β-TCP Scaffolds Research Articles

Inversely 3D-Printed β-TCP Scaffolds for Bone Replacement.

The aim of this study was to predefine the pore structure of β-tricalcium phosphate (β-TCP) scaffolds with different macro pore sizes (500, 750, and 1000 µm), to characterize β-TCP scaffolds, and to investigate the growth behavior of cells within these scaffolds. The lead structures for directional bone growth (sacrificial structures) were produced from polylactide (PLA) using the fused deposition modeling techniques. The molds were then filled with β-TCP slurry and sintered at 1250 °C, whereby the lead structures (voids) were burnt out. The scaffolds were mechanically characterized (native and after incubation in simulated body fluid (SBF) for 28 d). In addition, biocompatibility was investigated by live/dead, cell proliferation and lactate dehydrogenase assays. The scaffolds with a strand spacing of 500 µm showed the highest compressive strength, both untreated (3.4 ± 0.2 MPa) and treated with simulated body fluid (2.8 ± 0.2 MPa). The simulated body fluid reduced the stability of the samples to 82% (500), 62% (750) and 56% (1000). The strand spacing and the powder properties of the samples were decisive factors for stability. The fact that β-TCP is a biocompatible material is confirmed by the experiments. No lactate dehydrogenase activity of the cells was measured, which means that no cytotoxicity of the material could be detected. In addition, the proliferation rate of all three sizes increased steadily over the test days until saturation. The cells were largely adhered to or within the scaffolds and did not migrate through the scaffolds to the bottom of the cell culture plate. The cells showed increased growth, not only on the outer surface (e.g., 500: 36 ± 33 vital cells/mm² after three days, 180 ± 33 cells/mm² after seven days, and 308 ± 69 cells/mm² after 10 days), but also on the inner surface of the samples (e.g., 750: 49 ± 17 vital cells/mm² after three days, 200 ± 84 cells/mm² after seven days, and 218 ± 99 living cells/mm² after 10 days). This means that the inverse 3D printing method is very suitable for the presetting of the pore structure and for the ingrowth of the cells. The experiments on which this work is based have shown that the fused deposition modeling process with subsequent slip casting and sintering is well suited for the production of scaffolds for bone replacement.

Physical and Mechanical Properties of Composite Scaffolds with or without Collagen Impregnation

This in vitro study aimed at evaluating the physical and mechanical properties of newly developed scaffolds of poly (lactic-co-glycolic acid) (PLGA) and biphasic ceramic (Hydroxyapatite HA + beta-tricalciumphosphate β-TCP) with or without collagen impregnation to be used for bone regeneration in the oral and maxillofacial district. Solvent casting and particle leaching techniques were used to produce the scaffolds, which were then divided into six groups according to PLGA/HA + β-TCP ratio and impregnation with collagen: G1 (50/50) + collagen; G2 (60/40) + collagen; G3 (40/60) + collagen; G4 (50/50); G5 (60/40); G6 (40/60). As control group, inorganic xenogenous bone was used. Structure and porosity were evaluated by scanning electron microscopy, and a chemical analysis was performed through an energy-dispersive spectrometer. Moreover, to evaluate the hydrophilicity of the samples, a wettability test was conceived, and finally, mechanical properties were examined by a compression test. High porosity and interconnectivity, resulting in a large surface area and great fluid retention capacity, were presented by the PLGA/HA + β-TCP scaffolds. In the composite groups, collagen increased the wettability and the mechanical resistance, although the latter was not statistically affected by the percentage of HA + β-TCP added. Further in vitro and in vivo studies are needed for a deeper understanding of the influence of collagen on the biological behavior of the developed composite materials and their potential, namely biocompatibility and bioactivity, for bone tissue regeneration.

