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The aims are: (a) To develop the first low-shrinkage-stress nanocomposite with antibacterial and remineralization capabilities through the incorporation of dimethylaminododecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP); (b) to investigate the effects of the new composite on biofilm inhibition, mechanical properties, shrinkage stress, and calcium (Ca) and phosphate (P) ion releases. The low-shrinkage-stress resin consisted of urethane dimethacrylate and triethylene glycol divinylbenzyl ether. Composite was formulated with 3% DMAHDM and 20% NACP. Mechanical properties, shrinkage stress, and degree of conversion were evaluated. Streptococcus mutans biofilm growth on composites was assessed. Ca and P ion releases were measured. The shrinkage stress of the low-shrinkage-stress composite containing 3% DMAHDM and 20% NACP was 36% lower than that of traditional composite control (p < 0.05), with similar degrees of conversion of 73.9%. The new composite decreased the biofilm colony-forming unit by 4 log orders and substantially reduced biofilm lactic acid production compared to control composite (p < 0.05). Incorporating DMAHDM to the low-shrinkage-stress composite did not adversely affect the Ca and P ion release. A novel bioactive nanocomposite was developed with low shrinkage stress, strong antibiofilm activity, and high levels of ion release for remineralization, without undermining the mechanical properties and degree of conversion., (© 2021 Wiley Periodicals LLC.)

Infectious bone defects remain a significant challenge in orthopedics and dentistry. Calcium phosphate cement (CPC) have attracted significant interest in use as local drug delivery system, which with great potential to control release of antibiotics for the treatment of infectious bone defects. Within the current study, a novel antibacterial scaffold of chitosan-reinforced calcium phosphate cement delivering doxycycline hyclate (CPCC + DOX) was developed. Furthermore, the capacity of CPCC + DOX scaffolds for bone regeneration was enhanced by the human periodontal ligament stem cells (hPDLSCs) encapsulated in alginate beads. CPCC + DOX scaffolds were fabricated to contain different concentrations of DOX. Flexural strength of CPCC + DOX ranged from 5.56 ± 0.70 to 6.2 ± 0.72 MPa, which exceeded the reported strength of cancellous bone. Scaffolds exhibited continual DOX release, reaching 80% at 21 days. Scaffold with 5 mg/ml DOX (CPCC + DOX5mg) had a strong antibacterial effect, with a 4-log colony forming unit reduction against S. aureus and P. gingivalis. The proliferation and osteogenic differentiation of hPDLSCs encapsulated in alginate hydrogel microbeads were investigated in culture with CPCC + DOX scaffolds. CPCC + DOX5mg had no negative effect on proliferation of hPDLSCs. Alkaline phosphatase activity, mineral synthesis, and osteogenic gene expressions for CPCC + DOX5mg group were much higher than control group. DOX did not compromise the osteogenic induction. In summary, the novel CPCC + DOX scaffold exhibited excellent mechanical properties and strong antibacterial activity, while supporting the proliferation and osteogenic differentiation of hPDLSCs. The CPCC + DOX + hPDLSCs construct is promising to enhance bone regeneration and combat bone infections in dental, craniofacial, and orthopedic applications., (© 2021 John Wiley & Sons Ltd.)

Staphylococcus aureus (S. aureus) is a major pathogen for osteomyelitis. Calcium phosphate bone cement (CPC) paste is promising for orthopedic uses. Nanostructured graphene oxide (GO) showed antibacterial effect on Gram-positive bacteria. However, there has been no report of incorporating GO into CPC. The objectives of this study were to (a) develop an injectable and mechanically strong CPC-chitosan paste containing GO and (b) investigate the inhibition of S. aureus infection and the promotion of human umbilical cord mesenchymal stem cells (hUCMSCs) for bone regeneration. Injectable CPC-chitosan-GO paste was fabricated. Flexural strength, elastic modulus, and work-of-fracture of the CPC-chitosan and CPC-chitosan-GO bars were evaluated. Antibacterial effects against S. aureus biofilms were determined. hUCMSC growth and viability on disks were investigated. CPC-chitosan-GO bars had a flexural strength of 7.2 ± 1.6 MPa, matching that of CPC-chitosan control without GO. CPC-chitosan-GO had strong antibacterial effects on S. aureus, with an inhibition zone of 55.2 ± 2.5 mm, greater than that of CPC-chitosan control (30.1 ± 2.0 mm) (p < 0.05). CPC-chitosan-GO had potent antibacterial activity on S. aureus biofilms in vitro (p > 0.05). The injectable and antibacterial CPC-GO paste had no toxic effect, yielding excellent hUCMSC growth and viability on disks. The CPC-chitosan-GO had injectability, good strength, strong antibacterial effects, and excellent stem cell attachment and growth. CPC-chitosan-GO is promising for dental, craniofacial, and orthopedic applications to control infections and good biocompatibility to support stem cell viability to enhance bone regeneration., (© 2020 John Wiley & Sons, Ltd.)

Herein, we investigated a biointeractive tooth sealing material consisted of dimethylaminohexadecyl methacrylate (DMAHDM) and amorphous calcium phosphate nanoparticles (NACPs) to address the above issues simultaneously. Of note, 5% DMAHDM was incorporated into the resin blend, and 20% NACP was added to inorganic filler content of dental formulations intended as dental sealants. The sealing materials were used to seal human extracted teeth. The sealed teeth were subjected to an early childhood caries (ECC) key pathogen (Candida albicans and Streptococcus mutans) biofilm model using a dynamic caries tooth model (CDC reactor). The biofilm growth over the sealed teeth was assessed via colony-forming unit counting metabolic activity assays. The enamel surface hardness loss, degree of conversion, shear bond strength (SBS), and cytotoxicity were also investigated. Formulations having DMAHDM displayed antibacterial efficiency of 2.8-3.5 and 1.4-4.0 log inhibition for Streptococcus mutans and Candida albicans, respectively. Furthermore, the metabolic activity was reduced on the top of the sealed tooth with the biointeractive sealing materials (p < .05). The degree of conversion values was acceptable. The enamel surface hardness loss decreases (36 ± 9.8%) when in contact with the biointeractive tooth sealing material. The SBS of the combined formulation (5% DMAHDM + 20% NACP) was lower than commercial sealant but similar to experimental control. The investigated sealing material holds valuable dual antibacterial and antifungal activities associated with a reduced mineral loss against the cariogenic challenge promoted by ECC key pathogens., (© 2020 Wiley Periodicals LLC.)

