Jennifer L. Moreau

Mesenchymal stem cell proliferation and differentiation on an injectable calcium phosphate - chitosan composite scaffold

Calcium phosphate cement (CPC) can be molded or injected to form a scaffold in situ, has excellent osteoconductivity, and can be resorbed and replaced by new bone. However, its low strength limits CPC to non-stress-bearing repairs. Chitosan could be used to reinforce CPC, but mesenchymal stem cell (MSC) interactions with CPC-chitosan scaffold have not been examined. The objective of this study was to investigate MSC proliferation and osteogenic differentiation on high-strength CPC-chitosan scaffold. MSCs were harvested from rat bone marrow. At CPC powder/liquid (P/L) mass ratio of 2, flexural strength (mean+/-sd; n=5) was (10.0+/-1.1) MPa for CPC-chitosan, higher than (3.7+/-0.6) MPa for CPC (p<0.05). At P/L of 3, strength was (15.7+/-1.7)MPa for CPC-chitosan, higher than (10.2+/-1.8)MPa for CPC (p<0.05). Percentage of live MSCs attaching to scaffolds increased from 85% at 1 day to 99% at 14 days. There were (180+/-37) cells/mm(2) on scaffold at 1 day; cells proliferated to (1808+/-317) cells/mm(2) at 14 days. SEM showed MSCs with healthy spreading and anchored on nano-apatite crystals via cytoplasmic processes. Alkaline phosphatase activity (ALP) was (557+/-171) (pNPP mM/min)/(microg DNA) for MSCs on CPC-chitosan, higher than (159+/-47) on CPC (p<0.05). Both were higher than (35+/-32) of baseline ALP for undifferentiated MSCs on tissue-culture plastic (p<0.05). In summary, CPC-chitosan scaffold had higher strength than CPC. MSC proliferation on CPC-chitosan matched that of the FDA-approved CPC control. MSCs on the scaffolds differentiated down the osteogenic lineage and expressed high levels of bone marker ALP. Hence, the stronger CPC-chitosan scaffold may be useful for stem cell-based bone regeneration in moderate load-bearing maxillofacial and orthopedic applications.

Strong Nanocomposites with Ca, PO4, and F Release for Caries Inhibition

This article reviews recent studies on: (1) the synthesis of novel calcium phosphate and calcium fluoride nanoparticles and their incorporation into dental resins to develop nanocomposites; (2) the effects of key microstructural parameters on Ca, PO4, and F ion release from nanocomposites, including the effects of nanofiller volume fraction, particle size, and silanization; and (3) mechanical properties of nanocomposites, including water-aging effects, flexural strength, fracture toughness, and three-body wear. This article demonstrates that a major advantage of using the new nanoparticles is that high levels of Ca, PO4, and F release can be achieved at low filler levels in the resin, because of the high surface areas of the nanoparticles. This leaves room in the resin for substantial reinforcement fillers. The combination of releasing nanofillers with stable and strong reinforcing fillers is promising to yield a nanocomposite with both stress-bearing and caries-inhibiting capabilities, a combination not yet available in current materials.

Mechanical and acid neutralizing properties and bacteria inhibition of amorphous calcium phosphate dental nanocomposite

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.

Self-setting collagen-calcium phosphate bone cement: mechanical and cellular properties.

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.

Strength and fluoride release characteristics of a calcium fluoride based dental nanocomposite

Secondary caries and restoration fracture remain the two most common problems in restorative dentistry. Release of fluoride ions (F) could be a substantial benefit because F could enrich neighboring enamel or dentin to combat caries. The objective of this study was to incorporate novel CaF(2) nanoparticles into dental resin to develop stress-bearing, F-releasing nanocomposite. CaF(2) nanoparticles, prepared in our laboratories for the first time, were combined with reinforcing whisker fillers in a resin. Flexural strength (mean+/-sd; n=6) was 110+/-11 MPa for the composite containing 30% CaF(2) and 35% whiskers by mass. It matched the 108+/-19 MPa of a stress-bearing, non-releasing commercial composite (Tukeys at 0.05). The composite containing 20% CaF(2) had a cumulative F release of 2.34+/-0.26 mmol/L at 10 weeks. The initial F release rate was 2 microg/(hcm(2)), and the sustained release rate after 10 weeks was 0.29 microg/(hcm(2)). These values exceeded the reported releases of traditional and resin-modified glass ionomer materials. In summary, nanocomposites were developed with relatively high strength as well as sustained release of fluoride ions, a combination not available in current materials. These strong and F-releasing composites may yield restorations that can reduce the occurrence of both secondary caries and restoration fracture.

