Construction of Biomimetic Tissues with Anisotropic Structures via Stepwise Algorithm-Assisted Bioprinting.

Tissue-specific natural anisotropic microstructures play an important role in the normal functioning of tissues, yet they remain difficult to construct by current printing techniques. Herein, a stepwise algorithm-assisted bioprinting technology for the construction of biomimetic tissues with a customizable anisotropic microstructure by combining the Adaptive Mesh Generation algorithm and the Greedy Search algorithm is developed. Based on the mechanical topology optimization design mechanism, the Adaptive Mesh Generation algorithm can generate controllable anisotropic mesh patterns with the minimum free energy in plane models according to tissue-specific requirements. Subsequently, the Greedy Search algorithm can program the generated pattern data into optimized printing paths, effectively avoiding structural deformations caused by the multiple stacking of materials and reducing the printing time. The developed bioprinting technique is suitable for various types of bioinks including polymers, hydrogels, and organic/inorganic complexes. After combining with a calcium phosphorus bioink, the compound algorithm-assisted bioprinting technique successfully customizes femurs with biomimetic chemical compositions, anisotropic microstructures, and biological properties, demonstrating its effectiveness. Additionally, algorithm-assisted bioprinting is generally suitable for most commercial extrusion bioprinters that function in the geometric code (G-code) drive mode. Therefore, the algorithm-assisted extrusion bioprinting technology offers an intelligent manufacturing strategy for the customization of anisotropic microstructures in biomimetic tissues.

Inhibition of JNK and activation of the AMPK-Nrf2 axis by corosolic acid suppress osteolysis and oxidative stress.

The intracellular reactive oxygen species contribute to RANKL-induced osteoclastogenesis and osteolysis. Nuclear factor-erythroid 2-related factor 2 (Nrf2), a redox-sensitive transcription factor, is critical in the cellular defense against oxidative stress by induction of antioxidants and cytoprotective enzymes. In the current study, it was first demonstrated that RANKL-induced osteoclastogenesis and hydroxylapatite resorption were suppressed by Corosolic acid (CA) via inhibiting p-JNK and activating p-AMPK. Meanwhile, p-65, p-38, Akt, and GSK-3ß were partly inhibited during the treatment of CA. Osteoclastogenesis related genes, including NFATc1, c-fos, cathepsin K, and CTR were down-regulated by CA as well. Furthermore, the intracellular oxidative stress of CA-treated osteoclasts was dramatically decreased and Nrf2 was translocated into the nucleus to activate antioxidants including HO-1, NQO-1, and GCLC by CA. The LPS-induced mice calvarial osteolysis model was established for the in vivo investigation. Micro-CT morphometric analysis revealed that the treatment of CA restored LPS-induced bone loss and formation of osteoclasts. Besides, p-p65 and p-JNK were activated in the LPS group but inhibited by CA in vivo. The treatment of CA also activated p-AMPK during its attenuating LPS-induced osteolysis. Conclusively, CA effectively protects against LPS-induced osteolysis by suppressing osteoclastogenesis and oxidative stress through the inhibition of the JNK and activation of the AMPK-Nrf2 axis.

Modification of Cysteine 179 in IKKß by Ursolic Acid Inhibits Titanium-Wear-Particle-Induced Inflammation, Osteoclastogenesis, and Hydroxylapatite Resorption.

Aseptic loosening of artificial joints mainly accounts for the failure of arthroplasty. We previously reported that ursolic acid (UA) inhibited osteolysis caused by titanium (Ti) wear particles via suppression of NF-kB signaling. In the present study, that the suppressive effect of UA on Ti-particle-induced inflammation and osteoclastogenesis targets on IKKß cys-179 was demonstrated. A retrovirus packaged IKKßC179A plasmid with a Cys-179 mutation replaced by Ala was constructed. qRT-PCR, immunoblot, and immunofluorescence were used to evaluate the gene expressions. Secreted inflammatory cytokines were detected by ELISA. Formation and function of osteoclastogenesis were evaluated by TRAP stain and hydroxylapatite resorption assays. As a result, a mutation of IKKßC179A rescued the therapeutic effect of UA on Ti-particle-induced inflammation, including morphological transforms, upregulation of iNOS and COX-2, increased secretions of TNF-α, IL-1ß, and IL-6, and decreased secretion of IL-10. Meanwhile, inhibition of osteoclastogenesis and hydroxylapatite resorptions were restored by transfection of IKKßC179A. Phosphorylations of p65 and the IKKα/ß complex and translocation of p65 into the nucleus were suppressed by UA but rescued by a mutation of IKKßC179A. Conclusively, UA inhibits Ti-wear-particle-induced inflammation, osteoclastogenesis, and hydroxylapatite resorption via modifying cysteine 179 of IKKß.