Patients with dry mouth often have an acidic oral environment lacking saliva to provide calcium (Ca) and phosphate (P) ions. There has been no report on tooth remineralization in acidic pH4 and CaP ion-lacking solutions. The objective of this study was to develop a novel method of combining poly(amido amine) (PAMAM) with adhesive containing nanoparticles of amorphous calcium phosphate (NACP) for dentin remineralization in pH4 and CaP-lacking solution for the first time. Demineralized dentin was tested in four groups: (1) dentin control, (2) dentin with PAMAM, (3) dentin with NACP adhesive, (4) dentin with PAMAM + NACP adhesive. Dentin samples were examined by scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS) and hardness testing. Increasing the NACP filler level in adhesive from 0 to 40 wt% did not negatively affect the dentin bond strength (p > 0.1). NACP adhesive released CaP ions and neutralized the acid. PAMAM alone failed to achieve dentin remineralization in lactic acid. NACP alone induced slight dentin remineralization in lactic acid (p > 0.1). In contrast, the novel PAMAM + NACP group in the pH4 and CaP-lacking solution completely remineralized the predemineralized dentin, increasing its hardness which approached that of healthy dentin (p > 0.1). In conclusion, dentin remineralization via PAMAM + NACP adhesive in pH4 and CaP-lacking acid was achieved for the first time, when conventional remineralization methods such as PAMAM or NACP did not work. The novel PAMAM + NACP method is promising to increase the longevity of the composite-tooth bond, inhibit caries, remineralize lesions and protect tooth structures, even for patients with dry mouth and an acidic oral environment. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2414-2424, 2018., (© 2017 Wiley Periodicals, Inc.)

A main challenge for use of scaffolds in bone engineering involves non-invasive monitoring in vivo and enhanced bone regeneration. The tissue repair effect of superparamagnetic iron oxide nanoparticles (SPIONs) was demonstrated previously by our group. However, testing in vivo is needed to confirm in vitro results. Here, SPIONs loaded gelatin sponge (GS) was used as a scaffold (SPIONs-GS) and implanted in the incisor sockets of Sprague-Dawley rats. Incisor sockets filled with nothing and filled with GS served as controls. Rats were sacrificed at 2 and 4 weeks. A significant decrease in the signal intensity of T2-weighted magnetic resonance imaging (MRI) in the SPIONs-GS group was noted. Changes in image intensity of scaffolds (indicating scaffold degradation and interaction with host tissues) could be visually monitored over time. Microcomputed tomography showed that the SPIONs-GS group had more newly formed bone (64.44 ± 10.92 vs. 28.1 ± 4.49, p < .0001) and a better preserved alveolar ridge than blank control group at 4 weeks (0.962 ± 0.01 vs. 0.92 ± 0.01, p < .0001). Histology confirmed imaging results, showing good consistency in new bone formation and scaffold degradation. The number of SPIONs decreased rapidly with time due to quick degradation of GS, whereas the number of endocytic SPIONs in cells increased with time. These residual SPIONs, together with newly formed bone, could be detected by MRI at 4 weeks. Therefore, it was clear that SPIONs induced active osteogenesis. In conclusion, good visibility on MRI and enhanced regeneration of bone can be obtained by implanting SPIONs-GS in vivo without using an external magnetic field., (Copyright © 2018 John Wiley & Sons, Ltd.)

Metformin, a first-line antidiabetic drug used by millions of patients, has been shown to have potential osteogenic properties. The present study was performed to test the hypothesis that clinically relevant doses of metformin promote the osteogenic differentiation and mineralization of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). iPSC-MSCs were treated with metformin (10 μm) to assess cell viability, osteogenic differentiation, mineralization and activation of the LKB1/AMP-activated protein kinase (AMPK) signalling pathway, a surrogate marker of metformin action. To determine its potential application in MSC-based bone and periodontal tissue engineering, iPSC-MSCs were also treated with metformin when seeded on to calcium phosphate cement (CPC) scaffolds. Immunoblotting and cellular uptake assays showed that iPSC-MSCs express functional organic cation transporter-1 (OCT-1), a transmembrane protein that mediates the intracellular uptake of metformin. Although metformin treatment did not impair iPSC-MSC viability, it significantly stimulated alkaline phosphatase activity, enhanced mineralized nodule formation and increased expression of osteogenic markers, including Runt-related transcription factor 2 (RUNX2) and osterix. Inhibition of LKB1 activity, a common upstream AMPK kinase, markedly reversed metformin-induced AMPK activation, RUNX2 expression and nuclear localization. Moreover, metformin substantially increased mineralized nodule formation of iPSC-MSC seeded on CPC scaffolds. Collectively, functional OCT-expressing iPSC-MSCs responded to metformin by inducing an osteogenic effect in part mediated by the LKB1/AMPK pathway. Considering the widespread use of metformin in diabetics, this work may lead to novel tissue-engineering platforms where autogenous OCT-expressing iPSC-MSCs might be used to enhance bone and periodontal regeneration in diabetic patients prescribed with daily doses of metformin., (Copyright © 2017 John Wiley & Sons, Ltd.)

Macroporous calcium phosphate cement (CPC) with stem cell seeding is promising for bone regeneration. The objective of this study was to investigate the effects of co-delivering autologous bone marrow mesenchymal stem cells (BMSCs) and autologous platelet-rich plasma (PRP) in CPC scaffold for bone regeneration in minipigs for the first time. Twelve female adult Tibet minipigs (12-18 months old) were used. A cylindrical defect with 10 mm height and 8 mm diameter was prepared at the femoral condyle. Two bone defects were created in each minipig, one at each side of the femoral condyle. Three constructs were tested: (1) CPC scaffold (CPC control); (2) CPC seeded with BMSCs (CPC-BMSC); (3) CPC seeded with BMSCs and PRP (CPC-BMSC-PRP). Two time points were tested: 6 and 12 weeks (n = 4). Good integration of implant with surrounding tissues was observed in all groups. At 12 weeks, the CPC-BMSC-PRP group had significantly less residual CPC remaining in the defect than the CPC-BMSC group and the CPC control (p < 0.05). The residual CPC volume for the CPC-BMSC-PRP group was half that of the CPC control. New bone formation for CPC-BMSC-PRP was more than two-fold that of the CPC control (p < 0.05). CPC-BMSC-PRP had new blood vessel density that was nearly two-fold that of the CPC control (p < 0.05). In conclusion, CPC scaffold with autologous BMSC-PRP doubled the new bone regeneration and blood vessel density in minipigs compared with the CPC control. In the present study, the new macroporous CPC system with co-delivered BMSC-PRP has been shown to promote scaffold resorption and bone regeneration in large defects. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co-culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co-cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC-MSCs) and embryonic stem cells (hESC-MSCs). Constructs were implanted in 8 mm cranial defects of rats for 12 weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary-like structures were successfully formed on CPC in vitro in all four co-cultured groups. Microcapillary lengths increased with time (p < 0.05). Osteogenic and angiogenic gene expressions were highly elevated and mineralization by co-cultured cells increased with time (p < 0.05). New bone amount and blood vessel density of co-cultured groups were much greater than controls (p < 0.05) in an animal study. hUVECs co-cultured with hUCMSCs, hiPSC-MSCs and hESC-MSCs achieved new bone and vessel density similar to hUVECs co-cultured with hBMSCs (p > 0.1). Therefore, hUCMSCs, hiPSC-MSCs and hESC-MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co-cultures of hUVECs with hUCMSCs, hiPSC-MSCs, hESC-MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co-culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Fatigue caused by the cyclic loads of mastication and acid attack caused by the excretion of oral biofilms are two of the most critical challenges to the success of dental restorations and their clinical service life. The objective of this investigation was to evaluate the fatigue strength of human dentin when exposed to a simultaneous challenge of cyclic loading and acidic attack from oral bacteria. Rectangular beams of coronal dentin were obtained from third molars and subjected to cyclic flexural loading while exposed to an in-vitro microcosm biofilm model. Two different cariogenic protocols were considered and results were compared with those for control samples evaluated at neutral pH. According to the fatigue life distributions, dentin exposed to the biofilm model with 2.0% sucrose supplements pulsed twice per day caused a significant reduction in the fatigue strength (p < 0.001) with respect to 0.2% sucrose supplements pulsed once a day, and the control environment (without biofilm). The endurance limit after biofilm exposure was 20 MPa, which is 60% lower than that of the control environment without biofilm (50 MPa). Biofilm attack of dentin increases the likelihood of restored tooth failures by fatigue and after only modest periods of exposure. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1978-1985, 2017., (© 2016 Wiley Periodicals, Inc.)

