3D Printed Tricalcium Phosphate Scaffolds Research Articles

Osteogenically committed hUCMSCs-derived exosomes promote the recovery of critical-sized bone defects with enhanced osteogenic properties.

Low viability of seed cells and the concern about biosafety restrict the application of cell-based tissue-engineered bone (TEB). Exosomes that bear similar bioactivities to donor cells display strong stability and low immunogenicity. Human umbilical cord mesenchymal stem cells-derived exosomes (hUCMSCs-Exos) show therapeutic efficacy in various diseases. However, little is known whether hUCMSCs-Exos can be used to construct TEB to repair bone defects. Herein, PM-Exos and OM-Exos were separately harvested from hUCMSCs which were cultured in proliferation medium (PM) or osteogenic induction medium (OM). A series of in-vitro studies were performed to evaluate the bioactivities of human bone marrow mesenchymal stem cells (hBMSCs) when co-cultured with PM-Exos or OM-Exos. Differential microRNAs (miRNAs) between PM-Exos and OM-Exos were sequenced and analyzed. Furthermore, PM-Exos and OM-Exos were incorporated in 3D printed tricalcium phosphate scaffolds to build TEBs for the repair of critical-sized calvarial bone defects in rats. Results showed that PM-Exos and OM-Exos bore similar morphology and size. They expressed representative surface markers of exosomes and could be internalized by hBMSCs to promote cellular migration and proliferation. OM-Exos outweighed PM-Exos in accelerating the osteogenic differentiation of hBMSCs, which might be attributed to the differentially expressed miRNAs. Furthermore, OM-Exos sustainably released from the scaffolds, and the resultant TEB showed a better reparative outcome than that of the PM-Exos group. Our study found that exosomes isolated from osteogenically committed hUCMSCs prominently facilitated the osteogenic differentiation of hBMSCs. TEB grafts functionalized by OM-Exos bear a promising application potential for the repair of large bone defects.

In vivo and In vitro properties evaluation of curcumin loaded MgO doped 3D printed TCP scaffolds.

The lack of site-specific chemotherapeutic agents to treat bone malignancy throws a significant challenge in the design of a delivery vehicle. The major scientific question posed in this study is, can we utilize curcumin-loaded magnesium oxide (MgO) doped 3D printed tricalcium phosphate (TCP) bone grafts as a localized delivery system that improves early stage in vivo osseointegration and in vitro chemoprevention, antibacterial properties? We have utilized curcumin as an alternative natural chemopreventive agent for bone cancer-specific delivery after direct incorporation on the 3D printed tricalcium phosphate (TCP) bone grafts. The addition of MgO as a dopant to TCP leads to ∼1.3 times enhancement in compressive strength. The designed drug delivery system shows up to ∼22% curcumin release in a physiological pH of 7.4 after 30 days. The presence of curcumin leads to up to ∼8.5 times reduction in osteosarcoma viability. In vitro results indicate that these scaffolds significantly enhance bone-forming osteoblast cells while reducing the bone-resorbing osteoclast cells. The in vivo rat distal femur model surgery followed by histological assessment with H&E, vWF, and Movat pentachrome staining results show that the designed scaffolds lead to new bone formation (up to ∼2.5 times higher than the control) after successful implantation. The presence of MgO and curcumin results in up to ∼71% antibacterial efficacy against osteomyelitis causing S. aureus. These 3D printed osteogenic and chemopreventive scaffolds can be utilized in patient-specific low load-bearing defect sites.

Effects of Vitamin A (Retinol) Release from Calcium Phosphate Matrices and Porous 3D Printed Scaffolds on Bone Cell Proliferation and Maturation

Vitamin A is a fat-soluble compound widely known for vision health. Highly variable reports on its effects on bone health have necessitated further research to truly understand its role on bone cell proliferation. Retinol, one bioactive form of vitamin A, is incorporated into synthetic bone graft scaffolds for low load-bearing clinical bone treatment. The objective of this work is to understand the effects of retinol on osteoblast and osteoclast cells when embedded within calcium phosphate matrices, including interconnected porous 3D printed tricalcium phosphate scaffolds. Results show that hydrophobic retinol can be released from bone scaffolds when a combination of biodegradable polymers, polycaprolactone and polyethylene glycol, are employed as drug carriers. The release of retinol in vitro can support a 20 ± 1% increase in osteoblast (bone-forming) cell proliferation with proper cell adhesion and filopodial extensions. Osteoclast cell morphology is necrosed and torn with a reduction in proliferation at approximately 6 ± 1% when retinol is present. In addition, inhibition of osteoclastic resorption pit bays is noted using scanning electron microscopy. With the scaffolds' round pore interconnectivity facilitating retinol release, this system can provide an alternative to traditional bone grafts while additionally supporting bone healing through enhanced osteoblast cell proliferation and inhibition of osteoclast resorption activity.

