Loose Pre-Cross-Linking Mediating Cellulose Self-Assembly for 3D Printing Strong and Tough Biomimetic Scaffolds.

The lack of an effective printable ink preparation method and the usual mechanically weak performance obstruct the functional 3D printing hydrogel exploitation and application. Herein, we propose a gentle pre-cross-linking strategy to enable a loosely cross-linked cellulose network for simultaneously achieving favorable printability and a strong hydrogel network via mediating the cellulose self-assembly. A small amount of epichlorohydrin is applied to (i) slightly pre-cross-link the cellulose chains for forming the percolating network to regulate the rheological properties and (ii) form the loosely cross-linked points to mediate the cellulose chains' self-assembly for achieving superior mechanical properties. The fabrication of the complex 3D structures verifies the design flexibility. The printed cellulose hydrogels exhibit a biomimetic nanofibrous topology, remarkable tensile and compressive strength (5.22 and 11.80 MPa), as well as toughness (1.81 and 2.16 MJ/m3). As a demonstration, a bilayer scaffold (mimicking the osteochondral structure) consisting of a top pristine cellulose and a bottom cellulose/bioactive glass hydrogel is printed and exhibits superior osteochondral defect repair performance, showing a potential in tissue engineering. We anticipate that our loose pre-cross-linking 3D printing ink preparation concept can inspire the development of other polymeric inks and strong 3D printing functional hydrogels, eventually spreading the applications in diverse fields.

Conducting molybdenum sulfide/graphene oxide/polyvinyl alcohol nanocomposite hydrogel for repairing spinal cord injury.

A sort of composite hydrogel with good biocompatibility, suppleness, high conductivity, and anti-inflammatory activity based on polyvinyl alcohol (PVA) and molybdenum sulfide/graphene oxide (MoS2/GO) nanomaterial has been developed for spinal cord injury (SCI) restoration. The developed (MoS2/GO/PVA) hydrogel exhibits excellent mechanical properties, outstanding electronic conductivity, and inflammation attenuation activity. It can promote neural stem cells into neurons differentiation as well as inhibit the astrocytes development in vitro. In addition, the composite hydrogel shows a high anti-inflammatory effect. After implantation of the composite hydrogel in mice, it could activate the endogenous regeneration of the spinal cord and inhibit the activation of glial cells in the injured area, thus resulting in the recovery of locomotor function. Overall, our work provides a new sort of hydrogels for SCI reparation, which shows great promise for improving the dilemma in SCI therapy.

Zero-Dimensional Carbon Dots Enhance Bone Regeneration, Osteosarcoma Ablation, and Clinical Bacterial Eradication.

Zero-dimensional carbon dots (CD) and their effects on osteogenesis have been rarely studied in bone repair scaffolds. Here, we fabricate a novel CD doped chitosan/nanohydroxyapatite (CS/nHA/CD) scaffold with full potential to promote bone regeneration by a facile freeze-drying method. The CS/nHA/CD scaffolds enhanced cell adhesion and osteoinductivity in rat bone mesenchymal stem cells by up-regulating genes involved in focal adhesion and osteogenesis in vitro, which significantly improved the formation of vascularized new bone tissue at 4 weeks compared to pure CS/nHA scaffolds in vivo. Inspired by the excellent photothermal effect of CD, the scaffolds were applied in tumor photothermal therapy (PTT) under near-infrared (NIR) irradiation (808 nm, 1 W/cm2). The scaffolds significantly inhibited osteosarcoma cell proliferation in vitro and effectively suppressed tumor growth in vivo. Moreover, the CS/nHA/CD scaffolds possessed distinct antibacterial properties toward clinically collected S. aureus and E. coli, and their antibacterial activity was further enhanced under NIR irradiation. This work demonstrates that zero-dimensional CD can enhance the osteogenesis-inducing property of bone repair scaffolds and that CD doped scaffolds have potential for use in PTT for tumors and infections.

Osteon-mimetic 3D nanofibrous scaffold enhances stem cell proliferation and osteogenic differentiation for bone regeneration.