The objectives of this study were to develop the first protein-repellent resin-modified glass ionomer cement (RMGI) by incorporating 2-methacryloyloxyethyl phosphorylcholine (MPC) for orthodontic applications, and to investigate the MPC effects on protein adsorption, biofilm growth, and enamel bond strength. MPC was incorporated into RMGI at 0% (control), 1.5%, 3%, and 5% by mass. Specimens were stored in water at 37°C for 1 and 30 days. Enamel shear bond strength (SBS) was measured, and the adhesive remnant index (ARI) scores were assessed. Protein adsorption onto the specimens was determined by a micro bicinchoninic acid method. A dental plaque microcosm biofilm model with human saliva as inoculum was used. The results showed that adding 3% of MPC into RMGI did not significantly reduce the SBS (p > 0.1). There was no significant loss in SBS for RMGI containing 3% MPC after water-aging for 30 days, as compared to 1 day (p > 0.1). RMGI with 3% MPC had protein adsorption that was 1/10 that of control. RMGI with 3% MPC greatly reduced the bacterial adhesion, and lactic acid production and colony-forming units of biofilms, while substantially increasing the medium solution pH containing biofilms. The protein-repellent and biofilm-repellent effects were not decreased after water-aging for 30 days. In conclusion, the MPC-containing RMGI is promising to reduce biofilms and white spot lesions without compromising orthodontic bracket-enamel bond strength. The novel protein-repellent method may have applicability to other orthodontic cements, dental composites, adhesives, sealants, and cements to repel proteins and biofilms. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 949-959, 2016., (© 2015 Wiley Periodicals, Inc.)

The need for bone repair has increased as the population ages. Non-rigid calcium phosphate scaffolds could provide compliance for micro-motions within the tissues and yet have load-supporting strength. The objectives of this study were to: (a) develop a non-rigid calcium phosphate cement (CPC) with microbeads and fibre reinforcement; and (b) investigate human umbilical cord mesenchymal stem cell (hUCMSC) proliferation, osteodifferentiation and mineralization on non-rigid CPC for the first time. Non-rigid CPC was fabricated by adding extra tetracalcium phosphate in the traditional CPC and by incorporating chitosan, absorbable fibres and hydrogel microbeads. The non-rigid CPC-microbead scaffold possessed a strain-at-failure of 10.7%, much higher than the traditional CPC's strain of 0.05% which is typical for brittle bioceramics. Flexural strength of non-rigid CPC-microbead was 4-fold that of rigid CPC-microbead scaffold, while work-of-fracture (toughness) was increased by 20-fold. The strength of non-rigid CPC-microbead-fibre scaffold matched that of cancellous bone. hUCMSCs on non-rigid CPC proliferated from 100 cells/mm(2) at 1 day to 600 cells/mm(2) at 8 days. Alkaline phosphatase, osteocalcin and collagen gene expressions of hUCMSCs were greatly increased, and the cells synthesized bone minerals. hUCMSCs on non-rigid CPC-microbead-fibre constructs had higher bone markers and more mineralization than those on rigid CPC controls. In conclusion, this study developed the first non-rigid, in situ-setting calcium phosphate-microbead-fibre scaffold with a strain-at-failure exceeding 10%. hUCMSCs showed excellent proliferation, osteodifferentiation and mineralization on non-rigid CPC scaffold. The novel non-rigid CPC-hUCMSC construct with good strength, high strain-at-failure and toughness, as well as superior stem cell proliferation, osteodifferentiation and mineralization, is promising for load-bearing bone regeneration applications., (Copyright © 2012 John Wiley & Sons, Ltd.)

Dental resins containing 12-methacryloyloxydodecylpyridinium bromide (MDPB) showed potent antibacterial functions. Recent studies developed antibacterial resins containing nanoparticles of silver (NAg). The objectives of this study were to develop an adhesive containing dual agents of MDPB and NAg for the first time and to investigate the combined effects of antibacterial adhesive and primer on biofilm viability, metabolic activity, lactic acid, dentin bond strength, and fibroblast cytotoxicity. MDPB and NAg were incorporated into Scotchbond Multi-Purpose (SBMP) adhesive "A" and primer "P". Five systems were tested: SBMP adhesive A; A + MDPB; A+NAg; A + MDPB + NAg; P + MDPB + NAg together with A + MDPB + NAg. Dental plaque microcosm biofilms were cultured using mixed saliva from 10 donors. Metabolic activity, colony-forming units, and lactic acid production of biofilms were investigated. Human fibroblast cytotoxicity of bonding agents was determined. MDPB + NAg in adhesive/primer did not compromise dentin bond strength (p > 0.1). MDPB or NAg alone in adhesive substantially reduced the biofilm activities. Dual agents MDPB + NAg in adhesive significantly reduced the biofilm viability compared with each agent alone (p < 0.05). The greatest inhibition of biofilms was achieved when both adhesive and primer contained MDPB + NAg. Fibroblast viability of groups with dual antibacterial agents was similar to control using culture medium without resin eluents (p > 0.1). In conclusion, this study showed for the first time that the antibacterial potency of MDPB adhesive could be substantially enhanced via NAg. Adding MDPB + NAg into both primer and adhesive achieved the strongest antibiofilm efficacy. The dual agent (MDPB + NAg) method could have wide applicability to other adhesives, sealants, cements, and composites to inhibit biofilms and caries., (Copyright © 2013 Wiley Periodicals, Inc.)

