The development of collagen based composite scaffolds for bone regeneration

Bone is consisted of bone matrix,cells and bioactive factors,and bone matrix is the combination of inorganic minerals and organic polymers.Type I collagen fibril made of five triple-helical collagen chains is the main organic polymer in bone matrix.It plays an important role in the bone formation and remodeling process.Moreover,collagen is one of the most commonly used scaffold materials for bone tissue engineering due to its excellent biocompatibility and biodegradability.However,the low mechanical strength and osteoinductivity of collagen limit its wider applications in bone regeneration field.By incorporating different biomaterials,the properties such as porosity,structural stability,osteoinductivity,osteogenicity of collagen matrixes can be largely improved.This review summarizes and categorizes different kinds of biomaterials including bioceramic,carbon and polymer materials used as components to fabricate collagen based composite scaffolds for bone regeneration.Moreover,the possible directions of future research and development in this field are also proposed.

Synthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/chitin nanocrystals composite scaffolds for tissue engineering

Poly（3-hydroxybutyrate-co-3-hydroxyvalerate）（PHBV）/chitin nanocrystals（CNC） composite scaffolds were synthesized by the salt leaching and thermally induced phase separation（TIPS） technique.The scaffolds have porous structures with macro-pores（100-300 μm in diameters） and micro-pores（10μm）.The surface characteristics of the scaffolds were characterized by X-ray photoelectron spectroscopy（XPS） and static water contact angle measurement,and the mechanical properties were investigated by a compression test.Human adipose-derived stem cells（hADSCs） were seeded onto the PHBV/CNC scaffolds and in vitro cell culture results showed that the composite scaffolds enhanced the hADSCs adhesion,which implies that the material may have potential application in tissue engineering.

Comparing the regeneration potential between PLLA/Aragonite and PLLA/ Vaterite pearl composite scaffolds in rabbit radius segmental bone defects

Mussel-derived nacre and pearl,which are natural composites composed CaCO3 platelets and interplatelet organic matrix,have recently gained interest due to their osteogenic potential.The crystal form of CaCO3 could be either aragonite or vaterite depending on the characteristics of mineralization template within pearls.So far,little attention has been paid on the different osteogenic capacities between aragonite and vaterite pearl.In the current work,aragonite or vaterite pearl powders were incorporated into poly-L-lactic acid(PLLA)scaffold as bio-functional fillers for enhanced osteogenesis.In intro results revealed that PLLA/aragonite scaffold possessed stronger stimulatory effect on SaOS-2 cell proliferation and differentiation,evidenced by the enhanced cell viability,alkaline phosphatase activity,collagen synthesis and gene expressions of osteogenic markers including osteocalcin,osteopotin and bone sialoprotein.The bone regeneration potential of various scaffolds was evaluated in vivo employing a rabbit critical-sized radial bone defect model.The X-ray and micro-CT results showed that significant bone regeneration and bridging were achieved in defects implanted with composite scaffolds,while less bone formation and non-bridging were found for pure PLLA group.Histological evaluation using Masson's trichrome and hematoxylin/eosin(H&E)staining indicated a typical endochondral bone formation process conducted at defect sites treated with composite scaffolds.Through three-point bending test,the limbs implanted with PLLA/aragonite scaffold were found to bear significantly higher bending load compared to other two groups.Together,it is suggested that aragonite pearl has superior osteogenic capacity over vaterite pearl and PLLA/aragonite scaffold can be employed as a potential bone graft for bone regeneration.