Effects of surface area and topography on 3D printed tricalcium phosphate scaffolds for bone grafting applications.

Additive manufacturing (AM), or 3D printing, of bioceramic scaffolds promises personalized treatment options for patients with site-specific designability for repair and reconstruction of bone defects. Although the theory for creating these complex geometries has already been made possible through AM's advancement, such shapes' manufacturability is difficult due to printing with ceramics' inherent complexities. Ceramics have the added challenge of being highly brittle, poor handleability of green (pre-sintered) parts, making complex shape high strength parts challenging to manufacture. This has led to a significant literature gap regarding the feasibility of creating bioceramic scaffolds with unique architectures that can be used in site-specific, individualized patient treatment. This work aims to successfully create complex topographical surfaces of cylindrical bone-like scaffolds to understand the correlation of increasing the scaffold surface area on mechanical properties and in vitro osteoblast cell proliferation. An increase in osteoblast cell proliferation and facilitation in cellular attachment can ultimately lead to improved bone healing. This work explores the printing parameters within an Innovent+® ExOne binder jet 3D printer to produce scaffold designs from synthesized tricalcium phosphate powder. Mechanical testing reveals the designed structures enhance scaffold compressive strength by 30% compared to control dense cylindrical scaffolds. Osteoblast cell proliferation is also increased due to changes in surface topography with a nearly 2-fold increase. Our work incorporates macro-level topographical changes to increase surface area, which is another avenue that could be combined with other scaffold features such as porosity. Results show bulk surface topography modifications via 3D printing can increase surface area to support enhanced biological response without compromising mechanical properties. This discovery may enable a future generation of porous scaffolds with external structures for further progress towards proper defect-specific synthetic bone grafts.

Research on sintering process of tricalcium phosphate bone tissue engineering scaffold based on three-dimensional printing

Tricalcium phosphate (TCP) is one of the most widely used bioceramics for constructing bone tissue engineering scaffold. The three-dimensional (3D) printed TCP scaffold has precise and controllable pore structure, while with the limitation of insufficient mechanical properties. In this study, we investigated the effect of sintering temperature on the mechanical properties of 3D-printed TCP scaffolds in detail, due to the important role of the sintering process on the mechanical properties of bioceramic scaffolds. The morphology, mass and volume shrinkage, porosity, mechanical properties and degradation property of the scaffold was studied. The results showed that the scaffold sintered at 1 150℃ had the maximum volume shrinkage, the minimum porosity and optimal mechanical strength, with the compressive strength of (6.52 ± 0.84) MPa and the compressive modulus of (100.08 ± 18.6) MPa, which could meet the requirements of human cancellous bone. In addition, the 1 150℃ sintered scaffold degraded most slowly in the acidic environment compared to the scaffolds sintered at the other temperatures, demonstrating its optimal mechanical stability over long-term implantation. The scaffold can support bone mesenchymal stem cells (BMSCs) adherence and rapid proliferation and has good biocompatibility. In summary, this paper optimizes the sintering process of 3D printed TCP scaffold and improves its mechanical properties, which lays a foundation for its application as a load-bearing bone.

Cytotoxic and osteogenic effects of crocin and bicarbonate from calcium phosphates for potential chemopreventative and anti-inflammatory applications in vitro and in vivo.