Secondary caries remains the main reason for dental restoration failure. Replacement of failed restorations accounts for 50%-70% of all restorations performed. Antibacterial adhesives could inhibit biofilm acids at tooth-restoration margins, and calcium phosphate (CaP) ions could remineralize tooth lesions. The objectives of this study were to: (1) incorporate nanoparticles of silver (NAg), quaternary ammonium dimethacrylate (QADM), and nanoparticles of amorphous calcium phosphate (NACP) into bonding agent; and (2) investigate their effects on dentin bonding and microcosm biofilms. An experimental primer was made with pyromellitic glycerol dimethacrylate (PMGDM) and 2-hydroxyethyl methacrylate (HEMA). An adhesive was made with bisphenol-A-glycerolate dimethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA). NAg was incorporated into primer at 0.1 wt %. The adhesive contained 0.1% NAg and 10% QADM, and 0%-40% NACP. Incorporating NAg into primer and NAg-QADM-NACP into adhesive did not adversely affect dentin bond strength (p > 0.1). Scanning electron microscopy showed numerous resin tags, and transmission electron microscopy revealed NAg and NACP in dentinal tubules. Viability of human saliva microcosm biofilms on primer/adhesive/composite disks was substantially reduced via NAg and QADM. Metabolic activity, lactic acid, and colony-forming units of biofilms were much lower on the new bonding agents than control (p < 0.05). In conclusion, novel dental bonding agents containing NAg, QADM, and NACP were developed with the potential to kill residual bacteria in the tooth cavity and inhibit the invading bacteria along tooth-restoration margins, with NACP to remineralize tooth lesions. The novel method of combining antibacterial agents (NAg and QADM) with remineralizing agent (NACP) may have wide applicability to other adhesives for caries inhibition., (Copyright © 2012 Wiley Periodicals, Inc.)

Half of all dental restorations fail within 10 years, with secondary caries and restoration fracture being the main reasons. Calcium phosphate (CaP) composites can release Ca and PO(4) ions and remineralize tooth lesions. However, there has been no report on their long-term mechanical durability. The objective of this study was to investigate the wear, thermal-cycling, and water-aging of composites containing amorphous calcium phosphate nanoparticles (NACP). NACP of 112-nm and glass particles were used to fabricate four composites: (1) 0% NACP+75% glass; (2) 10% NACP+65% glass; (3) 15% NACP+60% glass; and (4) 20% NACP+50% glass. Flexural strength and elastic modulus of NACP nanocomposites were not degraded by thermal-cycling. Wear depth increased with increasing NACP filler level. Wear depths of NACP nanocomposites after 4 × 10(5) cycles were within the range for commercial controls. Mechanical properties of all the tested materials decreased with water-aging time. After 2 years, the strengths of NACP nanocomposites were moderately higher than the control composite, and much higher than the resin-modified glass ionomers. The mechanism of strength loss for resin-modified glass ionomer was identified as microcracking and air-bubbles. NACP nanocomposites and control composite were generally free of microcracks and air-bubbles. In conclusion, combining NACP nanoparticles with reinforcement glass particles resulted in novel nanocomposites with long-term mechanical properties higher than those of commercial controls, and wear within the range of commercial controls. These strong long-term properties, plus the Ca-PO(4) ion release and acid-neutralization capability reported earlier, suggest that the new NACP nanocomposites may be promising for stress-bearing and caries-inhibiting restorations., (Copyright © 2012 Wiley Periodicals, Inc.)

A dental composite containing amorphous calcium phosphate nanoparticles (NACP) was developed that released calcium (Ca) and phosphate (PO(4)) ions and possessed acid-neutralization capability. There has been little study on incorporation of antibacterial agents into calcium phosphate composites. The objective of this study was to investigate the effect of silver nanoparticle (NAg) mass fraction in NACP nanocomposite on mechanical properties and dental plaque microcosm biofilm for the first time. NACP nanoparticles of 116 nm were synthesized via a spray-drying technique. NAg nanoparticles were synthesized using Ag 2-ethylhexanoate and 2-(tert-butylamino)ethyl methacrylate, yielding NAg of particle size of 2.7 nm that were well-dispersed in the resin. Five NACP nanocomposites were fabricated with NAg mass fractions of 0, 0.028, 0.042, 0.088, and 0.175%, respectively. Mechanical properties of NACP nanocomposites containing 0-0.042% of NAg matched those of a commercial composite without antibacterial activity. Live/dead assay of dental plaque microcosm biofilms showed complete coverage with live bacteria on commercial composite. However, there were increasingly more dead bacteria with higher NAg content in the NACP nanocomposite. Colony-forming unit (CFU) counts for total microorganisms, total streptococci, and mutans streptococci for NACP nanocomposite with 0.042% NAg were about 1/4 those of commercial composite. Lactic acid production on NACP nanocomposite with 0.042% NAg was 1/3 that on commercial composite. In conclusion, novel NACP-NAg nanocomposites were developed which possessed good mechanical properties and potent antibacterial properties, with substantially reduced biofilm viability and lactic acid production. Hence, the NACP-NAg nanocomposites are promising for dental restorations with remineralizing and antibacterial capabilities., (Copyright © 2012 Wiley Periodicals, Inc.)

Tooth caries is a carbohydrate-modified bacterial infectious disease, and recurrent caries is a frequent reason for restoration failure. The objective of this study was to develop a novel antibacterial composite using tetracalcium phosphate (TTCP) fillers and bis(2-methacryloyloxy-ethyl) dimethyl-ammonium bromide, which is a quaternary ammonium dimethacrylate (QADM). QADM was synthesized using 2-(N,N-dimethylamino)ethyl methacrylate and 2-bromoethyl methacrylate and incorporated into a resin. The resin was filled with 40% TTCP and 30% glass particles. The following QADM mass fractions in the composite were tested: 0%, 6%, 12%, and 18%. Streptococcus mutans biofilms were formed on the composites and the colony-forming units (CFUs), metabolic activity, and lactic acid production were measured. The TTCP-QADM composite had flexural strength and elastic modulus similar to those of two commercial composites (p > 0.1). Increasing the QADM content in TTCP composite greatly decreased the bacteria growth and biofilm matrix production. There were significantly more dead bacteria with increasing QADM content. TTCP composite containing 18% QADM had biofilm CFU, metabolic activity, and acid production about half of those without QADM. Inversely linear relationships were established between QADM mass fraction and S. mutans biofilm CFU, metabolic activity, and acid production, with correlation coefficients R(2) ≥ 0.98. In conclusion, TTCP-QADM composites were developed and the effect of QADM mass fraction on the antibacterial properties of the composite was determined for the first time. The novel TTCP-QADM composites possessing a strong antibacterial capability, together with calcium phosphate ion release and good mechanical properties, are promising for dental restorations to reduce biofilm growth and recurrent caries., (Copyright © 2011 Wiley Periodicals, Inc.)

