Hockin H.K. Xu

An injectable calcium phosphate-alginate hydrogel-umbilical cord mesenchymal stem cell paste for bone tissue engineering.

The need for bone repair has increased as the population ages. Stem cell-scaffold approaches hold immense promise for bone tissue engineering. However, currently, preformed scaffolds for cell delivery have drawbacks including the difficulty to seed cells deep into the scaffold, and inability for injection in minimally-invasive surgeries. Current injectable polymeric carriers and hydrogels are too weak for load-bearing orthopedic applications. The objective of this study was to develop an injectable and mechanically-strong stem cell construct for bone tissue engineering. Calcium phosphate cement (CPC) paste was combined with hydrogel microbeads encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs). The hUCMSC-encapsulating composite paste was fully injectable under small injection forces. Cell viability after injection matched that in hydrogel without CPC and without injection. Mechanical properties of the construct matched the reported values of cancellous bone, and were much higher than previous injectable polymeric and hydrogel carriers. hUCMSCs in the injectable constructs osteodifferentiated, yielding high alkaline phosphatase, osteocalcin, collagen type I, and osterix gene expressions at 7 d, which were 50-70 fold higher than those at 1 d. Mineralization by the hUCMSCs at 14 d was 100-fold that at 1 d. In conclusion, a fully injectable, mechanically-strong, stem cell-CPC scaffold construct was developed. The encapsulated hUCMSCs remained viable, osteodifferentiated, and synthesized bone minerals. The new injectable stem cell construct with load-bearing capability may enhance bone regeneration in minimally-invasive and other orthopedic surgeries.

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.

Antibacterial amorphous calcium phosphate nanocomposites with a quaternary ammonium dimethacrylate and silver nanoparticles

OBJECTIVES Calcium and phosphate ion-releasing resin composites are promising for remineralization. However, there has been no report on incorporating antibacterial agents to these composites. The objective of this study was to develop antibacterial and mechanically strong nanocomposites incorporating a quaternary ammonium dimethacrylate (QADM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP). METHODS The QADM, bis(2-methacryloyloxyethyl) dimethylammonium bromide (ionic dimethacrylate-1), was synthesized from 2-(N,N-dimethylamino)ethyl methacrylate and 2-bromoethyl methacrylate. NAg was synthesized by dissolving Ag 2-ethylhexanoate salt in 2-(tert-butylamino)ethyl methacrylate. Mechanical properties were measured in three-point flexure with bars of 2 mm×2 mm×25 mm (n=6). Composite disks (diameter=9 mm, thickness=2 mm) were inoculated with Streptococcus mutans. The metabolic activity and lactic acid production of biofilms were measured (n=6). Two commercial composites were used as controls. RESULTS Flexural strength and elastic modulus of NACP+QADM, NACP+NAg, and NACP+QADM+NAg matched those of commercial composites with no antibacterial property (p>0.1). The NACP+QADM+NAg composite decreased the titer counts of adherent S. mutans biofilms by an order of magnitude, compared to the commercial composites (p<0.05). The metabolic activity and lactic acid production of biofilms on NACP+QADM+NAg composite were much less than those on commercial composites (p<0.05). Combining QADM and NAg rendered the nanocomposite more strongly antibacterial than either agent alone (p<0.05). SIGNIFICANCE QADM and NAg were incorporated into calcium phosphate composite for the first time. NACP+QADM+NAg was strongly antibacterial and greatly reduced the titer counts, metabolic activity, and acid production of S. mutans biofilms, while possessing mechanical properties similar to commercial composites. These nanocomposites are promising to have the double benefits of remineralization and antibacterial capabilities to inhibit dental caries.

