ObjectiveTo fabricate an injectable composite bone substitute with hyaluronic acid (HA) and calcium sulfate and to evaluate the biocompatibility and effect of the composite on cell proliferation, osteogenic differentiation in vitro and osteogenic capability in vivo. MethodsCalcium sulfate powder was mixed with HA solution, cross-linked HA solution, and phosphate buffer solution (PBS) in a ratio of 2∶1 (W/V) to get composites of CA+HA, CA+HAC, and CA. The standard extracts from above 3 materials were prepared according to ISO10993-5, and were used to culture mouse MC3T3-E1 cells. The composite biocompatibility and cell proliferation in different concentrations of extract were tested with cell counting kit-8 (CCK-8). The cells were cultured with standard medium as a control. The optimal concentration was selected for osteogenic differentiation test, and ELISA Kit was used to determine the alkaline phosphatase (ALP), collagen type I (COL-I), and osteocalcin (OCN). The femoral condylar bone defect was made on New Zealand white rabbits and repaired with CA+HA, CA+HAC, and CA. Micro-CT was done to evaluate new bone formation with bone volume/tissue volume (BV/TV) ratio at 6 and 12 weeks. HE staining was used to observe bone formation. ResultsCA+HA and CA+HAC were better in injectability and stability in PBS than CA. The biocompatibility test showed that absorbance (A) value of CA group was significantly lower than that of control group (P<0.05) at 6, 12, and 24 hours after culture, but no significant difference was found inA values between CA+HA group or CA+HAC group and control group (P>0.05). The proliferation test showed 25% and 50% extract of all 3 materials had significantly higherA value than control group (P<0.05). For 75% and 100% extract, only CA+HA group had significantly higherA value than control group (P<0.05). And 50% extract was selected for osteogenic differentiation test. At 14 and 21 days, ALP, COL-I and OCN concentrations of CA+HA group and CA+HAC group were significantly higher than those of CA group and control group (P<0.05). Micro-CT results showed higher BV/TV in CA+HA group and CA+HAC group than CA group at 6 and 12 weeks (P<0.05), but no significant difference was found between CA+HA group and CA+HAC group (P>0.05). HE staining revealed that a little bone tissue was seen in CA+HA group and CA+HAC group, but there was no bone formation in CA group at 6 weeks; more streak bone tissue in CA+HA group and CA+HAC group than CA group at 12 weeks. ConclusionComposites prepared with calcium sulfate and HA or with cross-linked HA are stable, injectable, and biocompatible. The materials have excellent effect on proliferation and differentiation of mouse MC3T3-E1 cells. They also show good osteogenic capability in vivo. So it is a potential bone substitutes for bone defective diseases.
Mussel foot proteins (MFp) could cure rapidly under water and adhere to different substrates. It has broad application prospects as an biocompatible bioglue. The soluble recombinant SUMO-MFp fusion protein (SFp3) was efficiently expressed inE.coli, and about 5% of tyrosine of SFp3 were converted into DOPA by using mushroom tyrosinase. The adhesion strength of the mixture of DOPA-containing SFp3 (DSFp3) and hyaluronic acid (MW = 1 500 kD) was more than twice that of the cyanoacrylate-based tissue adhesives, Dermabond®, and it reached 52% of its maximal strength within 5 minutes on cowhide. A layer-by-layer assembly of hyaluronic acid with DSFp3 was observed to form compact sheet structures through biofilm interferometry assay and scanning electron microscopy. This work provides a solution and theoretical basis for the low adhesion strength and slow curing of protein-based bioglue.
ObjectiveAfter using hyaluronic acid (HA) to modify curcumin (CUR), the effects of calcium phosphate cement (CPC) combined with HA/CUR on the proliferation and osteogenesis of osteoblasts were investigated.MethodsFirst, HA and CUR were esterified and covalently combined to prepare HA/CUR, and the characteristics were observed and the infrared spectrum was tested. Then, HA, CUR, and HA/CUR were mixed with CPC according to 5% (W/W) to prepare HA-CPC, CUR-CPC, and HA/CUR-CPC, respectively. Setting time detection, scanning electron microscope observation, injectable performance test, and compression strength test were conducted; and the CPC was used as a control. Osteoblasts were isolated and cultured from the skull of newborn Sprague Dawley rats, and the 2nd generation cells were cultured with the 4 types of bone cement, respectively. The effects of HA/CUR-CPC on the proliferation and osteogenesis of osteoblasts were estimated by the scanning electron microscopy observation, live/dead cell fluorescence staining, cell counting, osteopontin (OPN) immunofluorescence staining, alkaline phosphatase (ALP) staining,and alizarin red staining.ResultsInfrared spectroscopy test showed that HA and CUR successfully covalently combined. The HA/CUR-CPC group had no significant difference in initial setting time, final setting time, injectable rate, and compressive strength when compared with the other 3 groups (P>0.05); scanning electron microscope observation showed that HA/CUR was scattered on CPC surface. After co-culture of bone cement and osteoblasts, scanning electron microscopy observation showed that the osteoblasts, which had normal morphology and the growth characteristics of osteoblasts, clustered and adhered to HA/CUR-CPC. There was no significant difference in cell survival rate between HA/CUR-CPC group and other groups (P>0.05), and the number of cells significantly increased (P<0.05); the degrees of OPN immunofluorescence staining, ALP staining, and alizarin red staining were stronger than other groups.ConclusionHA/CUR-CPC has good biocompatibility and mechanical properties, which can promote the proliferation and osteogenesis of osteoblasts.
ObjectiveTo study the preparation and properties of the hyaluronic acid (HA)/α-calcium sulfate hemihydrate (α-CSH)/β-tricalcium phosphate (β-TCP) material (hereinafter referred to as composite material). Methods Firstly, the α-CSH was prepared from calcium sulfate dihydrate by hydrothermal method, and the β-TCP was prepared by wet reaction of soluble calcium salt and phosphate. Secondly, the α-CSH and β-TCP were mixed in different proportions (10∶0, 9∶1, 8∶2, 7∶3, 5∶5, and 3∶7), and then mixed with HA solutions with concentrations of 0.1%, 0.25%, 0.5%, 1.0%, and 2.0%, respectively, at a liquid-solid ratio of 0.30 and 0.35 respectively to prepare HA/α-CSH/ β-TCP composite material. The α-CSH/β-TCP composite material prepared with α-CSH, β-TCP, and deionized water was used as the control. The composite material was analyzed by scanning electron microscope, X-ray diffraction analysis, initial/final setting time, degradation, compressive strength, dispersion, injectability, and cytotoxicity. ResultsThe HA/α-CSH/β-TCP composite material was prepared successfully. The composite material has rough surface, densely packed irregular block particles and strip particles, and microporous structures, with the pore size mainly between 5 and 15 μm. When the content of β-TCP increased, the initial/final setting time of composite material increased, the degradation rate decreased, and the compressive strength showed a trend of first increasing and then weakening; there were significant differences between the composite materials with different α-CSH/β-TCP proportion (P<0.05). Adding HA improved the injectable property of the composite material, and it showed an increasing trend with the increase of concentration (P<0.05), but it has no obvious effect on the setting time of composite material (P>0.05). The cytotoxicity level of HA/α-CSH/β-TCP composite material ranged from 0 to 1, without cytotoxicity. Conclusion The HA/α-CSH/β-TCP composite materials have good biocompatibility. Theoretically, it can meet the clinical needs of bone defect repairing, and may be a new artificial bone material with potential clinical application prospect.