Stem cell-based tissue engineering offers immense promise for bone regeneration. The objective of this study was to develop a self-setting, mannitol-containing calcium phosphate cement (CPC) encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs) for bone tissue engineering. hUCMSCs could be an inexhaustible and low-cost alternative to the gold-standard bone marrow MSCs, which require an invasive procedure to harvest. hUCMSCs were encapsulated in alginate beads and mixed into the CPC paste. Water-soluble mannitol porogen was incorporated into CPC to create macropores. The porosity was increased from 49% for the hUCMSC-encapsulating CPC to 64% after adding mannitol and absorbable-fibres (p < 0.05). Flexural strength of the construct was increased from 0.3 MPa to 2.0 MPa via fibres. Live cell percentage was > 80% for all constructs. The ALP and OC gene expressions were low at 1 day and greatly increased at 14 days. The constructs that contained mannitol had significantly higher ALP and OC expressions than that without mannitol. ALP activity of hUCMSCs inside CPC with mannitol and fibre was significantly higher than that without mannitol. At 14 days, mineralization by the encapsulated hUCMSCs was eight-fold higher than that at 1 day. In conclusion, a novel mannitol-containing porous CPC-hUCMSC construct was developed for bone tissue engineering. Its advantages include cell delivery inside a load-bearing CPC that has injectable and in situ setting capabilities. hUCMSCs inside CPC had good viability and successfully osteodifferentiated. The self-setting and strong hUCMSC-encapsulating CPC scaffold is promising for bone tissue engineering in a wide range of orthopaedic and craniofacial applications., (Copyright © 2011 John Wiley & Sons, Ltd.)

Dental composites do not hinder bacteria colonization and plaque formation. Caries at the restoration margins is a frequent reason for replacement of existing restorations, which accounts for 50 to 70% of all restorations. The objectives of this study were to examine the filler level effect on nanocomposite containing nanoparticles of amorphous calcium phosphate (NACP) and investigate the load-bearing and acid-neutralizing properties and bacteria inhibition. NACP with 116-nm particle size were synthesized via a spray-drying technique and incorporated into a resin. Flexural strength of nanocomposite with 10 to 30% NACP fillers matched the strength of a commercial hybrid composite (p > 0.1). Nanocomposite with 40% NACP matched the strength of a microfill composite, which was 2-fold that of a resin-modified glass ionomer. Nanocomposite with 40% NACP neutralized a lactic acid solution of pH 4 by rapidly increasing the pH to 5.69 in 10 min. In contrast, the commercial controls had pH staying at near 4. Using Streptoccocus mutans, an agar disk-diffusion test showed no inhibition zone for commercial controls. In contrast, the inhibition zone was (2.5 ± 0.7) mm for nanocomposite with 40% NACP. Crystal violet staining showed that S. mutans coverage on nanocomposite was 1/4 that on commercial composite. In conclusion, novel calcium-phosphate nanocomposite matched the mechanical properties of commercial composite and rapidly neutralized lactic acid of pH 4. The nanocomposite appeared to moderately reduce the S. mutans growth, and further study is needed to obtain strong antimicrobial properties. The new nanocomposite may have potential to reduce secondary caries and restoration fracture, two main challenges facing tooth cavity restorations., (Copyright © 2011 Wiley Periodicals, Inc.)

Calcium phosphate cement (CPC) is osteoconductive and moldable, and it can conform to complex cavity shapes and set in situ to form hydroxyapatite. Chitosan could increase the strength and toughness of CPC, but there has been no investigation on recombinant human bone morphogenic protein-2 (rhBMP-2) delivery via CPC-chitosan composite and its effect on osteogenic induction of cells. The objective of this research was to investigate the mechanical properties and osteoblastic induction of MC3T3-E1 cells cultured on CPC-containing chitosan and rhBMP-2. Cell viability for CPC with chitosan and rhBMP-2 was comparable with that of control CPC, whereas the CPC-chitosan composite was stronger and tougher than CPC control. After 14 days, osteoblastic induction was quantified by measuring alkaline phosphatase (ALP) activity. ALP (mean +/- SD; n = 6) of cells seeded on conventional CPC without rhBMP-2 was (143 +/- 19) (mM pNpp/min)/(mug DNA). The addition of chitosan resulted in an ALP of 161 +/- 27. Further addition of rhBMP-2 to the CPC-chitosan composite increased the ALP to 305 +/- 111 (p < 0.05). All ALP activity on CPC composites was significantly higher when compared with the 10.0 +/- 3.3 of tissue culture polystyrene (p < 0.05). Flexural strength of CPC containing 15% (mass fraction) chitosan was 19.8 +/- 1.4 MPa, which is more than double the 8.0 +/- 1.4 MPa of conventional CPC (p < 0.05). The addition of chitosan to CPC increased the fracture toughness from 0.18 +/- 0.01 MPa.m(1/2) to 0.23 +/- 0.02 MPa.m(1/2) (p < 0.05). The relatively high strength, self-hardening CPC-chitosan composite scaffold is promising as a moderate load-bearing matrix for bone repair, with potential to serve as an injectable delivery vehicle for osteoinductive growth factors to promote osteoblastic induction and bone regeneration. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Because of its moldability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for craniofacial and orthopedic applications. The objectives of this study were to investigate the response of human mesenchymal stem cells (hMSCs) to a high-strength CPC-chitosan scaffold and to examine cell proliferation and osteogenic differentiation. hMSCs were seeded onto CPC-chitosan composite, CPC control, and tissue culture polystyrene (TCPS). Alkaline phosphatase activity (ALP) and mineralization of hMSCs were measured. CPC-chitosan had a flexural strength (mean + or - SD; n = 5) of (19.5 + or - 1.4) MPa, higher than (8.0 + or - 1.4) MPa of CPC control (p < 0.05). The percentage of live hMSCs on CPC-chitosan was (90.5 + or - 1.3)% at 8 days, matching (90.7 + or - 3.8)% of CPC control (p > 0.1). The CPC-chitosan surface area covered by the attached hMSCs increased from (51 + or - 11)% at 1 day to (90 + or - 4)% at 8 days (p < 0.05), matching those of CPC control (p > 0.1). Hence, the CPC strength was significantly increased via chitosan without compromising the hMSC response. At 8 days, there was a significant increase in ALP of cells in osteogenic media (10.99 + or - 0.93) [(mM pNpp/min)/(microg DNA)] versus control media (3.62 + or - 0.40) (p < 0.05). hMSCs in osteogenic media exhibited greater mineralization area of (47.5 + or - 19.7)% compared with (6.1 + or - 2.3)% in control medium on TCPS (p < 0.05). In conclusion, hMSCs showed excellent attachment and viability on the strong and tough CPC-chitosan scaffold, matching the hMSC response on CPC control. hMSCs were successfully differentiated down the osteogenic lineage. Hence, the strong, in situ hardening CPC-chitosan scaffold may be useful as a moderate load-bearing vehicle to deliver hMSCs for maxillofacial and orthopedic bone tissue engineering.