The scaffold microstructure is important for bone tissue engineering. Failure to synergistically imitate the hierarchical microstructure of the components of bone, such as an osteon with concentric multilayers assembled by nanofibers, hinders the performance for guiding bone regeneration. Here, a 2D bilayer nanofibrous membrane (BLM) containing poly(lactide-co-glycolide) (PLGA)/polycaprolactone (PCL) composite membranes in similar compositions (PCL15 and PCL20), but possessing different degrees of shrinkage, was fabricated via sequential electrospinning. Upon incubation in phosphate buffered saline (PBS) (37 °C), the 2D BLM spontaneously deformed into a 3D shape induced by PCL crystallization within the PLGA matrix, and the PCL15 and PCL20 layer formed a concave and convex surface, respectively. The 3D structure contained curved multilayers with an average diameter of 776 ± 169 µm, and on the concave and convex surface the nanofiber diameters were 792 ± 225 and 881 ± 259 nm, respectively. The initial 2D structure facilitated the even distribution of seeded cells. Adipose-derived stem cells from rats (rADSCs) proliferated faster on a concave surface than on a convex surface. For the 3D BLM, the osteogenic differentiation of rADSCs was significantly higher than that on 2D surfaces, even without osteogenic supplements, which resulted from the stretched cell morphology on the curved sublayer leading to increased expression of lamin-A. After being implanted into cranial defects in Sprague Dawley (SD) rats, 3D BLM significantly accelerated bone formation. In summary, 3D BLM with an osteon-like structure provides a potential strategy to repair bone defects.

Storage and release of rare earth elements in microsphere-based scaffolds for enhancing osteogenesis.

In osteoporosis and diabetes, it is essential to accelerate the bone repair and regeneration process. Trace rare earth elements such as lanthanum (La) ions (La3+) with appropriate concentrations are bioactive and can effectively regulate bone tissue performances. However, few well-established bone tissue engineering scaffolds can precisely and stably release La3+ to promote bone regeneration significantly. Based on the advantages of biodegradable microspheres and microsphere-based scaffolds for controlled drug release, we developed poly(lactide-co-glycolide) (PLGA)-based microsphere-based scaffolds as both three-dimensional (3D) porous scaffolds and La3+ storage and release systems for osteogenesis. So far, there is no study about microsphere-based scaffolds to release trace La3+ to induce osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs). PLGA microspheres co-embedded with La-doped mesoporous silica (LMS) with different amounts of doped La were sintered to prepare the LMS/PLGA (LMSP) microsphere-based scaffold. The La3+ release behavior of LMSP can be controlled by adjusting the doping amount of La in mesoporous silica (MS). All these scaffolds possessed a 3D network architecture. With the increase of La doping, LMSP can better compensate for the pH decrease caused by PLGA degradation. The combination of MS and PLGA can avoid the cytotoxicity of MS alone. All prepared LMSP scaffolds were non-cytotoxic. After BMSCs were implanted on scaffolds, LMSP could promote cells adhesion, proliferation, and osteogenic differentiation. Among these microsphere-based scaffolds, LMSP-3 with stable and higher dose La3+ release behavior showed the strongest ability to enhance the osteogenesis of BMSCs. The results showed that microsphere-based scaffolds with the ability to store and stably control the release of La3+ could effectively improve osteogenic performance, which provides a new idea for the construction of bone tissue engineering scaffolds.

Assembling Microgels via Dynamic Cross-Linking Reaction Improves Printability, Microporosity, Tissue-Adhesion, and Self-Healing of Microgel Bioink for Extrusion Bioprinting.

Extrusion bioprinting has been widely used to fabricate complicated and heterogeneous constructs for tissue engineering and regenerative medicine. Despite the remarkable progress acquired so far, the exploration of qualified bioinks is still challenging, mainly due to the conflicting requirements on the printability/shape-fidelity and cell viability. Herein, a new strategy is proposed to formulate a dynamic cross-linked microgel assembly (DC-MA) bioink, which can achieve both high printability/shape-fidelity and high cell viability by strengthening intermicrogel interactions through dynamic covalent bonds while still maintaining the relatively low mechanical modulus of microgels. As a proof-of-concept, microgels are prepared by cross-linking hyaluronic acid modified with methacrylate and phenylboric acid groups (HAMA-PBA) and methacrylated gelatin (GelMA) via droplet-based microfluidics, followed by assembling into DC-MA bioink with a dynamic cross-linker (dopamine-modified hyaluronic acid, HA-DA). As a result, 2D and 3D constructs with high shape-fidelity can be printed without post-treatment, and the encapsulated L929 cells exhibit high cell viability after extrusion. Moreover, the addition of the dynamic cross-linker (HA-DA) also improves the microporosity, tissue-adhesion, and self-healing of the DC-MA bioink, which is very beneficial for tissue engineering and regenerative medicine applications including wound healing. We believe the present work sheds a new light on designing new bioinks for extrusion bioprinting.