Calcium phosphate cement containing resorbable fibers for short-term reinforcement and macroporosity

Calcium phosphate cement (CPC) sets to form hydroxyapatite and has been used in medical and dental procedures. However, the brittleness and low strength of CPC prohibit its use in many stress-bearing locations, unsupported defects, or reconstruction of thin bones. Recent studies incorporated fibers into CPC to improve its strength. In the present study, a novel methodology was used to combine the reinforcement with macroporosity: large-diameter resorbable fibers were incorporated into CPC to provide short-term strength, then dissolved to create macropores suitable for bone ingrowth. Two types of resorbable fibers with 322 microm diameters were mixed with CPC to a fiber volume fraction of 25%. The set specimens were immersed in saline at 37 degrees C for 1, 7, 14, 28 and 56d, and were then tested in three-point flexure. SEM was used to examine crack-fiber interactions. CPC composite achieved a flexural strength 3 times, and work-of-fracture (toughness) nearly 100 times, greater than unreinforced CPC. The strength and toughness were maintained for 2-4 weeks of immersion, depending on fiber dissolution rate. Macropores or channels were observed in CPC composite after fiber dissolution. In conclusion, incorporating large-diameter resorbable fibers can achieve the needed short-term strength and fracture resistance for CPC while tissue regeneration is occurring, then create macropores suitable for vascular ingrowth when the fibers are dissolved. The reinforcement mechanisms appeared to be crack bridging and fiber pullout, the mechanical properties of the CPC matrix also affected the composite properties.

Strong and macroporous calcium phosphate cement: Effects of porosity and fiber reinforcement on mechanical properties†‡

Because of its excellent osteoconductivity and bone-replacement capability, self-setting calcium phosphate cement (CPC) has been used in a number of clinical procedures. For more rapid resorption and concomitant osseointegration, methods were desired to build macropores into CPC; however, this decreased its mechanical properties. The aims of this study, therefore, were to use fibers to strengthen macroporous CPC and to investigate the effects of the pore volume fraction on its mechanical properties. Water-soluble mannitol crystals were incorporated into CPC paste; the set CPC was then immersed in water to dissolve mannitol, producing macropores. Mannitol/(mannitol + CPC powder) mass fractions of 0, 10, 20, 30, and 40% were used. An aramid fiber volume fraction of 6% was incorporated into the CPC-mannitol specimens, which were set in 3 mm x 4 mm x 25 mm molds and then fractured in three-point flexure to measure the strength, work of fracture, and modulus. The dissolution of mannitol created well-formed macropores, with CPC at 40% mannitol having a total porosity of a 70.8% volume fraction. Increasing the mannitol content significantly decreased the properties of CPC without fibers (analysis of variance; p < 0.001). The strength (mean +/- standard deviation; n = 6) of CPC at 0% mannitol was 15.0 +/- 1.8 MPa; at 40% mannitol, it decreased to 1.4 +/- 0.4 MPa. Fiber reinforcement improved the properties, with the strength increasing threefold at 0% mannitol, sevenfold at 30% mannitol, and nearly fourfold at 40% mannitol. The work of fracture increased by 2 orders of magnitude, but the modulus was not changed as a result of fiber reinforcement. A scanning electron microscopy examination of specimens indicated crack deflection and bridging by fibers, matrix multiple cracking, and frictional pullout of fibers as the reinforcement mechanisms. Macroporous CPCs were substantially strengthened and toughened via fiber reinforcement. This may help extend the use of CPCs with macropores for bony ingrowth to the repair of larger defects in stress-bearing locations.

Bone tissue engineering via nanostructured calcium phosphate biomaterials and stem cells

Tissue engineering is promising to meet the increasing need for bone regeneration. Nanostructured calcium phosphate (CaP) biomaterials/scaffolds are of special interest as they share chemical/crystallographic similarities to inorganic components of bone. Three applications of nano-CaP are discussed in this review: nanostructured calcium phosphate cement (CPC); nano-CaP composites; and nano-CaP coatings. The interactions between stem cells and nano-CaP are highlighted, including cell attachment, orientation/morphology, differentiation and in vivo bone regeneration. Several trends can be seen: (i) nano-CaP biomaterials support stem cell attachment/proliferation and induce osteogenic differentiation, in some cases even without osteogenic supplements; (ii) the influence of nano-CaP surface patterns on cell alignment is not prominent due to non-uniform distribution of nano-crystals; (iii) nano-CaP can achieve better bone regeneration than conventional CaP biomaterials; (iv) combining stem cells with nano-CaP accelerates bone regeneration, the effect of which can be further enhanced by growth factors; and (v) cell microencapsulation in nano-CaP scaffolds is promising for bone tissue engineering. These understandings would help researchers to further uncover the underlying mechanisms and interactions in nano-CaP stem cell constructs in vitro and in vivo, tailor nano-CaP composite construct design and stem cell type selection to enhance cell function and bone regeneration, and translate laboratory findings to clinical treatments.