Three-dimensional substructure measurements for the differential diagnosis of ground glass nodules.

BACKGROUND: We analyzed the differences between maximum and peak computed tomography (CT) numbers (M-P), respectively representing the densities of the solid center and the main periphery of ground-glass nodules (GGNs), and the average change in M-P velocity (V(M-P)) during follow-up to differentiate between pre-invasive (PIA) and invasive adenocarcinoma (IAC). METHODS: Data of 102 patients were retrospectively collected and analyzed in our study including 43 PIAs and 59 IACs. Diameters, total volumes, and the maximum and peak CT numbers in CT number histograms were measured and followed for at least 3 months. This study was registered retrospectively. RESULTS: The M-P values for IACs were higher than those for PIAs (p = 0.001), with an area under the curve (AUC) of 0.810 and a threshold of 489.5 Hounsfield units (HU) in ROC analysis. The V(M-P) values for IACs were smaller than those for PIAs (p = 0.04), with an AUC of 0.805 and a threshold of 11.01 HU/day. CONCLUSIONS: M-P and V(M-P) values may help distinguish IACs from PIAs by representing the changes in the sub-structural densities of GGNs during follow-up.

Regulated macrophage immune microenvironment in 3D printed scaffolds for bone tumor postoperative treatment.

The 3D printing technique is suitable for patient-specific implant preparation for bone repair after bone tumor resection. However, improving the survival rate due to tumor recurrence remains a challenge for implants. The macrophage polarization induction to M2-type tumor-associated macrophages (TAMs) by the tumor microenvironment is a key factor of immunosuppression and tumor recurrence. In this study, a regenerative scaffold regulating the macrophage immune microenvironment and promoting bone regeneration in a dual-stage process for the postoperative treatment of bone tumors was constructed by binding a colony-stimulating factor 1 receptor (CSF-1R) inhibitor GW2580 onto in situ cosslinked hydroxybutylchitosan (HBC)/oxidized chondroitin sulfate (OCS) hydrogel layer covering a 3D printed calcium phosphate scaffold based on electrostatic interaction. The hydrogel layer on scaffold surface not only supplied abundant sulfonic acid groups for stable loading of the inhibitor, but also acted as the cover mask protecting the bone repair part from exposure to unhealthy growth factors in the microenvironment at the early treatment stage. With local prolonged release of inhibitor being realized via the functional material design, CSF-1R, the main pathway that induces polarization of TAMs, can be efficiently blocked, thus regulating the immunosuppressive microenvironment and inhibiting tumor development at a low therapeutic dose. At the later stage of treatment, calcium phosphate component of the scaffold can facilitate the repair of bone defects caused by tumor excision. In conclusion, the difunctional 3D printed bone repair scaffold regulating immune microenvironment in stages proposed a novel approach for bone tumor postoperative treatment.

Targeted Protein Fate Modulating Functional Microunits Promotes Intervertebral Fusion.