The two main challenges facing dental composite restorations are secondary caries and bulk fracture. Previous studies developed whisker-reinforced Ca-PO(4) composites that were relatively opaque. The objective of this study was to develop an esthetic glass particle-reinforced, photo-cured calcium phosphate composite. Tetracalcium phosphate (TTCP) particles were incorporated into a resin for Ca and PO(4) release, while glass particles provided reinforcement. Ion release and mechanical properties were measured after immersion in solutions with pH of 7, 5.5, and 4. For the composite containing 40% mass fraction of TTCP, incorporating glass fillers increased the strength (p < 0.05). Flexural strength (Mean +/- SD; n = 6) at 30% glass was 99 +/- 18 MPa, higher than 54 +/- 20 MPa at 0% glass (p < 0.05). Elastic modulus was 11 GPa at 30% glass, compared to 2 GPa without glass. At 28 days, the released Ca ion concentration was 4.61 +/- 0.18 mmol/L at pH of 4, much higher than 1.14 +/- 0.07 at pH of 5.5, and 0.27 +/- 0.01 at pH of 7 (p < 0.05). PO(4) release was also dramatically increased at cariogenic, acidic pH. The TTCP-glass composite had strength 2-3 fold that of a resin-modified glass ionomer control. In conclusion, the photo-cured TTCP-glass composite was "smart" and substantially increased the Ca and PO(4) release when the pH was reduced from neutral to a cariogenic pH of 4, when these ions are most needed to inhibit tooth caries. Its mechanical properties were significantly higher than previous Ca, PO(4), and fluoride releasing restoratives. Hence, the photo-cured TTCP-glass composite may have potential to provide the necessary combination of load-bearing and caries-inhibiting capabilities.

Calcium phosphate cement (CPC) can conform to complex bone cavities and set in-situ to form bioresorbable hydroxyapatite. The aim of this study was to develop a CPC-collagen composite with improved fracture resistance, and to investigate the effects of collagen on mechanical and cellular properties. A type-I bovine-collagen was incorporated into CPC. MC3T3-E1 osteoblasts were cultured. At CPC powder/liquid mass ratio of 3, the work-of-fracture (mean +/- sd; n = 6) was increased from (22 +/- 4) J/m(2) at 0% collagen, to (381 +/- 119) J/m(2) at 5% collagen (p < or = 0.05). At 2.5-5% of collagen, the flexural strength at powder/liquid ratios of 3 and 3.5 was 8-10 MPa. They matched the previously reported 2-11 MPa of sintered porous hydroxyapatite implants. SEM revealed that the collagen fibers were covered with nano-apatite crystals and bonded to the CPC matrix. Higher collagen content increased the osteoblast cell attachment (p < or = 0.05). The number of live cells per specimen area was (382 +/- 99) cells/mm(2) on CPC containing 5% collagen, higher than (173 +/- 42) cells/mm(2) at 0% collagen (p < or = 0.05). The cytoplasmic extensions of the cells anchored to the nano-apatite crystals of the CPC matrix. In summary, collagen was incorporated into in situ-setting, nano-apatitic CPC, achieving a 10-fold increase in work-of-fracture (toughness) and two-fold increase in osteoblast cell attachment. This moldable/injectable, mechanically strong, nano-apatite-collagen composite may enhance bone regeneration in moderate stress-bearing applications., ((c) 2008 Wiley Periodicals, Inc.)

The aim of this study was to develop a mechanically-strong calcium phosphate cement (CPC) with protein release. Chitosan was used to strengthen CPC and control protein release. Mass fraction of protein release = mass of released protein/mass of total protein incorporated into the specimen. Flexural strength (mean +/- sd; n = 6) of CPC containing 100 ng/mL of protein increased from 8.0 +/- 1.4 MPa with 0% chitosan, to 19.8 +/- 1.4 MPa with 15% chitosan (p < 0.05). The latter exceeded the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. When the chitosan mass fraction was increased from 0% to 10% and 15%, protein release varied from 0.60 +/- 0.03 to 0.41 +/- 0.04, and to 0.23 +/- 0.07, respectively (p < 0.05). When powder:liquid ratio increased from 2:1 to 3:1 and 4:1, protein release changed from 0.89 +/- 0.10 to 0.41 +/- 0.04, and to 0.23 +/- 0.07, respectively p < 0.05. Therefore, chitosan content and powder:liquid ratio successfully controlled the protein release. The protein release mass fraction, M, was related to CPC porosity P by: M = 16.9 P(4.5). In summary, a mechanically-strong CPC with controlled protein release was formulated. Protein release was proportional to CPC porosity. The in situ-hardening, nano-apatite composite may have potential for bone tissue engineering, especially when both mechanical strength and controlled release of therapeutic/bioactive agents are needed., (Copyright 2007 Wiley Periodicals, Inc.)

Calcium phosphate cement (CPC) sets in situ and forms apatite with excellent osteoconductivity and bone-replacement capability. The objectives of this study were to formulate an injectable tetracalcium phosphate-dicalcium phosphate cement (CPC(D)), and investigate the powder/liquid ratio and needle-size effects. The injection force (mean +/- SD; n = 4) to extrude the paste increased from (8 +/- 2) N using a 10-gauge needle to (144 +/- 17) N using a 21-gauge needle (p < 0.05). With the 10-gauge needle, the mass percentage of extruded paste was (95 +/- 4)% at a powder/liquid ratio of 3; it decreased to (70 +/- 12)% at powder/liquid = 3.5 (p < 0.05). A relationship was established between injection force, F, and needle lumen cross-sectional area, A: F = 5.0 + 38.7/A(0.8). Flexural strength, S, (mean +/- SD; n = 5) increased from (5.3 +/- 0.8) MPa at powder/liquid= 2 to (11.0 +/- 0.8) MPa at powder/liquid = 3.5 (p < 0.05). Pore volume fraction, P, ranged from 62.4% to 47.9%. A relationship was established: S = 47.7 x (1 - P)(2.3). The strength of the injectable CPC(D) matched/exceeded the reported strengths of sintered porous hydroxyapatite implants that required machining. The novel injectable CPC(D) with a relatively high strength may be useful in filling defects with limited accessibility such as periodontal repair and tooth root-canal fillings, and in minimally-invasive techniques such as percutaneous vertebroplasty to fill the lesions and to strengthen the osteoporotic bone., ((c) 2007 Wiley Periodicals, Inc.)