Dexamethasone-Loaded PLGA Microspheres Incorporated PLLA/PLGA/PCL Composite Scaffold for Bone Tissue Engineering

The combination of micro-carriers and polymer scaffolds as promising bone grafts have attracted considerable interest in recent decades.The poly(L-lactic acid)/poly(lactic-co-glycolic acid)/polycaprolactone(PLLA/PLGA/PCL)composite scaffold with porous structure was fabricated by thermally induced phase separation(TIPS).Dexamethasone(DEX)was incorporated into PLGA microspheres and then loaded on the PLLA/PLGA/PCL scaffoldtopreparethedesiredcompositescaffold.The physicochemical properties of the prepared composite scaffold were characterized.The morphology of rat bone marrow mesenchymal stem cells(BMSCs)grown on scaffolds was observed using scanning electron microscope(SEM)and fluorescence microscope.The resultsshowedthatthePLLA/PLGA/PCLscaffoldhad interconnected macropores and biomimetic nanofibrous structure.In addition,DEX can be released from scaffold in a sustained manner.More importantly,DEX loaded composite scaffold can effectively support the proliferation of BMSCs as indicated by fluorescence observation and cell proliferation assay.The results suggested that the prepared PLLA/PLGA/PCL composite scaffold incorporating drug-loaded PLGA microspheres could hold great potential for bone tissue engineering applications.

Magnesium incorporated chitosan based scaffolds for tissue engineering applications

Chitosan based porous scaffolds are of great interest in biomedical applications especially in tissue engineering because of their excellent biocompatibility in vivo,controllable degradation rate and tailorable mechanical properties.This paper presents a study of the fabrication and characterization of bioactive scaffolds made of chitosan(CS),carboxymethyl chitosan(CMC)and magnesium gluconate(MgG).Scaffolds were fabricated by subsequent freezing-induced phase separation and lyophilization of polyelectrolyte complexes of CS,CMC and MgG.The scaffolds possess uniform porosity with highly interconnected pores of 50-250 μm size range.Compressive strengths up to 400 kPa,and elastic moduli up to 5 MPa were obtained.The scaffolds were found to remain intact,retaining their original threedimensional frameworks while testing in in-vitro conditions.These scaffolds exhibited no cytotoxicity to 3T3 fibroblast and osteoblast cells.These observations demonstrate the efficacy of this new approach to preparing scaffold materials suitable for tissue engineering applications.

A novel akermanite/poly (lactic-co-glycolic acid) porous composite scaffold fabricated via a solvent casting-particulate leaching method improved by solvent self-proliferating process

Desirable scaffolds for tissue engineering should be biodegradable carriers to supply suitablemicroenvironments mimicked the extracellular matrices for desired cellular interactions and to providesupports for the formation of new tissues. In this work, a kind of slightly soluble bioactiveceramic akermanite (AKT) powders were aboratively selected and introduced in the PLGA matrix,a novel L-lactide modified AKT/poly (lactic-co-glycolic acid) (m-AKT/PLGA) composite scaffold wasfabricated via a solvent casting-particulate leaching method improved by solvent self-proliferatingprocess. The effects of m-AKT contents on properties of composite scaffolds and on MC3T3-E1 cellularbehaviors in vitro have been primarily investigated. The fabricated scaffolds exhibited threedimensionalporous networks, in which homogenously distributed cavities in size of 300–400 lmwere interconnected by some smaller holes in a size of 100–200 lm. Meanwhile, the mechanicalstructure of scaffolds was reinforced by the introduction of m-AKT. Moreover, alkaline ionic productsreleased by m-AKT could neutralize the acidic degradation products of PLGA, and the apatitemineralizationability of scaffolds could be largely improved. More valuably, significant promotionson adhesion, proliferation, and differentiation of MC3T3-E1 have been observed, which implied thecalcium, magnesium and especially silidous ions released sustainably from composite scaffoldscould regulate the behaviors of osteogenesis-related cells.

Article Preclinical evaluation of acute systemic toxicity of magnesium incorporated poly(lactic-co-glycolic acid)porous scaffolds by three-dimensional printing Jing