Doping lithium element to enhance compressive strength of β-TCP scaffolds manufactured by 3D printing for bone tissue engineering

The purpose of this study was to enhance the compressive strength of 3D printed beta-tricalcium phosphate (β-TCP) scaffolds for bone tissue engineering. Ca10Li(PO4)7 (CLP) is acquired by doping Li into β-TCP. The optimum sintering temperature of CLP was determined by the compressive strength, relative density, microstructure, grain size and behaviors of cracks. The dense structure and porous scaffolds of CLP were manufactured by 3D printing. Cell viability and cell morphology of mouse osteoblast cell line (MC3T3-E1) were evaluated by the cell counter kit-8 assay (CCK-8) and fluorescence microscope, respectively. The mineralization effects and degradation property in vitro of CLP scaffolds were investigated as well. The optimal sintering temperature of CLP is 1000 °C. The pores size of scaffold is about 400 × 400 μm and 400 × 300 μm in the Z-axis and XY-axis direction, correspondingly. The compressive strength of the dense structure (332.24 ± 17.81 MPa) and porous scaffold (69.12 ± 20.62 MPa) of CLP is approximately three times as large as that of β-TCP (100.89 ± 9.33 MPa, 24.14 ± 5.01 MPa). There is no statistical difference in cytotoxicity between CLP and β-TCP at the diverse culture days (p > 0.05). The scaffolds of CLP have the closer mineralization effects and degradation property in vitro to β-TCP. Possessing higher strength and excellent biocompatibility, CLP scaffolds manufactured by 3D printing have significant clinical potentials in bone tissue engineering.

Angiogenic effects of mesenchymal stem cells in combination with different scaffold materials

Tissue survival in regenerative tissue engineering requires rapid vascularization, which is influenced by scaffold material and seeded cell selection. Poly-l-lactide-co-glycolide (PLGA) and beta-tricalcium phosphate (β-TCP) are well-established biomaterials with angiogenic effects because of their material properties. Given the importance of the seeded cell type as a co-factor for vascularization, mesenchymal stem cells (MSCs) are known to have high angiogenic potential. We hypothesized that PLGA and β-TCP scaffolds seeded with MSCs would effectively induce a potent angiogenic response. Therefore, we studied the angiogenic effects after implanting PLGA and β-TCP scaffolds seeded with isogeneic MSCs in vivo. Fifty-six BALB/c mice were equally divided into seven groups and underwent implantation of the dorsal skinfold chambers. Two MSC groups were seeded on collagen-coated PLGA or β-TCP scaffolds, whereas groups 3–6 received collagen-coated or uncoated scaffolds without MSCs. No scaffold implantation was performed for group 7, which served as the control. Angiogenesis was assessed in vivo via intravital fluorescence microscopy. Angiogenic responses were noted on all scaffolds, whereupon MSC angiogenic response was significantly enhanced on days 6 and 10. Additionally, a comparison of biomaterials indicated increased angiogenic activity for β-TCP scaffolds compared with PLGA scaffolds. In conclusion, seeding β-TCP scaffolds with MSCs can accelerate vitalization and a combination of both significantly improves angiogenesis.

PLGA+HA/βTCP scaffolds embedding simvastatin induced mineral deposition by mesenchymal stem cells

PLGA+HA/βTCP scaffolds embedding simvastatin induced mineral deposition by mesenchymal stem cells

Sustained release of vitamin C from PCL coated TCP induces proliferation and differentiation of osteoblast cells and suppresses osteosarcoma cell growth.

The objective of this study is to understand the effect of sustained release of vitamin C from β-tricalcium phosphate (β-TCP) scaffold on proliferation, viability and differentiation of human fetal osteoblast cells (hFOB). The influence of pH, drug concentration, and presence of polymer on the sustained release of vitamin C from polycaprolactone (PCL) coated β-TCP scaffolds are studied. Prolonged and sustained release of vitamin C, over 60 days is observed in PCL coated β-TCP scaffolds compared to uncoated scaffolds. Presence of PCL helps to minimize the burst release of vitamin C from β-TCP scaffolds in the initial 24 h of release. To evaluate the osteogenic potential of vitamin C incorporated β-TCP scaffolds, osteoblast cells are cultured and cell morphology, proliferation, viability, and differentiation are assessed. Morphological characterization shows layer like osteoblast cell attachment in the presence of vitamin C compared to the control. MTT cell viability assay shows 2 folds increase in osteoblast cell density in the presence of vitamin C after 3,7 and 11 days of culture. Furthermore, increased ALP activity at 11 days of culture indicates the possible role of vitamin C on osteoblast differentiation. Additionally, a preliminary study shows vitamin C loaded scaffolds suppress osteosarcoma (MG-63) cell proliferation to 4 folds after 3 days compared to control. These results show a sustained release of vitamin C from PCL coated β-TCP scaffolds improve proliferation, viability, and differentiation of osteoblasts cell as well as mitigate osteosarcoma cell proliferation, suggesting its potential application as synthetic bone graft substitutes in tissue engineering application.