Ultrafast in-situ forming halloysite nanotube-doped chitosan/oxidized dextran hydrogels for hemostasis and wound repair.

A series of halloysite nanotube (HNT)-doped chitosan (CS)/oxidized dextran (ODEX) adhesive hydrogels were developed through a Schiff base reaction. The resultant CS/ODEX/HNT hydrogels could not only form in situ on wounds within only 1 s when injected, but could also adapt to wounds of different shapes and depths after injection. We established four rat and rabbit hemorrhage models and demonstrated that the hydrogels are better than the clinically used gelatin sponge for reducing hemostatic time and blood loss, particularly in arterial and deep noncompressible bleeding wounds. Moreover, the natural antibacterial features of CS and ODEX provided the hydrogels with strong bacteria-killing effects. Consequently, they significantly promoted methicillin-resistant Staphylococcus aureus -infected-wound repair compared to commercial gelatin sponge and silver-alginate antibacterial wound dressing. Hence, our multifunctional hydrogels with facile preparation process and utilization procedure could potentially be used as first-aid biomaterials for rapid hemostasis and infected-wound repair in emergency injury events.

3D printing of Cu-doped bioactive glass composite scaffolds promotes bone regeneration through activating the HIF-1α and TNF-α pathway of hUVECs.

The increasing insight into the molecular and cellular processes within the angiogenic cascade assists in enhancing the survival and integration of engineered bone constructs. Copper-doped bioactive glass (Cu-BG) is now a potential structural component of the novel scaffolds and implants used in orthopedic and dental repairs. However, it is difficult for BG, especially micro-nano particles, to be printed into scaffolds and still retain its biological activity and ability to biodegrade. Additionally, the mechanisms of the copper-stimulating autocrine and paracrine effects of human umbilical vein endothelial cells (hUVECs) during repair and regeneration of bone are not yet clear. Therefore, in this study, we created monodispersed micro-nano spherical Cu-BG particles with varying copper content through a sol-gel process. Through in vitro tests, we found that Cu-BG enhanced angiogenesis by activating the pro-inflammatory environment and the HIF-1α pathway of hUVECs. Furthermore, 2Cu-BG diluted extracts directly promoted the osteogenic differentiation of mouse bone mesenchymal stem cells (BMSCs) in vitro. Then, a new 3D-printed tyramine-modified gelatin/silk fibroin/copper-doped bioactive glass (Gel/SF/Cu-BG) scaffold for rat bone defects was constructed, and the mechanism of the profound angiogenesis effect regulated by copper was explored in vivo. Finally, we found that hydrogel containing 1 wt% 2Cu-BG effectively regulated the spatiotemporal coupling of vascularization and osteogenesis. Therefore, Cu-BG-containing scaffolds have great potential for a wide range of bone defect repairs.

Improving osteogenesis of calcium phosphate bone cement by incorporating with manganese doped ß-tricalcium phosphate.

Lack of osteogenic capacity limits the bone repair effect of calcium phosphate cement (CPC). In present work, bivalent manganese ion (Mn2+) doped ß-tricalcium phosphate (Mn-TCP) was incorporated into CPC to enhance its osteogenic ability. The incorporation of Mn-TCP promoted the hydration reaction of CPC. The presence of Mn2+ made the hydration products finer. When adding 10 wt% Mn-TCP in CPC (Mn-CPC-1), the setting time of CPC was shortened, whereas the strength and injectability were not changed. Mouse Bone marrow mesenchymal stem cells (mBMSCs) on Mn-CPC-1 and CPC with 20 wt% Mn-TCP (Mn-CPC-2) presented better adhesion and spreading behaviors. Besides, Mn-CPC-1 promoted the gene levels of ALP, Col-I and OC while Mn-CPC-2 promoted the gene levels of Runx2 and OC. Cellular behaviors were related to two points: one was the increase of adsorption capacity of proteins (e.g. BSA) after changing the surface properties of bone cements; and the other was the biological role of Mn2+ released from CPC in osteogenesis. All the results indicated that CPC incorporated with 10 wt% Mn-TCP has good osteogenesis and proper physicochemical properties, which will be a prospective biomaterial applying in the area of bone regeneration.