Novel CaF2 Nanocomposite with High Strength and Fluoride Ion Release

Secondary caries and restoration fracture remain common problems in dentistry. This study tested the hypothesis that combining nano-CaF2 and glass fillers would yield nanocomposites with high mechanical properties and F release. Novel CaF2 nanoparticles (56-nm) were synthesized via spray-drying and incorporated into resin. F release increased with increasing the nano-CaF2 content, or with decreasing pH (p < 0.05). F-release rates at 70-84 days were 1.13 µg/(cm2·day) and 0.50 µg/(cm2·day) for nanocomposites containing 30% and 20% nano-CaF2, respectively. They matched the 0.65 µg/(cm2·day) of resin-modified glass ionomer (p > 0.1). The nanocomposites had flexural strengths of 70-120 MPa, after 84-day immersion at pH 4, pH 5.5, and pH 7. These strengths were nearly three-fold that of resin-modified glass ionomer, and matched/exceeded a composite with little F release. In summary, novel CaF2 nanoparticles produced high F release at low filler levels, thereby making room in resin for reinforcement glass. This yielded nanocomposites with high F-release and stress-bearing properties, which may help reduce secondary caries and restoration fracture.

Long‐term mechanical durability of dental nanocomposites containing amorphous calcium phosphate nanoparticles

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.

Fluoride releasing restorative materials: Effects of pH on mechanical properties and ion release.

OBJECTIVES Secondary caries and restorative fracture are the two main reasons for restoration failures. Fluoride ion (F) release can help inhibit caries. Plaque pH after a sucrose rinse can decrease to a cariogenic pH of 4-4.5. The objective of this study was to investigate the effects of solution pH and immersion time on the mechanical properties and F release of restorative materials. METHODS Three resin-modified glass ionomers (Viremer, Fuji II LC, Ketac Nano), one compomer (Dyract Flow), and one composite (Heliomolar), were tested. Flexural strength and elastic modulus were measured before and after 84d of immersion in solutions of pH 4, 5.5, and 7. F release was measured as a function of pH and immersion time. RESULTS Immersion and material type had significant effects on mechanical properties. Vitremer had a flexural strength (mean±sd; n=6) of 99±25MPa before immersion; it decreased to 32±9MPa after 84d of immersion (p<0.05). In comparison, Heliomolar had a smaller strength loss, decreasing from 99±9MPa to 65±7MPa (p<0.05). Solution pH had little effect on mechanical properties. For example, Fuji II LC had a strength of 63±15MPa at pH 4, similar to 61±30MPa at pH 5.5, and 56±22MPa at pH 7 (p>0.1). In contrast, solution pH had a significant effect on F release. F release at 84d for Fuji was 609±25μg/cm(2) at pH 4, much higher than 258±36μg/cm(2) at pH 5.5, and 188±9μg/cm(2) at pH 7. SIGNIFICANCE The restoratives tested were able to greatly increase the F release at acidic, cariogenic pH, when these ions are most needed to inhibit caries. However, mechanical properties of these F-releasing restoratives degraded significantly in immersion. Efforts are needed to develop F-releasing restoratives with high levels of sustained F release, as well as improved durability of mechanical properties for large stress-bearing restorations.

Nanocomposite containing CaF2 nanoparticles: Thermal cycling, wear and long-term water-aging

OBJECTIVES Fluoride (F) releasing dental restoratives are promising to promote remineralization and combat caries. The objectives of this study were to develop nanocomposite containing calcium fluoride nanoparticles (nCaF(2)), and to investigate the long-term mechanical durability including wear, thermal-cycling and long-term water-aging behavior. METHODS Two types of fillers were used: nCaF(2) with a diameter of 53 nm, and glass particles of 1.4 μm. Four composites were fabricated with fillers of: (1) 0% nCaF(2)+65% glass; (2) 10% nCaF(2)+55% glass; (3) 20% nCaF(2)+45% glass; (4) 30% nCaF(2)+35% glass. Three commercial materials were also tested. Specimens were subjected to thermal-cycling between 5°C and 60°C for 10(5) cycles, three-body wear for 4×10(5) cycles, and water-aging for 2 years. RESULTS After thermal-cycling, the nCaF(2) nanocomposites had flexural strengths in the range of 100-150 MPa, five times higher than the 20-30 MPa for resin-modified glass ionomer (RMGI). The wear scar depth showed an increasing trend with increasing nCaF(2) filler level. Wear of nCaF(2) nanocomposites was within the range of wear for commercial controls. Water-aging decreased the strength of all materials. At 2 years, flexural strength was 94 MPa for nanocomposite with 10% nCaF(2), 60 MPa with 20% nCaF(2), and 48 MPa with 30% nCaF(2). They are 3-6 fold higher than the 15 MPa for RMGI (p<0.05). SEM revealed air bubbles and cracks in a RMGI, while composite control and nCaF(2) nanocomposites appeared dense and solid. SIGNIFICANCE Combining nCaF(2) with glass particles yielded nanocomposites with long-term mechanical properties that were comparable to those of a commercial composite with little F release, and much better than those of RMGI controls. These strong long-term properties, together with their F release being comparable to RMGI as previously reported, indicate that the nCaF(2) nanocomposites are promising for load-bearing and caries-inhibiting restorations.

Chapter 12 – Nano-Apatitic Composite Scaffolds for Stem Cell Delivery and Bone Tissue Engineering

The need for bone defect repair and regeneration arises from trauma, disease, congenital deformity, and tumor resection. Bone fracture occurs to seven million people each year in the United States, and musculoskeletal conditions cost