β-Tricalcium phosphate/ԑ-polycaprolactone composite scaffolds with a controllable gradient: Fabrication and characterization

Abstract β-tricalcium phosphate (β-TCP)/ԑ-polycaprolactone (PCL) composite scaffolds with a controllable gradient were developed with a two-step process: fabrication of the β-TCP scaffolds using digital light processing (DLP) 3D printing and then immersion of the β-TCP scaffolds into a PCL melt for different times. The gradient structure was controlled by the immersion time of the β-TCP scaffolds in the molten PCL. The composite scaffolds with a gradient exhibited a substantially higher compressive strength and toughness than the bare β-TCP scaffolds. Moreover, the increase in infiltration time also enhanced the compressive strength and toughness of the composite scaffolds because the infiltration thickness increased with infiltration time. The gradient structure resulted in a gradient degradation and may provide a better response to time-varying mechanical properties than pure β-TCP scaffolds during its absorption process. Therefore, composite scaffolds with a gradient are promising candidates for load-bearing bones or large segmental bone repair.

Use of transforming growth factor-β loaded onto β-tricalcium phosphate scaffold in a bone regeneration rat calvaria model.

Transforming growth factor-β (TGF-β1 ) enhances mesenchymal stem cell (MSC) differentiation into osteoblasts. The aim of the study was to assess whether TGF-β1 loaded onto β-tricalcium phosphate (β-TCP) synthetic scaffold enhances bone regeneration in a rat calvaria model. The release kinetics of TGF-β1 from β-TCP scaffold was evaluated in vitro. TGF-β1 in various concentrations (1-40 ng/mL) was loaded onto the β-TCP scaffold, and release kinetics was monitored by ELISA. The effect of TGF-β1 on the proliferation of MSCs was assessed using AlamarBlue, and MSC differentiation was evaluated by Alizarin Red quantification assay.Bone augmentation following transplantation of TGF-β1 loaded onto β-TCP in a rat calvaria model was evaluated in vivo. Greater TGF-β1 release from the 40 ng/mL concentration was found. A suppressive effect of TGF-β on the MSCs proliferation was observed with maximum inhibition obtained with 40 ng/mL compared to the control group (P = .028). A positive effect on MSCs osteogenic differentiation was found.Bone height and bone area fraction in vivo were similar with or without TGF-β1 ; however, blood vessel density and degradation of the scaffold were significantly higher in the TGF-β1 group. TGF-β1 adsorbed to β-TCP stimulated angiogenesis and scaffold degradation that may enhance bone formation.

Allogenic chondrocyte/osteoblast-loaded β-tricalcium phosphate bioceramic scaffolds for articular cartilage defect treatment

The medical community has expressed significant interest in the treatment of cartilage defect. Successful repair of articular cartilage defects remains a challenge in clinics. Due to the huge advantages of 3D micro/nanomaterials, 3D artificial micro/nano scaffolds have been widely developed and explored in the tissue repair of articular joints. In this study, chondrocyte/osteoblast-loaded β-tricalcium phosphate (β-TCP) bioceramic scaffold and chondrocyte-loaded β-TCP bioceramic scaffold were prepared by micromass stem cell culture and bioreactor-based cells-loaded scaffold culture for articular cartilage defect treatment. The results demonstrate chondrocyte and osteoblast can be successfully induced from allogeneic bone marrow stromal cells using micromass stem cell culture. Further, chondrocyte-loaded β-TCP scaffold and osteoblast-loaded β-TCP scaffold can be successfully prepared by bioreactor-based cells-loaded scaffold culture. Finally, the scaffolds are applied for Beagle articular cartilage defect treatment. The relative cartilage regeneration abilities on Beagle femoral trochleae were as follows: Chondrocyte/osteoblast-loaded β-TCP bioceramic scaffold group > chondrocyte-loaded β-TCP bioceramic scaffold group > β-TCP bioceramic scaffold. Therefore, micromass stem cell culture and bioreactor-based cells-loaded scaffold culture can be applied to prepare chondrocyte/osteoblast-loaded β-TCP bioceramic scaffold for articular cartilage defect treatment. It suggests allogenic chondrocyte/osteoblast-loaded β-TCP bioceramic scaffold could be potentially used in the treatment of patients with cartilage defects.

