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.

3D additive manufactured composite scaffolds with antibiotic-loaded lamellar fillers for bone infection prevention and tissue regeneration

Bone infections following open bone fracture or implant surgery remain a challenge in the orthopedics field.In order to avoid high doses of systemic drug administration,optimized local antibiotic release from scaffolds is required.3D additive manufactured(AM)scaffolds made with biodegradable polymers are ideal to support bone healing in non-union scenarios and can be given antimicrobial properties by the incorporation of antibiotics.In this study,ciprofloxacin and gentamicin intercalated in the interlamellar spaces of magnesium aluminum layered double hydroxides(MgAl)andα-zirconium phosphates(ZrP),respectively,are dispersed within a thermoplastic polymer by melt compounding and subsequently processed via high temperature melt extrusion AM(~190◦C)into 3D scaffolds.The inorganic fillers enable a sustained antibiotics release through the polymer matrix,controlled by antibiotics counterions exchange or pH conditions.Importantly,both antibiotics retain their functionality after the manufacturing process at high temperatures,as verified by their activity against both Gram+and Gram-bacterial strains.Moreover,scaffolds loaded with filler-antibiotic do not impair human mesenchymal stromal cells osteogenic differentiation,allowing matrix mineralization and the expression of relevant osteogenic markers.Overall,these results suggest the possibility of fabricating dual functionality 3D scaffolds via high temperature melt extrusion for bone regeneration and infection prevention.

Biomimetic three-layered membranes comprising(poly)-e-caprolactone,collagen and mineralized collagen for guided bone regeneration

The distinct structural properties and osteogenic capacity are important aspects to be taken into account when developing guided bone regeneration membranes.Herein,inspired by the structure and function of natural periosteum,we designed and fabricated using electrospinning a fibrous membrane comprising(poly)-e-caprolactone(PCL),collagen-I(Col)and mineralized Col(MC).The three-layer membranes,having PCL as the outer layer,PCL/Col as the middle layer and PCL/Col/MC in different ratios(5/2.5/2.5(PCM-1);3.3/3.3/3.3(PCM-2);4/4/4(PCM-3)(%,w/w/w))as the inner layer,were produced.The physiochemical properties of the different layers were investigated and a good integration between the layers was observed.The three-layeredmembranes showed tensile properties in the range of those of natural periosteum.Moreover,the membranes exhibited excellent water absorption capability without changes of the thickness.In vitro experiments showed that the inner layer of the membranes supported attachment,proliferation,ingrowth and osteogenic differentiation of human bone marrowderived stromal cells.In particular cells cultured on PCM-2 exhibited a significantly higher expression of osteogenesis-related proteins.The three-layered membranes successfully supported new bone formation inside a critical-size cranial defect in rats,with PCM-3 being the most efficient.The membranes developed here are promising candidates for guided bone regeneration applications.

Assessing the response of human primary macrophages to defined fibrous architectures fabricated by melt electrowriting

The dual role of macrophages in the healing process depends on macrophage ability to polarize into phenotypes that can propagate inflammation or exert anti-inflammatory and tissue-remodeling functions.Controlling scaf-fold geometry has been proposed as a strategy to influence macrophage behavior and favor the positive host response to implants.Here,we fabricated Polycaprolactone(PCL)scaffolds by Melt Electrowriting(MEW)to investigate the ability of scaffold architecture to modulate macrophage polarization.Primary human macrophages unpolarized(M0)or polarized into M1,M2a,and M2c phenotypes were cultured on PCL films and MEW scaffolds with pore geometries(square,triangle,and rhombus grid)characterized by different angles.M0,M2a,and M2c macrophages wrapped along the fibers,while M1 macrophages formed clusters with rounded cells.Cell bridges were formed only for angles up to 90◦.No relevant differences were found among PCL films and 3D scaffolds in terms of surface markers.CD206 and CD163 were highly expressed by M2a and M2c macrophages,with M2a macrophages presenting also high levels of CD86.M1 macrophages expressed moderate levels of all markers.The rhombus architecture promoted an increased release by M2a macrophages of IL10,IL13,and sCD163 compared to PCL films.The proangiogenic factor IL18 was also upregulated by the rhombus configuration in M0 and M2a macrophages compared to PCL films.The interesting findings obtained for the rhombus architecture represent a starting point for the design of scaffolds able to modulate macrophage phenotype,prompting investigations addressed to verify their ability to facilitate the healing process in vivo.

Mesoporous bioactive glass composition effects on degradation and bioactivity

Mesoporous bioactive glasses(MBGs)are promising materials for regenerative medicine,due to their favorable properties including bioactivity and degradability.These key properties,but also their surface area,pore structure and pore volume are strongly dependent on synthesis parameters and glass stoichiometry.However,to date no systematic study on MBG properties covering a broad range of possible compositions exists.Here,24 MBG compositions in the SiO_(2)-CaO-P_(2)O_(5) system were synthesized by varying SiO_(2)(60-90 mol%),CaO and P_(2)O_(5) content(both 0 to 40 mol-%),while other synthesis parameters were kept constant.Mesopore characteristics,degradability and bioactivity were analysed.The results showed that,within the tested range of compositions,mesopore formation required a molar SiO_(2) content above 60%but was independent of CaO and P_(2)O_(5) content.While mesopore size did not depend on glass stoichiometry,mesopore arrangement was influenced by the SiO_(2) content.Specific surface area and pore volume were slightly altered by the SiO_(2) content.All materials were degradable;however,degradation as well as bioactivity,i.e.the ability to form a CaP mineral on the surface,depended on stoichiometry.Major differences were found in early surface reactions in simulated body fluid:where some MBGs induced direct hydroxyapatite crystallization,high release of calcium in others resulted in calcite formation.In summary,degradation and bioactivity,both key parameters of MBGs,can be controlled by glass stoichiometry over a broad range while leaving the unique structural parameters of MBGs relatively unaffected.This allows targeted selection of material compositions for specific regenerative medicine applications.