Delayed healing and nonhealing of bone defects or resected bone sites remains an important clinical concern in the biomedical field. Osteosarcoma is one of the most common types of primary bone cancers. Among calcium phosphates, hydroxyapatite (HA) and tricalcium phosphate (TCP) are the most widely used in various biomedical applications for bone reconstruction and replacement. In this study, crocin, saffron's natural bioactive and anti-inflammatory molecule, and bicarbonate, a neutralizing agent, were directly loaded onto HA disks to evaluate their in vitro release and effect on human osteoblast and osteosarcoma cell lines. This was assessed through release, initial toxicity, drug optimization, final toxicity studies and in vivo anti-inflammatory assessment through H&E indexing. It is hypothesized that the release of crocin, bicarbonate, and the dual release of both agents will decrease osteosarcoma cellular viability with no effect on osteoblast cells. A plateaued release of crocin and bicarbonate was achieved over seven weeks in physiological and acidic environments, where bicarbonate was shown to modulate the release of crocin. Through morphological characterization and MTT assay analysis, bicarbonate showed no toxicity to human fetal osteoblast (hFOB) cells and crocin significantly enhanced osteoblast proliferation. Through drug concentration optimization, all drug loaded samples decreased human osteosarcoma (MG-63) viability by 50% compared to control samples by Day 11, with clear changes in cell spreading and morphology. Moreover, 3D printed TCP scaffolds loaded with crocin and bicarbonate were tested in vivo in order to assess their preliminary effects on inflammation in a rat distal femur model at 4 days. Lower inflammatory cellular recruitment was achieved in the presence of crocin and bicarbonate, compared to the control. These results suggest a pro-apoptotic mechanism against osteosarcoma as well as anti-inflammatory properties of crocin and bicarbonate, elucidating a potential application for osteosarcoma regulation and wound healing for bone tissue regeneration applications.

Enhanced In Vivo Bone and Blood Vessel Formation by IronOxide and Silica Doped 3D Printed Tricalcium Phosphate Scaffolds.

Calcium phosphate (CaP) ceramics show significant promise towards bone graft applications because of the compositional similarity to inorganic materials of bone. With 3D printing, it is possible to create ceramic implants that closely mimic the geometry of human bone and can be custom-designed for unusual injuries or anatomical sites. The objective of the study was to optimize the 3D-printing parameters for the fabrication of scaffolds, with complex geometry, made from synthesized tricalcium phosphate (TCP) powder. This study was also intended to elucidate the mechanical and biological effects of the addition of Fe+3 and Si+4 in TCP implants in a rat distal femur model for 4, 8, and 12weeks. Doped with Fe+3 and Si+4 TCP scaffolds with 3D interconnected channels were fabricated to provide channels for micronutrients delivery and improved cell-material interactions through bioactive fixation. Addition of Fe+3 into TCP enhanced early-stage new bone formation by increasing type I collagen production. Neovascularization was observed in the Si+4 doped samples after 12 weeks. These findings emphasize that the additive manufacturing of scaffolds with complex geometry from synthesized ceramic powder with modified chemistry is feasible and may serve as a potential candidate to introduce angiogenic and osteogenic properties to CaPs, leading to accelerated bone defect healing.

Effects of pore distribution and chemistry on physical, mechanical, and biological properties of tricalcium phosphate scaffolds by binder-jet 3D printing

Porous tricalcium phosphate (TCP) scaffolds are becoming more and more important for treating musculoskeletal diseases. With the maturation of 3D printing (3DP) technology in the past two decades, porous TCP scaffolds can also be easily prepared with complex design and high dimensional accuracy. However, the mechanical and biological properties of porous TCP scaffolds prepared by 3D printing still need improvements. In this study, novel 3D printed TCP and MgO/ZnO-TCP scaffolds were prepared by an binder-jet 3D printer. Scaffolds had a dense core and porous surface feature with a designed pore size of 500 μm and a designed porosity of 18%. After printing, scaffolds were sintered in a muffle furnace at 1250 °C. The presence of MgO and ZnO increased the surface area of TCP from 1.18 ± 0.01 m2/g to 2.65 ± 0.02 m2/g, the bulk density from 37.89 ± 0.83% to 50.82 ± 1.10%, and the compressive strength from 17.94 ± 1.65 MPa to 27.46 ± 2.63 MPa. Enhanced osteoblast proliferation was shown in doped 3D printed TCP scaffolds compared to the pure 3DP TCP. In addition, the use of 3D printing as well as dense core and porous surface design enhanced the surface roughness and osteoblast proliferation of TCP scaffolds. This novel 3D printed MgO/ZnO-TCP scaffold shows enhanced mechanical and biological properties, which is promising for orthopedic and dental applications.

Mechanical and biological effects of infiltration with biopolymers on 3D printed tricalciumphosphate scaffolds.