Research on corrosion behavior and biocompatibility of a porous Mg-3%Zn/5%β-Ca3(PO4)2 composite scaffold for bone tissue engineering.

Rapid corrosion rates are a major impediment to the use of magnesium alloys in bone tissue engineering despite their good mechanical properties and biodegradability. Zinc is a promising alloy element, and it is an effective grain refiner for magnesium. β-Ca3(PO4)2 (β-TCP) is widely used for bone regeneration because of its good biocompatibility, and it also has a similar chemical and crystal structure to human bone. In this research, the magnesium alloy was reinforced by adding 3%Zn (wt.%) and 5%β-TCP (wt.%) particles in order to improve the corrosion resistance and biocompatibility. Furthermore, the biomaterial was prepared through powder metallurgy technology using NH4HCO3 as space-holding particles to construct porous Mg-3%Zn/5%β-TCP scaffolds. The results revealed that the magnesium-zinc phase and calcium phosphate phase were uniformly distributed in the α-magnesium matrix. Mechanical and corrosion tests indicated that the scaffolds had mechanical strengths similar to that of human bone, and their corrosion resistance decreased with an increase in the porosity. The scaffolds had cytotoxicity grades of 0-1 against MG63 cells, SaoS2 cells, and HK-2 cells, which suggested that they were appropriate for cellular applications. In addition, the scaffolds demonstrated excellent biocompatibility when tested in rabbits. These results indicate that porous Mg-3%Zn/5%β-TCP scaffolds are promising biodegradable implants for bone tissue engineering.

3D printed β-TCP scaffold with sphingosine 1-phosphate coating promotes osteogenesis and inhibits inflammation

Traditional treatments for bone repair with allografts and autografts are limited by the source of bone substitutes. Bone tissue engineering via a cell-based bone tissue scaffold is a new strategy for treatment against large bone defects with many advantages, such as the accessibility of biomaterials, good biocompatibility and osteoconductivity; however, the inflammatory immune response is still an issue that impacts osteogenesis. Sphingosine 1-phosphate (S1P) is a cell-derived sphingolipid that can mediate cell proliferation, immunoregulation and bone regeneration. We hypothesised that coating S1P on a β-Tricalcium phosphate (β-TCP) scaffold could regulate the immune response and increase osteogenesis. We tested the immunoregulation capability on macrophages and the osteogenic capability on rat bone marrow stromal cells of the coated scaffolds, which showed good biocompatibility. Additionally, the coated scaffolds exhibited dose-dependent inhibition of inflammatory-related gene expression. A high concentration of S1P (0.5 μM) upregulated osteogenic-related gene expression of OPN, OCN and RUNX2, which also significantly increased the alkaline phosphatase activity, as compared with the control group. In conclusion, S1P coated β-TCP scaffold could inhibit inflammation and promote bone regeneration.

Comparative characterization of the hydrogel added PLA/β-TCP scaffolds produced by 3D bioprinting

Designing the 3D bioprinted scaffolds has been a promising tool which assists cells to adhere and supports the self-repair mechanism of native tissues. In order to practice this hypothesis, we used 3D bioprinting methods and fabricated Polylactic acid (PLA) and β-tricalcium phosphate (β-TCP) scaffolds and incorporated them with four different hydrogels (Collagen (Col), Sodium Alginate (SAlg), Halamomas Levan (HLh), Chitosan (Ch). Furthermore, PLA/β-TCP Scaffolds incorporated with chitosan were further enhanced with Amoxicillin (AMX). These scaffolds were further characterized for their chemical, morphological, antibacterial and cellular biocompatibility. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry, compressive strength test, agar diffusion test and in vitro cellular behaviour tests were performed to reveal structural, mechanical and biological characteristics of scaffolds. Among others, chitosan including samples demonstrated significantly superior and beneficial features such as the highest biocompatibility and structural integrity regarding morphology, compared to the other samples. Therefore, chitosan including samples were loaded with amoxicillin (AMX) to increase antimicrobial activity of the scaffolds. All together, these data show that PLA/β-TCP 3D printed scaffolds incorporated with different hydrogels has the ability to assist the tissue to repair and regeneration.