Clinical data indicate that secondary caries and restoration fracture are the most common problems facing tooth restorations. Our ultimate goal was to develop mechanically-strong and caries-inhibiting dental composites. The specific goal of this pilot study was to understand the relationships between composite properties and the ratio of reinforcement filler/releasing filler. Nanoparticles of monocalcium phosphate monohydrate (MCPM) were synthesized and incorporated into a dental resin for the first time. Silicon carbide whiskers were fused with silica nanoparticles and mixed with the MCPM particles at MCPM/whisker mass ratios of 1:0, 2:1, 1:1, 1:2, and 0:1. The composites were immersed for 1-56 days to measure Ca and PO4 release. When the MCPM/whisker ratio was changed from 0:1 to 1:2, the composite flexural strength (mean +/- SD; n = 5) decreased from 174 +/- 26 MPa to 138 +/- 9 MPa (p < 0.05). A commercial nonreleasing composite had a strength of 112 +/- 14 MPa. When the MCPM/whisker ratio was changed from 1:2 to 1:1, the Ca concentration at 56 days increased from 0.77 +/- 0.04 mmol/L to 1.74 +/- 0.06 mmol/L (p < 0.05). The corresponding PO4 concentration increased from 3.88 +/- 0.21 mmol/L to 9.95 +/- 0.69 mmol/L (p < 0.05). Relationships were established between the amount of release and the MCPM volume fraction v(MCPM) in the resin: [Ca]= 42.9 v(MCPM) (2.7), and [PO4] = 48.7 v(MCPM) (1.4). In summary, the method of combining nanosized releasing fillers with reinforcing fillers yielded Ca- and PO4-releasing composites with mechanical properties matching or exceeding a commercial stress-bearing, nonreleasing composite. This method may be applicable to the use of other Ca-PO4 fillers in developing composites with high stress-bearing and caries-preventing capabilities, a combination not yet available in any dental materials., ((c) 2006 Wiley Periodicals, Inc.)

Calcium phosphate cement (CPC) can conform to complex cavity shapes and set in situ to form bioresorbable hydroxyapatite. The aim of this study was to introduce cell-encapsulating alginate hydrogel beads into CPC and to improve the mechanical properties using chitosan and fiber mesh reinforcement. Because the CPC setting was harmful to the MC3T3-E1 osteoblast cells, alginate was used to encapsulate and protect the cells in CPC. Cells were encapsulated into alginate beads, which were then mixed into three pastes: conventional CPC, CPC-chitosan, and CPC-chitosan-mesh. After 1 day culture inside the setting cements, there were numerous live cells and very few dead cells, indicating that the alginate beads adequately protected the cells. Cell viability was assessed by measuring the mitochondrial dehydrogenase activity, using a Wst-1 colorimetric assay. Absorbance at 450 nm (arbitrary units) (mean +/- SD; n = 5) was 1.36 +/- 0.41 for cells inside conventional CPC, 1.29 +/- 0.24 for cells inside CPC-chitosan composite, and 0.73 +/- 0.22 for cells inside CPC-chitosan-mesh composite. All three values were similar to 1.00 +/- 0.14 for the control with cells in beads in the cell culture medium without any CPC (Tukey's at p = 0.05). Flexural strength for conventional CPC containing cell-encapsulating beads was 1.3 MPa. It increased to 2.3 MPa when chitosan was incorporated. It further increased to 4.3 MPa with chitosan and the reinforcement from one fiber mesh, and 9.5 MPa with chitosan and three sheets of fiber mesh. The latter two strengths matched reported strengths for sintered porous hydroxyapatite implants and cancellous bone. In summary, cell-encapsulated-alginate-CPC constructs showed favorable cell viability. The use of chitosan and mesh progressively improved the mechanical properties. These strong, in situ hardening, and cell-seeded hydroxyapatite cements may have potential for bone tissue engineering in moderate stress-bearing applications.

Calcium phosphate cement (CPC) sets in situ with intimate adaptation to the contours of defect surfaces, and forms an implant having a structure and composition similar to hydroxyapatite, the putative mineral in teeth and bones. The objective of the present study was to develop an injectable CPC using dicalcium phosphate dihydrate (DCPD) with a high solubility for rapid setting. Two agents were incorporated to impart injectability and fast-hardening to the cement: a hardening accelerator (sodium phosphate) and a gelling agent (hydroxypropyl methylcellulose, HPMC). The cement with DCPD was designated as CPC(D), and the conventional cement was referred to as CPC(A). Using water without sodium phosphate, CPC(A) had a setting time of 82 +/- 6 min. In contrast, CPC(D) exhibited rapid setting with a time of 17 +/- 1 min. At 0.2 mol/L sodium phosphate, setting time for CPC(D) was 15 +/- 1 min, significantly faster than 40 +/- 2 min for CPC(A) (Tukey's at 0.95). Sodium phosphate decreased the paste injectability (measured as the paste mass extruded from the syringe divided by the original paste mass inside the syringe). However, the addition of HPMC dramatically increased the paste injectability. For CPC(D), the injectability was increased from 65% +/- 12% without HPMC to 98% +/- 1% with 1% HPMC. Injectability of CPC(A) was also doubled to 99% +/- 1%. The injectable and rapid-setting CPC(D) possessed flexural strength and elastic modulus values overlapping the reported values for sintered porous hydroxyapatite implants and cancellous bone. In summary, the rapid setting and relatively high strength and elastic modulus of CPC(D) should help the graft to quickly attain strength and geometrical integrity within a short period of time postoperatively. Furthermore, the injectability of CPC(D) may have potential for procedures involving defects with limited accessibility or narrow cavities, when there is a need for precise placement of the paste, and when using minimally invasive surgical techniques.

Approximately a million bone grafts are performed each year in the United States, and this number is expected to increase rapidly as the population ages. Calcium phosphate cement (CPC) can intimately adapt to the bone cavity and harden to form resorbable hydroxyapatite with excellent osteoconductivity and bone-replacement capability. The objective of this study was to develop a strong CPC using synergistic reinforcement via suture fibers and chitosan, and to determine the fiber strength-CPC composite strength relationship. Biopolymer chitosan and cut suture filaments were randomly mixed into CPC. Both suture filaments and composite were immersed in a physiological solution. After 1-day immersion, cement flexural strengths (mean +/- SD; n = 6) were: (2.7 +/- 0.8) MPa for CPC control; (11.2 +/- 1.0) MPa for CPC-chitosan; (17.7 +/- 4.4) MPa for CPC-fiber composite; and (40.5 +/- 5.8) MPa for CPC-chitosan-fiber composite. They are significantly different from each other (Tukey's at 0.95). The strength increase from chitosan and fiber together in CPC was much more than that from either fiber or chitosan alone. The composite strength became (9.8 +/- 0.6) MPa at 35-day immersion and (4.2 +/- 0.7) MPa at 119 days, comparable to reported strengths for sintered porous hydroxyapatite implants and cancellous bone. After suture fiber dissolution, long macropore channels were formed in CPC suitable for cell migration and tissue ingrowth. A semiempirical relationship between suture fiber strength S(F) and composite strength S(C) were obtained: S(C) = 14.1 + 0.047 S(F), with R = 0.92. In summary, this study achieved substantial synergistic effects by combining random suture filaments and chitosan in CPC. This may help extend the use of the moldable, in situ hardening hydroxyapatite to moderate stress-bearing orthopedic applications. The long macropore channels in CPC should be advantageous for cell infiltration and bone ingrowth than conventional random pores and spherical pores.