Stable regulation of protein fate is a prerequisite for successful bone tissue repair. As a ubiquitin-specific protease (USP), USP26 can stabilize the protein fate of ß-catenin to promote the osteogenic activity of mesenchymal cells (BMSCs) and significantly increased bone regeneration in bone defects in aged mice. However, direct transfection of Usp26 in vivo is inefficient. Therefore, improving the efficient expression of USP26 in target cells is the key to promoting bone tissue repair. Herein, 3D printing combined with microfluidic technology is applied to construct a functional microunit (protein fate regulating functional microunit, denoted as PFFM), which includes GelMA microspheres loaded with BMSCs overexpressing Usp26 and seeded into PCL 3D printing scaffolds. The PFFM provides a microenvironment for BMSCs, significantly promotes adhesion, and ensures cell activity and Usp26 supplementation that stabilizes ß-catenin protein significantly facilitates BMSCs to express osteogenic phenotypes. In vivo experiments have shown that PFFM effectively accelerates intervertebral bone fusion. Therefore, PFFM can provide new ideas and alternatives for using USP26 for intervertebral fusion and other hard-to-repair bone defect diseases and is expected to provide clinical translational potential in future treatments.

3D Biomimetic Calcified Cartilaginous Callus that Induces Type H Vessels Formation and Osteoclastogenesis.

The formation of a calcified cartilaginous callus (CACC) is crucial during bone repair. CACC can stimulate the invasion of type H vessels into the callus to couple angiogenesis and osteogenesis, induce osteoclastogenesis to resorb the calcified matrix, and promote osteoclast secretion of factors to enhance osteogenesis, ultimately achieving the replacement of cartilage with bone. In this study, a porous polycaprolactone/hydroxyapatite-iminodiacetic acid-deferoxamine (PCL/HA-SF-DFO) 3D biomimetic CACC is developed using 3D printing. The porous structure can mimic the pores formed by the matrix metalloproteinase degradation of the cartilaginous matrix, HA-containing PCL can mimic the calcified cartilaginous matrix, and SF anchors DFO onto HA for the slow release of DFO. The in vitro results show that the scaffold significantly enhances angiogenesis, promotes osteoclastogenesis and resorption by osteoclasts, and enhances the osteogenic differentiation of bone marrow stromal stem cells by promoting collagen triple helix repeat-containing 1 expression by osteoclasts. The in vivo results show that the scaffold significantly promotes type H vessels formation and the expression of coupling factors to promote osteogenesis, ultimately enhancing the regeneration of large-segment bone defects in rats and preventing dislodging of the internal fixation screw. In conclusion, the scaffold inspired by biological bone repair processes effectively promotes bone regeneration.

Engineered Customizable Microvessels for Progressive Vascularization in Large Regenerative Implants.

Inspired by the rapid angiogenesis of natural microvessels in vivo, engineered customizable microvessels (ECMVs) are developed which can naturally angiogenic sprout and induce vascular network formation via combing a celluar coaxial microfluidic extrusion technique with microsurgery post-process. ECMVs can be used for customization of primarily pre-vascularized soft tissue regenerative implants with personalized shape and vascular density with the aid of sacrificial printing technology. After collaborating with surrounding cells, ECMVs angiogenic sprouted and formed daughter vascular networks. Through techniques such as injection and suturing, ECMVs can also be introduced into large bone repair implants for pre-vascularization and osteogenesis promotion. Furthermore, the microvessel networks with personalized shapes are customized by connecting the coaxial microfluidic system to a 3D printer. It is further demonstrated that the vascularization promotion and anastomose with host vessels of the ECMVs in vivo. Thus, ECMVs provide a simple engineering strategy for rapid vascularization of clinically large regenerative soft/hard tissue implants.

Synergy effects of Asperosaponin VI and bioactive factor BMP-2 on osteogenesis and anti-osteoclastogenesis.

Osteoporosis is a reduction in skeletal mass due to the decrease of osteogenic ability and the activation of the osteoclastic function. Inhibiting bone resorption and accelerating the new bone formation is a promising strategy to repair the bone defect of osteoporosis. In this study, we first systematically investigated the roles of Chinese medicine Asperosaponin VI (ASP VI) on osteogenic mineralization of BMSCs and osteoclastogenesis of BMMs, and then explored the synergistic effect of ASP VI and BS (BMP-2 immobilized in 2-N, 6-O-sulfated chitosan) on bone formation. The result showed that ASP VI with the concentration lower than 10-4 M contributed to the expression of osteogenic gene and inhibited osteoclastic genes RANKL of BMSCs. Simultaneously, ASP VI significantly reduced the differentiation of mononuclear osteoclasts in the process of osteoclast formation induced by M-CSF and RANKL. Furthermore, by stimulating the SMADs, TGF-ß1, VEGFA, and OPG/RANKL signaling pathways, ASBS (ASP VI and BS) substantially enhanced osteogenesis, greatly promoted angiogenesis, and suppressed osteoclastogenesis. The findings provide a new perspective on osteoporosis care and prevention.