Calcium phosphate scaffolds with defined interconnecting channel structure provide a mimetic 3D niche for bone marrow metastasized tumor cell growth

Metastasis of tumor cells in the bone marrow (BM) is a multi-step and highly dynamic process during which cells succumb important phenotypic changes. Behavior of disseminated tumor cells in BM is strictly regulated by three-dimensional (3D) cell-cell and cell-matrix interactions. In this study, we explored whether the β-tricalcium-phosphate (β-TCP) scaffolds with a tailored interconnecting channel structure could enable appropriate 3D mimetic BM microenvironment for the growth of metastatic neuroblastoma cells. The scaffolds provided the mechanical support for human mesenchymal stromal cells (hMSC) allowing them to proliferate, differentiate towards osteoblasts, and produce the deposits of extracellular matrix inside the interconnected channels. The in vitro microenvironment shaped by stromal cells was then tailored by neuroblastoma tumor cells. Immunohistological analyses confirmed the organization of tumor cells into the forms of spheres only when co-cultured with hMSC-derived osteoblasts. The growing rate of tumor cells in 3D conditions was less marked comparing to the one of the cells grown as 2D monolayer as confirmed by decreased Ki-67 expression. Instead, the 3D culturing of neuroblastoma cells inside supportive stroma promoted cell quiescence as sustained by increased p27 level. A balance between cell proliferation, survival, and differentiation was more evident for tumor cells grown inside the 3D scaffolds, thus mirroring better the situation that occurs in vivo where the cells do not follow the exponential growth rate. We conclude that the proposed 3D β-TCP scaffold type provides a mimetic 3D in vitro niche suitable for studying behavior of BM metastasized tumor cells. Statement of SignificanceBone marrow (BM) niche is a favorite target of metastatic neuroblastoma cells. To better address the molecular mechanisms that sustain spatiotemporal organization of neuroblastoma cells in the marrow we mimicked the three-dimensional (3D) assembly of stromal and tumor cells inside β-tricalcium-phosphate (β-TCP) scaffolds. β-TCP scaffolds with a tailored interconnecting channel structure provided mechanical support to mesenchymal stromal cells allowing them to differentiate towards osteoblasts and to produce extracellular matrix. A dynamic cell-matrix interplay favored the characteristic rosette-like growth of metastatic neuroblastoma cells and triggered their quiescence. With our study, we confirmed the potential of β-TCP scaffolds with reproduced BM niche as a cost-effective in vitro model for the growth of disseminated tumor cells, and for related biological and pharmacological surveys.

Engineering Porous β-Tricalcium Phosphate (β-TCP) Scaffolds with Multiple Channels to Promote Cell Migration, Proliferation, and Angiogenesis.

Inadequate oxygen and nutrient diffusion in a porous scaffold often resulted in insufficient formation of branched vasculatures, which hindered bone regeneration. In this study, interconnected porous β-tricalcium phosphate (β-TCP) scaffolds with different geometric designs of channels were fabricated and compared to discover the functionality of structure on facilitating nutrient diffusion for angiogenesis. In vitro fluid transportation and degradation of the scaffolds were performed. Cell infiltration, migration, and proliferation of human umbilical vein endothelial cells (HUVECs) on the scaffolds were carried out under both static and dynamic culture conditions. A computational simulation model and a series of immunofluorescent staining were implemented to understand the mechanism of cell behavior in response to different types of scaffolds. Results showed that geometry with multiple channels significantly accelerated the release of Ca2+ and increased the fluid diffusion efficiency. Moreover, multiple channels promoted HUVECs' infiltration and migration in vitro. The ex vivo implantation results showed that the channels promoted cells from the rats' calvarial bone explants to infiltrate into the implanted scaffold. Multiple channels also stimulated HUVECs' proliferation prominently at both static and dynamic culturing conditions. The expression of both cell migration-related protein α5 and angiogenesis-related protein CD31 on multiple-channeled scaffolds was upregulated compared to that on the other two types of scaffolds, implying that multiple channels reinforced cell migration and angiogenesis. All the findings suggested that the geometric design of multiple channels in the porous β-TCP scaffold has promising potential to promote cell infiltration, migration, and further vascularization when implanted in vivo.