Calcium phosphate cement (CPC) sets in situ to form resorbable hydroxyapatite with chemical and crystallographic similarity to the apatite in human bones, hence it is highly promising for clinical applications. The objective of the present study was to develop a CPC that is fast setting and has high strength in the early stages of implantation. Two approaches were combined to impart high early strength to the cement: the use of dicalcium phosphate dihydrate with a high solubility (which formed the cement CPC(D)) instead of anhydrous dicalcium phosphate (which formed the conventional cement CPC(A)), and the incorporation of absorbable fibers. A 2 x 8 design was tested with two materials (CPC(A) and CPC(D)) and eight levels of cement reaction time: 15 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 8 h, and 24 h. An absorbable suture fiber was incorporated into cements at 25% volume fraction. The Gilmore needle method measured a hardening time of 15.8 min for CPC(D), five-fold faster than 81.5 min for CPC(A), at a powder:liquid ratio of 3:1. Scanning electron microscopy revealed the formation of nanosized rod-like hydroxyapatite crystals and platelet crystals in the cements. At 30 min, the flexural strength (mean +/- standard deviation; n = 5) was 0 MPa for CPC(A) (the paste did not set), (4.2 +/- 0.3) MPa for CPC(D), and (10.7 +/- 2.4) MPa for CPC(D)-fiber specimens, significantly different from each other (Tukey's at 0.95). The work of fracture (toughness) was increased by two orders of magnitude for the CPC(D)-fiber cement. The high early strength matched the reported strength for cancellous bone and sintered porous hydroxyapatite implants. The composite strength S(c) was correlated to the matrix strength S(m): S(c) = 2.16S(m). In summary, substantial early strength was imparted to a moldable, self-hardening and resorbable hydroxyapatite via two synergistic approaches: dicalcium phosphate dihydrate, and absorbable fibers. The new fast-setting and strong cement may help prevent catastrophic fracture or disintegration in moderate stress-bearing bone repairs.

Calcium phosphate cement (CPC) self-hardens to form hydroxyapatite, has excellent osteoconductivity and bone-replacement ability, and is promising for craniofacial and orthopedic repair. However, its low strength limits CPC to only nonstress repairs. This study aimed to reinforce CPC with meshes to increase strength, and to form macropores in CPC for bone ingrowth after mesh dissolution. A related aim was to evaluate the biocompatibility of the new CPC-mesh composite. Absorbable polyglactin meshes, a copolymer of poly(glycolic) and poly(lactic) acids, were incorporated into CPC to provide strength and then form interconnected cylindrical macropores suitable for vascular ingrowth. The composite flexural strength, work-of-fracture, and elastic modulus were measured as a function of the number of mesh sheets in CPC ranging from 1 (a mesh on the tensile side of the specimen) up to 13 (mesh sheets throughout the entire specimen), and as a function of immersion time in a physiological solution from 1 to 84 days. Cell culture was performed with osteoblast-like cells and the cell viability was quantified using an enzymatic assay. The strengths (mean +/- SD; n = 6) of CPC containing 13 or 6 meshes were 24.5 +/- 7.8 and 19.7 +/- 4.3 MPa, respectively, not significantly different from each other; both were significantly higher than 8.8 +/- 1.9 MPa of CPC without mesh (Tukey's at 0.95). The work-of-fracture of CPC with 13 or 6 meshes was 3.35 +/- 0.80 and 2.95 +/- 0.58 kJ/m(2), respectively, two orders of magnitude higher than 0.021 +/- 0.006 kJ/m(2) of CPC without mesh. Interconnected macropores were formed in CPC at 84 days' immersion. The new CPC-mesh formulation supported the adhesion, spreading, proliferation, and viability of osteoblast-like cells in vitro. In conclusion, absorbable meshes in CPC increased the implant strength by three-fold and work-of-fracture by 150 times; interconnected macropores suitable for bone ingrowth were created in CPC after mesh dissolution. The higher strength may help extend the use of CPC to larger stress-bearing repairs, and the macropores may facilitate tissue ingrowth and integration of CPC with adjacent bone., (Copyright 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 267-278, 2004)

Calcium phosphate cement (CPC) is highly promising for craniofacial and orthopedic repair because of its ability to self-harden in situ to form hydroxyapatite with excellent osteoconductivity. However, its low strength, long hardening time, and lack of macroporosity limit its use. This study aimed to develop fast-setting and antiwashout CPC scaffolds with high strength and tailored macropore formation rates. Chitosan, sodium phosphate, and hydroxypropyl methylcellulose (HPMC) were used to render CPC fast-setting and resistant to washout. Absorbable fibers and mannitol porogen were incorporated into CPC for strength and macropores for bone ingrowth. Flexural strength, work-of-fracture, and elastic modulus were measured vs. immersion time in a physiological solution. Hardening time (mean +/- SD; n = 6) was 69.5 +/- 2.1 min for CPC-control, 9.3 +/- 2.8 min for CPC-HPMC-mannitol, 8.2 +/- 1.5 min for CPC-chitosan-mannitol, and 6.7 +/- 1.6 min for CPC-chitosan-mannitol-fiber. The latter three compositions were resistant to washout, whereas the CPC-control paste showed washout in a physiological solution. Immersion for 1 day dissolved mannitol and created macropores in CPC. CPC-chitosan-mannitol-fiber scaffold had a strength of 4.6 +/- 1.4 MPa, significantly higher than 1.2 +/- 0.1 MPa of CPC-chitosan-mannitol scaffold and 0.3 +/- 0.2 MPa of CPC-HPMC-mannitol scaffold (Tukey's). The strength of CPC-chitosan-mannitol-fiber scaffold was maintained up to 42 days and then decreased because of fiber degradation. Work-of-fracture and elastic modulus showed similar trends. Long cylindrical macropore channels were formed in CPC after fiber dissolution. The resorbable, fast-setting, anti-washout and strong CPC scaffold should be useful in craniofacial and orthopedic repairs. The novel method of combining fast- and slow-dissolution porogens/fibers to produce scaffolds with high strength and tailored macropore formation rates to match bone healing rates may have wide applicability to other biomaterials., (Copyright 2004 Wiley Periodicals, Inc. J Biomed Mater Res 68A: 725-734, 2004)