Transcriptome Analysis Revealed the Symbiosis Niche of 3D Scaffolds to Accelerate Bone Defect Healing.

Three dimension (3D) printed scaffolds have been shown to be superior in promoting tissue repair, but the cell-level specific regulatory network activated by 3D printing scaffolds with different material components to form a symbiosis niche have not been systematically revealed. Here, three typical 3D printed scaffolds, including natural polymer hydrogel (gelatin-methacryloyl, GelMA), synthetic polymer material (polycaprolactone, PCL), and bioceramic (ß-tricalcium phosphate, ß-TCP), are fabricated to explore the regulating effect of the symbiotic microenvironment during bone healing. Enrichment analysis show that hydrogel promotes tissue regeneration and reconstruction by improving blood vessel generation by enhancing oxygen transport and red blood cell development. The PCL scaffold regulates cell proliferation and differentiation by promoting cellular senescence, cell cycle and deoxyribonucleic acid (DNA) replication pathways, accelerating the process of endochondral ossification, and the formation of callus. The ß-TCP scaffold can specifically enhance the expression of osteoclast differentiation and extracellular space pathway genes to promote the differentiation of osteoclasts and promote the process of bone remodeling. In these processes, specific biomaterial properties can be used to guide cell behavior and regulate molecular network in the symbiotic microenvironment to reduce the barriers of regeneration and repair.

Biological homeostasis-inspired light-excited multistage nanocarriers induce dual apoptosis in tumors.

In the microenvironment of an organism, each element always regulates and compensates for each other's defects, finally achieving biostable equilibrium. Herein, inspired by the balance of biological homeostasis and the interconstraint of elements, light-responsive nanoparticle with anti-vascularization and oxygen-supplying ability such like a homeostasis body is constructed by the electrostatic adsorption of reactive oxygen species (ROS)-responsive copolymers with photosensitizers and oxygen donors, which act as the elements of homeostasis body can interact through multistage reactions forming a balance that induces double apoptosis including those caused by the photosensitizer itself and those induced after oxygenation. In this homeostasis body, the element photosensitizer can simultaneously generate hyperthermia and ROS. The former can not only inhibit the growth of blood vessels and promote cell necrosis, but induce the thermally responsive release of oxygen to alleviate tumor hypoxia for enhanced PDT. And the latter will induce rapid depolymerization of nanoparticles, promote the penetration and finally induce double apoptosis through multistage reactions. Immunofluorescence data further demonstrate that the nanoparticles significantly alleviated tumor hypoxia upon photoexcitation. Thus, such nanoparticles with multistage synergistic effects have demonstrated excellent effects in achieving biostable equilibrium to induce dual apoptosis and may also be a good strategy in hypoxic tumors therapy.

Tissue-specific engineering: 3D bioprinting in regenerative medicine.

Despite its complexity, the human body is composed of only four basic tissue types, namely epithelial, connective, muscular and nervous tissues. Notably, each tissue is an assemblage of similarly functional cells united in performing a specific function. Instead of mimicking functionality mechanically, three-dimensional (3D) bioprinting based on histological categories is a strategy designed with multiple materials and techniques, which is a versatile technology able to form functional organ structures in line with simplicity. This review aims to provide an overview of tissue-specific 3D bioprinting based on the biological characteristics of four tissue types, including the histological features, biomaterials and corresponding applications. It first briefly introduces the goals of tissue-specific bioprinting and then summarizes the major techniques and identification of particular material development. Moreover, its remarkable regenerative power in replacement therapy and novel outbreak in particular tissues are assembled by epithelial, connective, nerve and muscle tissues. Finally, we discuss challenges and future prospects of tissue-specific based 3D bioprinting in biomedicine, hoping to further inspire the development.