Melatonin decorated 3D-printed beta-tricalcium phosphate scaffolds promoting bone regeneration in a rat calvarial defect model

3D-printed β-TCP scaffolds decorated with melatonin via dopamine mussel-inspired chemistry enhance the osteogenesis and in vivo bone regeneration.

Characterization and degradation analyses of PLGA+HA βTCP scaffolds incorporating simvastatin for bone regeneration

Characterization and degradation analyses of PLGA+HA βTCP scaffolds incorporating simvastatin for bone regeneration

Study on cytotoxicity of three-dimensional printed β-tricalcium phosphate loaded poly (lactide-co-glycolide) anti-tuberculosis drug sustained release microspheres and its effect on osteogenic differentiation of bone marrow mesenchymal stem cells

To study the effect of three-dimensional (3D) printed β-tricalcium phosphate (β-TCP) scaffold loaded poly (lactide-co-glycolide) (PLGA) anti-tuberculosis drug sustained release microspheres on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its cytotoxicity. Isoniazid and rifampicin/PLGA sustained release microspheres were prepared by W/O/W multiple emulsion method. The β-TCP scaffolds were prepared by 3D printing technique. The microspheres were loaded on the scaffolds by centrifugal oscillation method to prepare composite materials. The BMSCs of Sprague Dawley rat were isolated and cultured by whole bone marrow adherent method, and the third generation cells were used for the following experiments. BMSCs were co-cultured with osteogenic induction medium (group A), PLGA anti-tuberculosis drug sustained release microsphere extract (group B), 3D printed β-TCP scaffold extract (group C), and 3D printed β-TCP scaffold loaded PLGA anti-tuberculosis drug sustained release microsphere composite extract (group D), respectively. Cytotoxicity was detected by cell counting kit 8 (CCK-8) method; the calcium deposition was observed by alizarin red staining; and the mRNA expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were detected by real-time fluorescence quantitative PCR (RT-qPCR). CCK-8 assay showed that the absorbance ( A) value of groups A, B, C, and D increased gradually with the culture time prolonging. After cultured for 24, 48, and 72 hours, the A value decreased in the order of groups A, C, B, and D. There was no significant difference between groups B and D ( P>0.05), but there were significant differences between other groups ( P<0.05). The cytotoxicity was evaluated as grade 0-2, and the toxicity test was qualified. Alizarin red staining showed that red mineralized nodules were formed in all groups at 21 days after osteogenic induction, but the number of mineralized nodules decreased sequentially in groups C, D, A, and B. RT-qPCR test results showed that the relative expressions of OCN and BSP genes in groups A, B, C, and D increased gradually with the culture time prolonging. The relative expression of ALP gene increased at 7 and 14 days, and decreased at 21 days. After cultured for 7, 14, and 21 days, the relative expressions of ALP, OCN, and BSP genes decreased sequentially in groups C, D, A, and B; the differences were significant between groups at different time points ( P<0.05). 3D printed β-TCP loaded PLGA anti-tuberculosis drug sustained release microsphere composites have no obvious cytotoxicity to BMSCs, and can promote BMSCs to differentiate into osteoblasts to a certain extent.

Form and functional repair of long bone using 3D-printed bioactive scaffolds.