Bilayer pifithrin-α loaded extracellular matrix/PLGA scaffolds for enhanced vascularized bone formation.

Small intestinal submucosa extracellular matrix (SIS-ECM) composite materials are catching eyes in tissue engineering but have been rarely studied in bone repair. In this study, we developed the unique bilayer bone scaffolds by assembling decellularized SIS-ECM and poly(lactic-co-glycolic acid) (PLGA) nanofibers through the electrospinning technique. To strengthen the bioactivity of the scaffolds, pifithrin-α (PFTα), a p53 inhibitor that can reduce the repressive function of p53 in osteogenesis, was preloaded in the PLGA electrospinning solution. We found that the resultant SIS-ECM/PLGA/PFTα scaffolds exhibited porous morphology, good biocompatibility, and enhanced osteoinductivity. Specifically, the SIS-ECM/PLGA/PFTα scaffolds could promote the osteogenic differentiation and mineralization of the preosteoblasts MC3T3-E1 in a PFTα does dependent manner in vitro. Furthermore, the SIS-ECM/PLGA/PFTα scaffolds were better than the pure SIS-ECM and SIS-ECM/PLGA scaffolds in terms of vessel and new bone tissue formation after 4 weeks post-implantation in vivo. These overall findings indicated that the bilayer PFTα loaded SIS-ECM/PLGA scaffolds facilitated vascularized bone regeneration, showing promising potential for bone tissue engineering.

Improved osteogenesis and angiogenesis of a novel copper ions doped calcium phosphate cement.

Improving the angio1genesis potential of bone-repairing materials is vital for the repair of cancerous bone defects. It can further facilitate the delivery of active substances with osteogenesis and anti-tumor functions, ultimately promoting the formation of new bone tissues. Copper ions (Cu2+) have been proved to be beneficial to angiogenesis. This study developed a new type of Cu-containing calcium phosphate cement (Cu-CPC) by incorporating with copper phosphate (CuP) nanoparticles with a photothermal anti-tumor effect. The results revealed that the main phases of all hydrated CPCs were hydroxyapatite, unreacted tricalcium phosphate and calcium carbonate. But the hydration products of CPC became thinner after the incorporation of Cu2+. With the increase of CuP concentration, the setting time of CPC was prolonged while the injectability and the compressive strength were increased. The release concentration of Cu2+in vitro was among 0.01 to 0.74 mg/mL, which showed a positive relation with CuP content. Mouse bone marrow stromal cells (mBMSCs) displayed higher adhesion activity, proliferation performance and expression of osteogenic genes and proteins on CPC with 0.01 wt% CuP (0.01Cu-CPC) and 0.05 wt% CuP (0.05Cu-CPC). When human umbilical vein endothelial cells were co-cultured with 0.01Cu-CPC and 0.05Cu-CPC extracts, the proliferation and angiogenesis-related gene and protein expression were significantly increased, and the in vitro tube formation capacity was promoted. However, higher CuP content inhibited the proliferation of mBMSCs. In conclusion, CPC with 0.01 wt% and 0.05 wt% CuP nanoparticles has the potential to promote bone formation around cancerous bone defects, which would be promising for bone regeneration and treatment of bone tumors.

Engineered macroporous hydrogel scaffolds via pickering emulsions stabilized by MgO nanoparticles promote bone regeneration.