Patient-specific Scaffolds with a Biomimetic Gradient Environment for Articular Cartilage-Subchondral Bone Regeneration.

Functional articular repair is known to be hampered by tissue degradation, which occurs in the defective local inflammatory environment that is also characterized by disrupted angiogenesis. The advanced fabrication of scaffolds with designed chemical and physical cues, which provide a biomimetic environment for tissue regeneration, holds considerable promise to circumvent the problem and thus allows functional articular repair. Herein, we developed scaffolds with controllable shapes with hydroxybutyl chitosan (HBC) and oxidized chondroitin sulfate (OCS) hydrogels, whose chemical composition was similar to that of the cartilage extracellular matrix (ECM). By optimizing the concentration of OCS, the functional cross-linker, we achieved a hydrogel promoting proliferation, adhesion, and ECM formation of chondrocytes and inhibiting tube formation of endothelial cells. Using a hydration procedure and bioactivation of mesenchymal stem cells (MSCs), we obtained mesoporous silicate-doped calcium phosphate cement (MS/CPC) scaffolds with a bioactive surface similar to that of bones, with improved osteogenesis and vascularization properties. Personalized cartilage-subchondral repair scaffolds with stable combination were successfully fabricated based on the self-cross-linking properties of the Schiff-based HBC/OCS hydrogel and the macroporous structure of MS/CPC scaffolds with the aid of a 3D printing technique. This study proposes a strategy to design individualized tissue repair biomimetic gradient scaffolds. Further assessments of their osteochondral defect repair properties in vivo should be performed.

IL-35 ameliorates collagen-induced arthritis by promoting TNF-α-induced apoptosis of synovial fibroblasts and stimulating M2 macrophages polarization.

Synovitis, the chronic inflammation of the synovial membranes, is a hallmark of rheumatoid arthritis, a chronic disease with profound impact on human health. Recently, interleukin-35 (IL-35), a new member of the IL-12 family, was identified as an anti-inflammatory and immunosuppressive cytokine and was shown to ameliorate collagen-induced arthritis (CIA) in mice. However, the mechanism by which IL-35 alleviates CIA remains unknown. In this study, we investigated the effect of IL-35 on the CIA microenvironment and, specifically, the tumor necrosis factor alpha (TNF-α)-induced macrophage inflammatory response and apoptosis of fibroblast-like synoviocytes (FLSs). Firstly, using RT-PCR, western blot, and flow cytometry, we found that IL-35 suppressed TNF-α-induced inflammatory responses by down-regulating iNOS and COX-2 in peripheral blood monocyte-derived macrophages. IL-35 also activated alternative M2 macrophage polarization, as determined by evaluation of CCR7 and CD206 expression. Moreover, we showed that IL-35 enhanced TNF-α-induced FLS apoptosis. Using a panel of immunohistochemical and immunofluorescence analyses in a CIA model established in 18 DBA/1J mice, we demonstrated that IL-35 promotes synoviocyte apoptosis and alternative activation of macrophages to alleviate arthritis in vivo. Taken together, our results show that IL-35 promotes TNF-α-induced FLS apoptosis and modulates M2 macrophage polarization to ameliorate CIA inflammation both in vitro and in vivo.

Controllable fabrication of hydroxybutyl chitosan/oxidized chondroitin sulfate hydrogels by 3D bioprinting technique for cartilage tissue engineering.

Biological regeneration of articular cartilage continues to be a challenge at present. Functional engineered implants with patient-specific sizes are difficult to achieve. The aim of this study is to fabricate a biocompatible cell-laden hydrogel with a designable structure. Covalent hydrogels were prepared with water soluble hydroxybutyl chitosan (HBC) and oxidized chondroitin sulfate (OCS) via a Schiff-base reaction. With the aid of three-dimensional (3D) bioprinted sacrificial molds, HBC/OCS hydrogel with various structures were obtained. After the material constituent optimization process, an injectable hydrogel with a uniform porous structure of 100 µm average pore size was developed to form macroporous hydrogel. In vitro and in vivo biocompatibility of optimized HBC/OCS hydrogel were also carefully assessed. The results indicated that human adipose-derived mesenchymal stem cells could be 3D cultured in HBC/OCS hydrogel maintaining good viability. Moreover, the hydrogels were found to trigger the least amount of pro-inflammatory gene expression of macrophage and to inhibit acute immune responses in 7 d. These results demonstrate the potential of HBC/OCS hydrogels as a cell delivery system for cartilage tissue engineering.