The aim of this study was to evaluate the influence of infiltrating 3D printed (TCP) scaffolds with different biodegradable polymers on their mechanical and biological properties. 3D printed TCP scaffolds with interconnecting channels measuring 450±50 µm were infiltrated with four different biodegradable copolymers. To determine the average compressive strength, a uniaxial testing system was used. Additionally, scaffolds were seeded with MC3T3 cells and cell viability was assessed by live/dead-assay. Uninfiltrated TCP had an average compression strength of 1.92±0.38 MPa. Mechanical stability was considerably increased in all infiltrated scaffolds up to a maximum of 7.36±0.57 MPa. All scaffolds demonstrated high cell survival rates with a maximum of 94±10 % living cells. In conclusion, infiltration of 3D printed tricalcium phosphate scaffolds with biodegradable polymers significantly improved mechanical properties and biological properties were comparable to those of uninfiltrated TCP scaffolds.

SrO- and MgO-doped microwave sintered 3D printed tricalcium phosphate scaffolds: mechanical properties and in vivo osteogenesis in a rabbit model.

The presence of interconnected macro pores allows guided tissue regeneration in tissue engineering scaffolds. However, highly porous scaffolds suffer from having poor mechanical strength. Previously, we showed that microwave sintering could successfully be used to improve mechanical strength of macro porous tricalcium phosphate (TCP) scaffolds. This study reports the presence of SrO and MgO as dopants in TCP scaffolds improves mechanical and in vivo biological performance. We have used direct three dimensional printing (3DP) technology for scaffold fabrication. These 3DP scaffolds possessed multiscale porosity, that is, 3D interconnected designed macro pores along with intrinsic micro pores. A significant increase in mechanical strength, between 37 and 41%, was achieved due to SrO and MgO doping in TCP as compared with pure TCP. Maximum compressive strengths of 9.38 ± 1.86 MPa and 12.01 ± 1.56 MPa were achieved by conventional and microwave sintering, respectively, for SrO-MgO-doped 3DP scaffolds with 500 μm designed pores. Histomorphological and histomorphometric analysis revealed a significantly higher osteoid, bone and haversian canal formation induced by the presence of SrO and MgO dopants in 3DP TCP as compared with pure TCP scaffolds when tested in rabbit femoral condyle defect model. Increased osteon and thus enhanced network of blood vessel formation, and osteocalcin expression were observed in the doped TCP scaffolds. Our results show that these 3DP SrO-MgO-doped TCP scaffolds have the potential for early wound healing through accelerated osteogenesis and vasculogenesis.

Polycaprolactone-coated 3D printed tricalcium phosphate scaffolds for bone tissue engineering: in vitro alendronate release behavior and local delivery effect on in vivo osteogenesis.

The aim of this work was to evaluate the effect of in vitro alendronate (AD) release behavior through polycaprolactone (PCL) coating on in vivo bone formation using PCL-coated 3D printed interconnected porous tricalcium phosphate (TCP) scaffolds. Higher AD and Ca2+ ion release was observed at lower pH (5.0) than that at higher pH (7.4). AD and Ca2+ release, surface morphology, and phase analysis after release indicated a matrix degradation dominated AD release caused by TCP dissolution. PCL coating showed its effectiveness for controlled and sustained AD release. Six different scaffold compositions, namely, (i) TCP (bare TCP), (ii) TCP + AD (AD-coated TCP), (iii) TCP + PCL (PCL-coated TCP), (iv) TCP + PCL + AD, (v) TCP + AD + PCL, and (vi) TCP + AD + PCL + AD were tested in the distal femoral defect of Sprague–Dawley rats for 6 and 10 weeks. An excellent bone formation inside the micro and macro pores of the scaffolds was observed from histomorphology. Histomorphometric analysis revealed maximum new bone formation in TCP + AD + PCL scaffolds after 6 weeks. No adverse effect of PCL on bioactivity of TCP and in vivo bone formation was observed. All scaffolds with AD showed higher bone formation and reduced TRAP (tartrate resistant acid phosphatase) positive cells activity compared to bare TCP and TCP coated with only PCL. Bare TCP scaffolds showed the highest TRAP positive cells activity followed by TCP + PCL scaffolds, whereas TCP + AD scaffolds showed the lowest TRAP activity. A higher TRAP positive cells activity was observed in TCP + AD + PCL compared to TCP + AD scaffolds after 6 weeks. Our results show that in vivo local AD delivery from PCL-coated 3DP TCP scaffolds could further induce increased early bone formation.