Modular operation of microfluidic chips for highly parallelized cell culture and liquid dosing via a fluidic circuit board

Microfluidic systems enable automated and highly parallelized cell culture with low volumes and defined liquid dosing.To achieve this,systems typically integrate all functions into a single,monolithic device as a“one size fits all”solution.However,this approach limits the end users’(re)design flexibility and complicates the addition of new functions to the system.To address this challenge,we propose and demonstrate a modular and standardized plug-and-play fluidic circuit board(FCB)for operating microfluidic building blocks(MFBBs),whereby both the FCB and the MFBBs contain integrated valves.A single FCB can parallelize up to three MFBBs of the same design or operate MFBBs with entirely different architectures.The operation of the MFBBs through the FCB is fully automated and does not incur the cost of an extra external footprint.We use this modular platform to control three microfluidic large-scale integration(mLSI)MFBBs,each of which features 64 microchambers suitable for cell culturing with high spatiotemporal control.We show as a proof of principle that we can culture human umbilical vein endothelial cells(HUVECs)for multiple days in the chambers of this MFBB.Moreover,we also use the same FCB to control an MFBB for liquid dosing with a high dynamic range.Our results demonstrate that MFBBs with different designs can be controlled and combined on a single FCB.Our novel modular approach to operating an automated microfluidic system for parallelized cell culture will enable greater experimental flexibility and facilitate the cooperation of different chips from different labs.

Magnetically responsive nanofibrous ceramic scaffolds for on-demand motion and drug delivery

Smart biomaterials,featuring not only bioactivity,but also dynamic responsiveness to external stimuli,are desired for biomedical applications,such as regenerative medicine,and hold great potential to expand the boundaries of the modern clinical practice.Herein,a magnetically responsive three-dimensional scaffold with sandwich structure is developed by using hydroxyapatite(HA)nanowires and ferrosoferric oxide(Fe_(3)O_(4))nanoparticles as building blocks.The magnetic HA/Fe_(3)O_(4) scaffold is fully inorganic in nature,but shows polymeric hydrogel-like characteristics including a 3D fibrous network that is highly porous(>99.7%free volume),deformable(50%deformation)and elastic,and tunable stiffness.The magnetic HA/Fe3O4 scaffold has been shown to execute multimodal motion upon exposure to an external magnetic field including shape transformation,rolling and somersault.In addition,we have demonstrated that the magnetic scaffold can serve as a smart carrier for remotely controlled,on-demand delivery of compounds including an organic dye and a protein.Finally,the magnetic scaffold has exhibited good biocompatibility,supporting the attachment and proliferation of human mesenchymal stromal cells,thereby showing great potential as smart biomaterials for a variety of biomedical applications.

Human osteoclast formation and resorptive function on biomineralized collagen

Biomineralized collagen composite materials pose an intriguing alternative to current synthetic bone graft substitutes by offering a biomimetic composition that closely resembles native bone.We hypothesize that this composite can undergo cellular resorption and remodeling similar to natural bone.We investigate the formation and activity of human osteoclasts cultured on biomineralized collagen and pure collagen membranes in comparison to cortical bone slices.Human monocytes/macrophages from peripheral blood differentiate into multinucleated,tartrate-resistant alkaline phosphatase(TRAP)-positive osteoclast-like cells on all substrates.These cells form clear actin rings on cortical bone,but not on biomineralized collagen or pure collagen membranes.Osteoclasts form resorption pits in cortical bone,resulting in higher calcium ion concentration in cell culture medium;however,osteoclast resorption of biomineralized collagen and collagen membranes does not measurably occur.Activity of osteoclast enzymes-TRAP,carbonic anhydrase II(CA-II),and cathepsin-K(CTS-K)-is similar on all substrates,despite phenotypic differences in actin ring formation and resorption.The mesh-like structure,relatively low stiffness,and lack of RGD-containing binding domains are likely the factors responsible for preventing formation of stable actin rings on and resorption of(biomineralized)collagen membranes.This insight helps to guide further research toward the optimized design of biomineralized collagen composites as a more biomimetic bone-graft substitute.

3D-printed bioactive scaffolds from nanosilicates and PEOT/PBT for bone tissue engineering

Additive manufacturing(AM)has shown promise in designing 3D scaffold for regenerative medicine.However,many synthetic biomaterials used for AM are bioinert.Here,we report synthesis of bioactive nanocomposites from a poly(ethylene oxide terephthalate)(PEOT)/poly(butylene terephthalate)(PBT)(PEOT/PBT)copolymer and 2D nanosilicates for fabricating 3D scaffolds for bone tissue engineering.PEOT/PBT have been shown to support calcification and bone bonding ability in vivo,while 2D nanosilicates induce osteogenic differentiation of human mesenchymal stem cells(hMSCs)in absence of osteoinductive agents.The effect of nanosilicates addition to PEOT/PBT on structural,mechanical and biological properties is investigated.Specifically,the addition of nanosilicate to PEOT/PBT improves the stability of nanocomposites in physiological conditions,as nanosilicate suppressed the degradation rate of copolymer.However,no significant increase in the mechanical stiffness of scaffold due to the addition of nanosilicates is observed.The addition of nanosilicates to PEOT/PBT improves the bioactive properties of AM nanocomposites as demonstrated in vitro.hMSCs readily proliferated on the scaffolds containing nanosilicates and resulted in significant upregulation of osteo-related proteins and production of mineralized matrix.The synergistic ability of nanosilicates and PEOT/PBT can be utilized for designing bioactive scaffolds for bone tissue engineering.