Interleukin-35 Inhibits TNF-α-Induced Osteoclastogenesis and Promotes Apoptosis via Shifting the Activation From TNF Receptor-Associated Death Domain (TRADD)-TRAF2 to TRADD-Fas-Associated Death Domain by JAK1/STAT1.

Over-activated osteoclasts derived from myeloid or peripheral blood monocytes by inflammatory cytokines results in osteoporosis, osteoarthritis, and other bone erosion-related diseases. Interleukin 35 (IL-35) is a novel anti-inflammatory and immunosuppressive factor. This study investigated the effect of IL-35 on TNF-α-induced osteoclastogenesis. In the presence of IL-35, this process was detected by Tartrate-Resistant Acid Phosphatase (TRAP) staining, F-actin staining, and bone resorption assays. The effects of IL-35 on TNF-α-induced apoptosis were demonstrated by TUNEL staining, cell viability assays, and flow cytometry. Moreover, a microarray was performed to detect the effect of IL-35 on TNF-α-activated phosphatase kinase. The effect of IL-35 on the TNF-α-mediated activation of NF-κB, MAPK, TRAF2, RIP1, Fas-associated death domain (FADD), and caspase3 was further investigated. In addition, a murine calvarial osteolysis model was established via the subcutaneous injection of TNF-α onto the calvaria, and histological analysis was subsequently performed. As a result, IL-35 inhibited TNF-α-induced osteoclast formation and bone resorption in vitro and osteolysis calvaria in vivo. NFATc1, c-fos, and TRAP were downregulated by IL-35 through the inhibition of NF-κB and MAPK, during which JAK1/STAT1 was activated. Moreover, based on TUNEL staining and flow cytometry, IL-35 was shown to enhance TNF-α-induced osteoclast apoptosis. Meanwhile, FADD and cleaved-caspase 3 were increased in cells treated with TNF-α and IL-35, whereas the DNA-binding activity of NF-κB was increased in TNF-α-treated cells, but was decreased in cells treated with both TNF-α and IL-35. In conclusion, IL-35 inhibits TNF-α-induced osteoclastogenesis and promotes apoptosis by activating JAK1/STAT1 and shifting activation from TNF receptor-associated death domain (TRADD)-TRAF2/RIP1-NF-κB to TRADD-FADD-caspase 3 signaling.

Proinflammatory and osteolysis-inducing effects of 3D printing Ti6Al4V particles in vitro and in vivo.

Ti6Al4V printing particles have been recently used for fabricating orthopedic implants. Removing these particles completely from fabricated implants is challenging. Furthermore, recycled particles are commonly used in fabrication without additional analysis. Ti6Al4V wear particles derived from orthopedic implants are known to induce inflammatory responses and osteolysis. However, the biosafety of printing particles remains unknown. Here, we investigated the proinflammatory and osteolysis-inducing effects of commonly used original and recycled Ti6Al4V printing particles in vitro and in vivo. Our results indicated that although less serious effects were induced compared to wear particles, inflammatory responses and osteoclast-mediated bone resorption were induced by the original printing particles in a particle size-dependent manner. Recycled particles were found to more strongly stimulate bone resorption and inflammatory responses than the original particles; the in vivo effect was enhanced with an increase in particle concentration. Furthermore, the results of our in vitro experiments verified that the printing particles activate macrophages to secrete inflammatory cytokines and promote osteoclastogenesis, which is closely related to particle size and concentration. Taken together, our findings provide a valuable reference for the use of raw printing materials and examination of recycling procedures for implant fabrication.

RhBMP-2 loaded 3D-printed mesoporous silica/calcium phosphate cement porous scaffolds with enhanced vascularization and osteogenesis properties.