Hydrogels are appealing biomaterials for regenerative medicine since biomimetic modifications of their polymeric network can provide unique physical properties and emulate the native extracellular matrix (ECM). Meanwhile, therapeutic metal ions, such as magnesium ions (Mg2+), not only regulate cellular behaviours but also stimulate local bone formation and healing. However, the absence of a meaningful macroporous structure and the uncompromising mechanical strength are still challenges. Herein, we designed a macroporous composite hydrogel based on mild and fast thiol-ene click reactions. The Pickering emulsion method was adopted to form a macroporous structure and introduce MgO nanoparticles (NPs). The results show that the composite hydrogel possesses good mechanical strength and an evenly distributed macroporous structure. MgO NPs stabilized at the oil/water interface not only function as effective emulsion stabilizers, but also enhance the mechanical properties of hydrogels and mediate the sustained release of Mg2+. In vitro cell experiments demonstrated that the composite hydrogel displays good biocompatibility. More importantly, the release of Mg2+ ions from hydrogels can effectively promote the osteogenic differentiation of BMSCs. Furthermore, an in vivo study showed that macroporous hydrogels can provide a good extracellular matrix microenvironment for in situ osteogenesis and accelerate bone tissue regeneration.

Comparative in vitro study on binary Mg-RE (Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu) alloy systems.

Correct selection of alloying elements is important for developing novel biodegradable magnesium alloys with superior mechanical and biological performances. In contrast to various reports on nutrient elements (Ca, Zn, Sr, etc.) as alloying elements of biomedical magnesium alloys, there is limited information about how to choose the right rare earth elements (REEs) as alloying elements of magnesium. In this work, 16 kinds of REEs were individually added into Mg, including Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Du, Ho, Er, Tm, Yb and Lu, to fabricate binary Mg-RE model alloys with different composition points. Under the same working history, comparative studies were undertaken and the impact of each kind of rare earth element on the microstructure, mechanical property, corrosion behavior and biocompatibility of Mg were investigated. The corresponding influence level for the 16 kinds of REEs were ranked. The results showed that the second phases were detected in some Mg-RE alloys, which were mainly composed of Mg12RE. By adding different REEs into Mg with proper contents, the mechanical properties of resulting Mg-RE binary alloys could be adjusted in wide range. The corrosion resistance of Mg-light REE alloys was generally better than Mg-heavy REE alloys. As for biocompatibility, Mg-RE model alloys showed no cytotoxic effect on MC3T3-E1 cells. The hemolysis rates of all experimental Mg-RE model alloys were lower than 5% except for Mg-Lu alloy model. In general, the addition of different REEs into Mg could improve its performance from different aspects. This work provides a better understanding on suitable REEs as alloying elements for magnesium, and the future R&D direction on biomedical Mg-RE alloys was proposed. STATEMENT OF SIGNIFICANCE: In contrast to various reports on nutrient elements (Ca, Zn, Sr, etc.) as alloying elements of biomedical magnesium alloys, until now there is limited information about how to choose the right rare earth elements (REEs) as alloying elements of magnesium. In this work, comparative studies were undertaken by individually adding 16 kinds of REEs, including Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Du, Ho, Er, Tm, Yb and Lu, into Mg to fabricate binary Mg-RE model alloys, with different composition points, then the impact of each kind of rare earth element on the microstructure, mechanical property, corrosion behavior and biocompatibility of Mg under the same working history were investigated, and the corresponding influence level for the 16 kinds of REEs were ranked. This work provides a better understanding on suitable REEs as alloying elements for magnesium, and the future R&D direction on biomedical Mg-RE alloys was proposed.

Strontium ranelate simultaneously improves the radiopacity and osteogenesis of calcium phosphate cement.

In a minimally invasive surgery of osteoporotic fractures, high radiopacity is necessary to monitor the delivery and positioning of injectable cements and good osteogenesis is indispensable. In this work, strontium ranelate (SrR), an agent for treating osteoporosis, is firstly used as a radiopaque agent for calcium phosphate cement (CPC). The addition of SrR does not affect the hydration products of CPC, but prolonged the setting time and decreased the compressive strength. The injectability of the cement was higher than 85% when SrR content is more than 10 wt%. The radiopacity of CPC is significantly improved by SrR and higher than cortical bone when the content of SrR is more than 5 wt%. The concentration of Sr ions released from CPC is increased by the increasing content of SrR, which is among 17-1329 µM. Moreover, CPCs with SrR significantly promote the osteogenic differentiation of mouse bone marrow mesenchymal stem cells and inhibit the osteoclastogenic differentiation of RAW264.7 cells. Based on its good radiopacity and osteogenesis, suppressed osteoclastogenesis and appropriate physicochemical properties, the radiopaque CPC with more than 10 wt% SrR is prospective to be a promising biomaterial for osteoporotic fracture repairing in minimal invasive surgery.

