructs before preservation.In contrast,the use ofα-MEM led to a clear reduction in prevascular structures,with concurrent induction of high levels of apoptosis and autophagy at the cellular level.In vivo evaluation using a chorioallantoic membrane model demonstrated that,in opposition toα-MEM,Hypothermosol preservation retained the angiogenic potential of constructs before preservation by recruiting a similar number of blood vessels from the host and presenting similar integration with host tissue.These results emphasize the need of studying the impact of preservation techniques on key properties of tissue engineering constructs such as prevascularization,in order to validate and streamline their clinical application.