A major limitation in the development of effective scaffolds for bone regeneration has been the limited vascularization of the regenerating tissue. Here, we propose the development of a novel calcium phosphate cement (CPC)-based scaffold combining the properties of mesoporous silica (MS) with recombinant human bone morphogenic protein-2 (rhBMP-2) to facilitate vascularization and osteogenesis. Specifically, the development of a custom MS/CPC paste allowed the three-dimensional (3D) printing of scaffolds with a defined macroporous structure and optimized silicon (Si) ions release profile to promote the ingrowth of vascular tissue at an early stage after implantation in support of tissue viability and osteogenesis. In addition, the scaffold microstructure allowed the prolonged release of rhBMP-2, which in turn significantly stimulated the osteogenesis of human bone marrow stromal cells in vitro and of bone regeneration in vivo as shown in a rabbit femur defect repair model. Thus, the combination MS/CPC/rhBMP-2 scaffolds might provide a solution to issues of tissue necrosis during the regeneration process and therefore might be able to be readily developed into a useful tool for bone repair in the clinic.

3D printed scaffolds of calcium silicate-doped ß-TCP synergize with co-cultured endothelial and stromal cells to promote vascularization and bone formation.

Synthetic bone scaffolds have potential application in repairing large bone defects, however, inefficient vascularization after implantation remains the major issue of graft failure. Herein, porous ß-tricalcium phosphate (ß-TCP) scaffolds with calcium silicate (CS) were 3D printed, and pre-seeded with co-cultured human umbilical cord vein endothelial cells (HUVECs) and human bone marrow stromal cells (hBMSCs) to construct tissue engineering scaffolds with accelerated vascularization and better bone formation. Results showed that in vitro ß-TCP scaffolds doped with 5% CS (5%CS/ß-TCP) were biocompatible, and stimulated angiogenesis and osteogenesis. The results also showed that 5%CS/ß-TCP scaffolds not only stimulated co-cultured cells angiogenesis on Matrigel, but also stimulated co-cultured cells to form microcapillary-like structures on scaffolds, and promoted migration of BMSCs by stimulating co-cultured cells to secrete PDGF-BB and CXCL12 into the surrounding environment. Moreover, 5%CS/ß-TCP scaffolds enhanced vascularization and osteoinduction in comparison with ß-TCP, and synergized with co-cultured cells to further increase early vessel formation, which was accompanied by earlier and better ectopic bone formation when implanted subcutaneously in nude mice. Thus, our findings suggest that porous 5%CS/ß-TCP scaffolds seeded with co-cultured cells provide new strategy for accelerating tissue engineering scaffolds vascularization and osteogenesis, and show potential as treatment for large bone defects.

Fabrication and characterization of toughness-enhanced scaffolds comprising ß-TCP/POC using the freeform fabrication system with micro-droplet jetting.

A novel elastomeric material, poly(1,8-octanediol-co-citrate) (POC), has demonstrated tremendous versatility because of its advantageous toughness, tunable degradation properties, and efficient drug release capability. In this study, POC was used to improve the mechanical performance of ß-tricalcium phosphate (ß-Ca3(PO4)2, ß-TCP). (3D) ß-TCP/POC composite scaffolds were fabricated by a 3D printing technique based on the freeform fabrication system with micro-droplet jetting (FFS-MDJ). The physiochemical properties, compressive modulus, drug release behavior, and cell response of ß-TCP/POC composite scaffolds were systematically investigated. The results showed that ß-TCP/POC scaffolds had uniform macropores of 300-400 µm, porosity of approximately 45%, biodegradability in phosphate-buffered saline, and high compressive modulus of 50-75 MPa. With the incorporation of POC into ß-TCP, the toughness of the composite scaffolds was improved significantly. Moreover, ß-TCP/POC scaffolds exhibited sustained drug (ibuprofen (IBU)) release capability. Additionally, ß-TCP/POC scaffolds facilitated C2C12 cell attachment and proliferation. It was indicated that the 3D-printed porous ß-TCP/POC scaffolds with high compressive modulus and good drug delivery performance might be a promising candidate for bone defect repair.