Injuries to the extremities often require resection of necrotic hard tissue. For large-bone defects, autogenous bone grafting is ideal but, similar to all grafting procedures, is subject to limitations. Synthetic biomaterial-driven engineered healing offers an alternative approach. This work focuses on three-dimensional (3D) printing technology of solid-free form fabrication, more specifically robocasting/direct write. The research hypothesizes that a bioactive calcium-phosphate scaffold may successfully regenerate extensive bony defects in vivo and that newly regenerated bone will demonstrate mechanical properties similar to native bone as healing time elapses. Robocasting technology was used in designing and printing customizable scaffolds, composed of 100% beta tri-calcium phosphate (β-TCP), which were used to repair critical sized long-bone defects. Following full thickness segmental defects (~11mm×full thickness) in the radial diaphysis in New Zealand white rabbits, a custom 3D-printed, 100% β-TCP, scaffold was implanted or left empty (negative control) and allowed to heal over 8, 12, and 24weeks. Scaffolds and bone, en bloc, were subjected to micro-CT and histological analysis for quantification of bone, scaffold and soft tissue expressed as a function of volume percentage. Additionally, biomechanical testing at two different regions, (a) bone in the scaffold and (b) in native radial bone (control), was conducted to assess the newly regenerated bone for reduced elastic modulus (Er ) and hardness (H) using nanoindentation. Histological analysis showed no signs of any adverse immune response while revealing progressive remodelling of bone within the scaffold along with gradual decrease in 3D-scaffold volume over time. Micro-CT images indicated directional bone ingrowth, with an increase in bone formation over time. Reduced elastic modulus (Er ) data for the newly regenerated bone presented statistically homogenous values analogous to native bone at the three time points, whereas hardness (H) values were equivalent to the native radial bone only at 24weeks. The negative control samples showed limited healing at 8weeks. Custom engineered β-TCP scaffolds are biocompatible, resorbable, and can directionally regenerate and remodel bone in a segmental long-bone defect in a rabbit model. Custom designs and fabrication of β-TCP scaffolds for use in other bone defect models warrant further investigation.

Preclinical Studies of the Biosafety and Efficacy of Human Bone Marrow Mesenchymal Stem Cells Pre-Seeded into β-TCP Scaffolds after Transplantation.

Background: Cell-Based Therapies (CBT) constitute a valid procedure for increasing the quantity and quality of bone in areas with an inadequate bone volume. However, safety and efficacy should be investigated prior to clinical application. The objective of this study was to evaluate the biodistribution, safety and osteogenic capacity of bone marrow-derived human mesenchymal stem cells (hBMMSCs) pre-seeded into β-tricalcium phosphate (TCP) and implanted into NOD/SCID mice at subcutaneous and intramuscular sites. Methods: hBMMSCs were isolated, characterized and then cultured in vitro on a porous β-TCP scaffold. Cell viability and attachment were analyzed and then hBMMSCs seeded constructs were surgically placed at subcutaneous and intramuscular dorsal sites into NOD/SCID mice. Acute and subchronic toxicity, cell biodistribution and efficacy were investigated. Results: There were no deaths or adverse events in treated mice during the 48-hour observation period, and no toxic response was observed in mice. In the 12-week subchronic toxicity study, no mortalities, abnormal behavioral symptoms or clinical signs were observed in the saline control mice or the hBMMSCs/β-TCP groups. Finally, our results showed the bone-forming capacity of hBMMSCs/β-TCP since immunohistochemical expression of human osteocalcin was detected from week 7. Conclusions: These results show that transplantation of hBMMSCs/β-TCP in NOD/SCID mice are safe and effective, and might be applied to human bone diseases in future clinical trials.

Gel casting of β-TCP scaffolds: Effects of foaming time and polymer coating

Three-dimensional interconnected porous structure and ideal mechanical strength are essential for biological scaffold for bone tissue engineering application. However, ceramic scaffold is very brittle and exhibit low strength. The main aim of this study is to investigate the effect of foaming time on the microstructure and mechanical properties of porous beta tricalcium phosphate (β-TCP) scaffold. The effect of polymer coating on the β-TCP scaffold was also investigated. Porous β-TCP scaffolds were fabricated using the gel casting method by varying the foaming time (60s and 90s) followed by sintering at 1100°C. Porous β-TCP scaffold was coated with polymer coating by using dipping method. Results indicate that increased foaming time results in higher porosity and lower compression strength. It is observed that polymer coating improved around 116% compressive strength in comparison to that of uncoated scaffold. Fourier transform infrared (FTIR) confirm the presence of gelatin on scaffold walls. Based on the morphology observed by scanning electron microscope (SEM), it is found that the scaffold fabricated by gel casting method showed open pores characteristic.