Repair of Complex Craniofacial Bone Defects Using 3D-Printed Tricalcium Phosphate Scaffolds

Repair of bone lost to trauma, disease, or birth defect requires regeneration of large volumes of structurally complex bone. Current bone repair methods, like bone grafts or particulate materials, are imperfect for repair of complex craniofacial defects which require formation of large amounts of natural, mechanically strong bone. 3D-printed, Direct Write (DW), scaffolds composed of tricalcium phosphate (TCP) with temporary calcium sulfate filler can serve as a better option to repair large complex bone defects. Such scaffolds are mechanically stable and can be custom printed to match the exact defect shape and size. Current literature debates scaffold pore sizes for optimal bone repair, stating scaffold pore size should be from 100-400μm. The objective of this study is to determine how pore size dictates the quality of ingrowing bone. This will allow the design of scaffolds that can regenerate the natural architecture and mechanical properties of bone in complex craniofacial defects.

3D printed tricalcium phosphate scaffolds: Effect of SrO and MgO doping on in vivo osteogenesis in a rat distal femoral defect model.

The presence of interconnected macro pores is important in tissue engineering scaffolds for guided tissue regeneration. This study reports in vivo biological performance of interconnected macro porous tricalcium phosphate (TCP) scaffolds due to the addition of SrO and MgO as dopants in TCP. We have used direct three dimensional printing (3DP) technology for scaffold fabrication followed by microwave sintering. Mechanical strength was evaluated by scaffolds with 500 µm, 750 µm, and 1000 µm interconnected designed pore sizes. Maximum compressive strength of 12.01 ± 1.56 MPa was achieved for 500 µm interconnected designed pore size Sr-Mg doped scaffold. In vivo biological performance of the microwave sintered pure TCP and Sr-Mg doped TCP scaffolds was assessed by implanting 350 µm designed interconnected macro porous scaffolds in rat distal femoral defect. Sintered pore size of these 3D printed scaffolds were 311 ± 5.9 µm and 245 ± 7.5 µm for pure and SrO-MgO doped TCP scaffolds, respectively. These 3D printed scaffolds possessed multiscale porosity, i.e., 3D interconnected designed macro pores along with intrinsic micro pores. Histomorphology and histomorphometric analysis revealed a significant increase in osteoid like new bone formation, and accelerated mineralization inside SrO and MgO doped 3D printed TCP scaffolds as compared to pure TCP scaffolds. An increase in osteocalcin and type I collagen level was also observed in rat blood serum with SrO and MgO doped TCP scaffolds compared to pure TCP scaffolds. Our results show that these 3D printed SrO and MgO doped TCP scaffolds with multiscale porosity contributed to early healing through accelerated osteogenesis.

Microwave-sintered 3D printed tricalcium phosphate scaffolds for bone tissue engineering

This study reports the manufacturing process of 3D interconnected macroporous tricalcium phosphate (TCP) scaffolds with controlled internal architecture by direct 3D printing (3DP), and high mechanical strength obtained by microwave sintering. TCP scaffolds with 27%, 35% and 41% designed macroporosity with pore sizes of 500 μm, 750 μm and 1000 μm, respectively, were manufactured by direct 3DP. These scaffolds were then sintered at 1150 °C and 1250 °C in conventional electric muffle and microwave furnaces, respectively. Total open porosity between 42% and 63% was obtained in the sintered scaffolds due to the presence of intrinsic micropores along with designed pores. A significant increase in compressive strength between 46% and 69% was achieved by microwave compared to conventional sintering as a result of efficient densification. Maximum compressive strengths of 10.95 ± 1.28 MPa and 6.62 ± 0.67 MPa were achieved for scaffolds with 500 μm designed pores (~ 400 μm after sintering) sintered in microwave and conventional furnaces, respectively. An increase in cell density with a decrease in macropore size was observed during in vitro cell-material interactions using human osteoblast cells. Histomorphological analysis revealed that the presence of both micro- and macropores facilitated osteoid-like new bone formation when tested in femoral defects of Sprague-Dawley rats. Our results show that bioresorbable 3D-printed TCP scaffolds have great potential in tissue engineering applications for bone tissue repair and regeneration.