Biodegradable polymer scaffolds combined with bioactive components which accelerate osteogenesis and angiogenesis have promise for use in clinical bone defect repair.The preclinical acute toxicity evaluation is an essential assay of implantable biomaterials to assess the biosafety for accelerating clinical translation.We have successfully developed magnesium(Mg)particles and beta-tricalcium phosphate(β-TCP)for incorporation into poly(lactic-co-glycolic acid)(PLGA)porous composite scaffolds(PTM)using low-temperature rapid prototyping three-dimensional-printing technology.The PTM scaffolds have been fully evaluated and found to exhibit excellent osteogenic capacity for bone defect repair.The preclinical evaluation of acute systemic toxicities is essential and important for development of porous scaffolds to facilitate their clinical translation.In this study,acute systemic toxicity of the PTM scaffolds was evaluated in mice by intraperitoneal injection of the extract solutions of the scaffolds.PTM composite scaffolds with different Mg andβ-TCP content(denoted as PT5M,PT10M,and PT15M)were extracted with different tissue culture media,including normal saline,phosphate-buffered saline,and serum-free minimum essential medium,to create the extract solutions.The evaluation was carried out following the National Standard.The acute toxicity was fully evaluated through the collection of extensive data,including serum/organs ion concentration,fluorescence staining,and in vivo median lethal dose measurement.Mg in major organs(heart,liver,and lung),and Mg ion concentrations in serum of mice,after intraperitoneal injection of the extract solutions,were measured and showed that the extract solutions of PT15M caused significant elevation of serum Mg ion concentrations,which exceeded the safety threshold and led to the death of the mice.In contrast,the extract solutions of PT5M and PT10M scaffolds did not cause the death of the injected mice.The median lethal dose of Mg ions in vivo for mice was determined for the first time in this study to be 110.66 mg/kg,and the safety level of serum magnesium toxicity in mice is 5.4 mM,while the calcium serum safety level is determined as 3.4 mM.The study was approved by the Animal Care and Use Committee of Shenzhen Institute of Advanced Technology,Chinese Academy of Sciences(approval No.SIAT-IRB-170401-YGS-LYX-A0346)on April 5,2017.All these results showed that the Mg ion concentration of intraperitoneally-injected extract solutions was a determinant of mouse survival,and a high Mg ion concentration(more than 240 mM)was the pivotal factor contributing to the death of the mice,while changes in pH value showed a negligible effect.The comprehensive acute systemic toxicity evaluation for PTM porous composite scaffolds in this study provided a reference to guide the design and optimization of this composite scaffold and the results demonstrated the preclinical safety of the as-fabricated PTM scaffold with appropriate Mg content,strongly supporting the official registration process of the PTM scaffold as a medical device for clinical translation.

Osteogenic and antibacterial dual functions of a novel levofloxacin loaded mesoporous silica microspheres/nano-hydroxyapatite/ polyurethane composite scaffold

Lev/MSNs/n-HA/PU has been proved to be a novel scaffold material to treat bone defect caused by chronic osteomyelitis.We have previously identified that this material can effectively treat chronic osteomyelitis caused by Staphylococcus aureus in vivo.However,the potential mechanisms of antibacterial and osteogenic induction properties remain unclear.Thus,for osteogenesis property,immunohistochemistry,PCR,and Western blot were performed to detect the expression of osteogenic markers.Furthermore,flow cytometry and TUNEL were applied to analyze MC3T3-E1 proliferation and apoptosis.For antibacterial property,the material was co-cultivated with bacteria,bacterial colony forming units was counted and the release time of the effective levofloxacin was assayed by agar disc-diffusion test.Moreover,scanning electron microscope was applied to observe adhesion of bacteria.In terms of osteogenic induction,we found BMSCs adherently grew more prominently on Lev/MSNs/n-HA/PU.Lev/MSNs/n-HA/PU also enhanced the expression of osteogenic markers including OCN and COL1a1,as well as effectively promoted the transition from G1 phase to G2 phase.Furthermore,Lev/MSNs/n-HA/PU could reduce apoptosis of MC3T3-E1.Besides,both Lev/MSNs/n-HA/PU and n-HA/PU materials could inhibit bacterial colonies,while Lev/MSNs/n-HA/PU possessed a stronger antibacterial activities,and lower bacterial adhesion than n-HA/PU.These results illustrated that Lev/MSNs/n-HA/PU composite scaffold possess favorable compatibility in vitro,which induce osteogenic differentiation of MSCs,promote proliferation and differentiation of MC3T3-E1,and inhibit apoptosis.Moreover,clear in vitro antibacterial effect of Lev/MSNs/n-HA/PU was also observed.In summary,this study replenishes the potential of Lev/MSNs/n-HA/PU composite scaffold possess dual functions of anti-infection and enhanced osteogenesis for future clinical application.