In vitro and in vivo studies of Mg-30Sc alloys with different phase structure for potential usage within bone.

Proper alloying magnesium with element scandium (Sc) could transform its microstructure from α phase with hexagonal closed-packed (hcp) structure into ß phase with body-cubic centered (bcc) structure. In the present work, the Mg-30 wt% Sc alloy with single α phase, dual phases (α + ß) or ß phase microstructure were developed by altering the heat-treatment routines and their suitability for usage within bone was comprehensively investigated. The ß phased Mg-30 wt% Sc alloy showed the best mechanical performance with ultimate compressive strength of 603 ±â€¯39 MPa and compressive strain of 31 ±â€¯3%. In vitro degradation test showed that element scandium could effectively incorporate into the surface corrosion product layer, form a double-layered structure, and further protect the alloy matrix. No cytotoxic effect was observed for both single α phased and ß phased Mg-30 wt% Sc alloys on MC3T3 cell line. Moreover, the ß phased Mg-30 wt%Sc alloy displayed acceptable corrosion resistance in vivo (0.06 mm y-1) and maintained mechanical integrity up to 24 weeks. The degradation process did not significantly influence the hematology indexes of inflammation, hepatic or renal functions. The bone-implant contact ratio of 75 ±â€¯10% after 24 weeks implied satisfactory integration between ß phased Mg-30 wt%Sc alloy and the surrounding bone. These findings indicate a potential usage of the bcc-structured Mg-Sc alloy within bone and might provide a new strategy for future biomedical magnesium alloy design. STATEMENT OF SIGNIFICANCE: Scandium is the only rare earth element that can transform the matrix of magnesium alloy into bcc structure, and Mg-30 wt%Sc alloy had been recently reported to exhibit shape memory effect. The aim of the present work is to study the feasibility of Mg-30 wt%Sc alloy with different constitutional phases (single α phase, single ß phase or dual phases (α + ß)) as biodegradable orthopedic implant by in vitro and in vivo testings. Our findings showed that ß phased Mg-30 wt%Sc alloy which is of bcc structure exhibited improved strength and superior in vivo degradation performance (0.06 mm y-1). No cytotoxicity and systematic toxicity were shown for ß phased Mg-30 wt%Sc alloy on MC3T3 cell model and rat organisms. Moreover, good osseointegration, limited hydrogen gas release and maintained mechanical integrity were observed after 24 weeks' implantation into the rat femur bone.

Integrating 3D Printing and Biomimetic Mineralization for Personalized Enhanced Osteogenesis, Angiogenesis, and Osteointegration.

Titanium (Ti) alloy implants can repair bone defects at load-bearing sites. However, they mechanically mismatch with the natural bone and lack customized adaption with the irregularly major-sized load-bearing bone defects, resulting in the failure of implant fixation. Mineralized collagen (MC), a building block in bone, can induce angiogenesis and osteogenesis, and 3D printing technology can be employed to prepare scaffolds with an overall shape customized to the bone defect. Hence, we induced the formation of MC, made of hydroxyapatite (HAp) nanocrystals and collagen fibers, in 3D-printed porous Ti6Al4V (PT) scaffolds through in situ biomimetic mineralization. The resultant MC/PT scaffolds exhibited a bone-like Young's modulus and were customized to the anatomical contour of actual bone defects of rabbit model. We found that the biocompatibility and osteogenic differentiation are best when the mass ratio between HAp nanocrystals and collagen fibers is 1 in MC. We then implanted the MC/PT scaffolds into the customized radius defect rabbit model and found that the MC/PT scaffolds significantly improved the vascularized bone tissue formation and integration between new bone and the implants. Therefore, a combination of 3D printing and biomimetic mineralization could lead to customized 3D PT scaffolds for enhanced angiogenesis, osteogenesis, and osteointegration. Such scaffolds represent novel patient-specific implants for precisely repairing irregular major-sized load-bearing bone defects.