Novel magnetic silk fibroin scaffolds with delayed degradation for potential long-distance vascular repair

Although with the good biological properties,silk fibroin(SF)is immensely restrained in long-distance vascular defect repair due to its relatively fast degradation and inferior mechanical properties.It is necessary to construct a multifunctional composite scaffold based on SF.In this study,a novel magnetic SF scaffold(MSFCs)was prepared by an improved infiltration method.Compared with SF scaffold(SFC),MSFCs were found to have better crystallinity,magnetocaloric properties,and mechanical strength,which was ascribed to the rational introduction of iron-based magnetic nanoparticles(MNPs).Moreover,in vivo and in vitro experiments demonstrated that the degradation of MSFCs was significantly extended.The mechanism of delayed degradation was correlated with the dual effect that was the newly formed hydrogen bonds between SFC and MNPs and the complexing to tyrosine(Try)to inhibit hydrolase by internal iron atoms.Besides,theβ-crystallization of protein in MSFCs was increased with the rise of iron concentration,proving the beneficial effect after MNPS doped.Furthermore,although macrophages could phagocytose the released MNPs,it did not affect their function,and even a reasonable level might cause some cytokines to be upregulated.Finally,in vitro and in vivo studies demonstrated that MSFCs showed excellent biocompatibility and the growth promotion effect on CD34-labeled vascular endothelial cells(VECs).In conclusion,we confirm that the doping of MNPs can significantly reduce the degradation of SFC and thus provide an innovative perspective of multifunctional biocomposites for tissue engineering.

Titanium alloy composited with dual-cytokine releasing polysaccharide hydrogel to enhance osseointegration via osteogenic and macrophage polarization signaling pathways

Titanium alloy has been widely used in orthopedic surgeries as bone defect filling.However,the regeneration of high-quality new bones is limited due to the pro-inflammatory microenvironment around implants,resulting in a high occurrence rate of implant loosening or failure in osteological therapy.In this study,extracellular matrix-mimetic polysaccharide hydrogel co-delivering BMP-2 and interleukin(IL)-4 was composited with 3D printed titanium alloy to promote the osseointegration and regulate macrophage response to create a pro-healing microenvironment in bone defect.Notably,it is discovered from the bioinformatics data that IL-4 and BMP-2 could affect each other through multiple signal pathways to achieve a synergistic effect toward osteogenesis.The composite scaffold significantly promoted the osteoblast differentiation and proliferation of human bone marrow mesenchyme stem cells(hBMSCs).The repair of large-scale femur defect in rat indicated that the dual-cytokinedelivered composite scaffold could manipulate a lower inflammatory level in situ by polarizing macrophages to M2 phenotype,resulting in superior efficacy of mature new bone regeneration over the treatment of native titanium alloy or that with an individual cytokine.Collectively,this work highlights the importance of M2-type macrophages-enriched immune-environment in bone healing.The biomimetic hydrogel–metal implant composite is a versatile and advanced scaffold for accelerating in vivo bone regeneration,holding great promise in treating orthopedic diseases.

Biphasic mineralized collagen-based composite scaffold for cranial bone regeneration in developing sheep

Appropriate mechanical support and excellent osteogenic capability are two essential prerequisites of customized implants for regenerating large-sized cranial bone defect.Although porous bone scaffolds have been widely proven to promote bone regeneration,their weak mechanical properties limit the clinical applications in cranioplasty.Herein,we applied two previously developed mineralized collagen-based bone scaffolds(MC),porous MC(pMC)and compact MC(cMC)to construct a biphasic MC composite bone scaffold(bMC)to repair the large-sized cranial bone defect in developing sheep.A supporting frame composed of cMC phase in the shape of tic–tac–toe structure was fabricated first and then embedded in pMC phase.The two phases had good interfacial bond,attributing to the formation of an interfacial zone.The in vivo performance of the bMC scaffold was evaluated by using a cranial bone defect model in 1-month-old sheep.The computed tomography imaging,X-ray scanning and histological evaluation showed that the pMC phase in the bMC scaffold,similar to the pMC scaffold,was gradually replaced by the regenerative bone tissues with comprehensively increased bone mineral density and complete connection of bone bridge in the whole region.The cMC frame promoted new bone formation beneath the frame without obvious degradation,thus providing appropriate mechanical protection and ensuring the structural integrity of the implant.In general,the sheep with bMC implantation exhibited the best status of survival,growth and the repair effect.The biphasic structural design may be a prospective strategy for developing new generation of cranioplasty materials to regenerate cranial bone defect in clinic.

Decellularized small intestine submucosa/polylactic-co-glycolic acid composite scaffold for potential application in hypopharyngeal and cervical esophageal tissue repair

There has been an increase in the incidence of hypopharyngeal and cervical esophageal cancer worldwide,and hence growing needs for hypopharyngeal and cervical esophageal tissue repair.This work produced a bi-layer composite scaffold with decellularized small intestine submucosa and polylactic-co-glycolic acid,which resembled the layered architectures of its intended tissues.The decellularized small intestine submucosa contained minimal residual DNA(52.5±61.2 ng/mg)and the composite scaffold exhibited satisfactory mechanical properties(a tensile modulus of 21.1±64.8 MPa,an ultimate tensile strength of 14.0±62.9MPa and a failure strain of 26.9±65.1%).The interactions between cells and the respective layers of the scaffold were characterized by CCK-8 assays,immunostaining and Western blotting.Desirable cell proliferation and phenotypic behaviors were observed.These results have provided an important basis for the next-step in vivo studies of the scaffold,and bode well for its future clinical applications.

Preparation and Characterization of 3D Printed Hydroxyapatite-Whisker-Strengthened Hydroxyapatite Scaffold Coated with Biphasic Calcium Phosphate

This study investigates the in vitro degradation of calcium-deficient hydroxyapatite powder after heat treatment at different temperatures and analyzes the calculated phase composition,particle size distribution,degradation rate,and bioactivity of the powder after heat treatment.A mixture of hydroxyapatite and𝛽-tricalcium phosphate(BCP)coatings was prepared on the surface of a 3D-printed hydroxyapatite-whisker-strengthened hydroxyapatite scaf-fold(HAw/HA)by vacuum impregnation and ultraviolet light curing combined with an optimized heat treatment process.The performance of the coatings under different methods was characterized.The composite scaffolds with highly interconnected pores and excellent mechanical properties were prepared,and their biodegradation performance,bioactivity,osteoconductivity,and osteoinductivity of the scaffolds were improved.The results showed that calcium-deficient hydroxyapatite began to transform into BCP between 600℃and 800℃,and the powder treated at 800℃has better bioactivity.The BCP coating prepared by light curing was more uniform,resulting in a higher interfacial bonding strength,and has better osteoconductivity and osteoinductivity than that prepared by vacuum impregnation.

Synthesis and characterization of collagen/PLGA biodegradable skin scaffold fibers

The aim of this study is to investigate the applicability of poly(lactic-co-glycolic acid)(PLGA)/collagen composite scaffold for skin tissue engineering.PLGA and collagen were dissolved in HFIP as a common solvent and fibrous scaffolds were prepared by electrospinning method.The scaffolds were characterized by scanning electron microscopy(SEM),FTIR spectroscopy,mercury porosimetry,tensile strength,biocompatibility assays and Biodegradation.Cytotoxicity and cell adhesion were tested for two cell line groups,human dermal fibroblast(HDF)and human keratinocyte(HaCat).SEM images showed appropriate cell adhesion to the scaffold for both cell lines.MTT assays indicated that the cell viability of HDF cells increased with time,but the number of HaCat cells decreased after 14 days.The ultimate tensile strength was suitable for skin substitute application,but its elongation at break was rather low.For successful clinical application of the PLGA/collagen scaffold,some properties especially mechanical strain needs to be improved.