Synergistic reinforcement of in situ hardening calcium phosphate composite scaffold for bone tissue engineering

Calcium phosphate cement (CPC) hardens in situ to form solid hydroxyapatite, can conform to complex cavity shapes without machining, has excellent osteoconductivity, and is able to be resorbed and replaced by new bone. Therefore, CPC is promising for use in craniofacial and orthopaedic repairs. However, the low strength and lack of macroporosity of CPC limit its use. The aim of the present study was to increase the strength and toughness of CPC while creating macropores suitable for cell infiltration and bone ingrowth, and to investigate the effects of chitosan and mesh reinforcement on the composite properties. Specimens were self-hardened in 3 mm x 4 mm x 25 mm molds, immersed in a physiological solution for 1-84 d, and tested in three-point flexure. After 1d, the unreinforced CPC control had a flexural strength (mean+/-s.d.; n=6) of (3.3+/-0.4)MPa. The incorporation of chitosan or mesh into CPC increased the strength to (11.9+/-0.8) and (21.3+/-2.7)MPa, respectively. The incorporation of both chitosan and mesh synergistically into CPC dramatically increased the strength to (43.2+/-4.1)MPa. The work-of-fracture (WOF) (toughness) was also increased by two orders of magnitude. After 84 d immersion in a simulated physiological solution, the meshes in CPC dissolved and formed interconnected cylindrical macropores. The novel CPC scaffold had a flexural strength 39% higher, and WOF 256% higher than the conventional CPC without macropores. The new composite had an elastic modulus within the range for cortical bone and cancellous bone, and a flexural strength higher than those for cancellous bone and sintered porous hydroxyapatite implants. In conclusion, combining two different reinforcing agents together in self-hardening CPC resulted in superior synergistic strengthening compared to the traditional use of a single reinforcing agent. The strong and macroprous CPC scaffold may be useful in stress-bearing craniofacial and orthopaedic repairs.

Reinforcement of a self‐setting calcium phosphate cement with different fibers

A water-based calcium phosphate cement (CPC) has been used in a number of medical and dental procedures due to its excellent osteoconductivity and bone replacement capability. However, the low tensile strength of CPC prohibits its use in many unsupported defects and stress-bearing locations. Little investigation has been carried out on the fiber reinforcement of CPC. The aims of the present study, therefore, were to examine whether fibers would strengthen CPC, and to investigate the effects of fiber type, fiber length, and volume fraction. Four different fibers were used: aramid, carbon, E-glass, and polyglactin. Fiber length ranged from 3-200 mm, and fiber volume fraction ranged from 1.9-9.5%. The fibers were mixed with CPC paste and placed into molds of 3 x 4 x 25 mm. A flexural test was used to fracture the set specimens and to measure the ultimate strength, work-of-fracture, and elastic modulus. Scanning electron microscopy was used to examine specimen fracture surfaces. Fiber type had significant effects on composite properties. The composite ultimate strength in MPa (mean +/- SD; n = 6) was (62+/-16) for aramid, (59+/-11) for carbon, (29+/-8) for E-glass, and (24+/-4) for polyglactin, with 5.7% volume fraction and 75 mm fiber length. In comparison, the strength of unreinforced CPC was (13+/-3). Fiber length also played an important role. For composites containing 5.7% aramid fibers, the ultimate strength was (24+/-3) for 3 mm fibers, (36+/-13) for 8 mm fibers, (48 +/-14) for 25 mm fibers, and (62+/-16) for 75 mm fibers. At 25 mm fiber length, the ultimate strength of CPC composite was found to be linearly proportional to fiber strength. In conclusion, a self-setting calcium phosphate cement was substantially strengthened via fiber reinforcement. Fiber length, fiber volume fraction, and fiber strength were found to be key microstructural parameters that controlled the mechanical properties of CPC composites.

The fast release of stem cells from alginate-fibrin microbeads in injectable scaffolds for bone tissue engineering

Stem cell-encapsulating hydrogel microbeads of several hundred microns in size suitable for injection, that could quickly degrade to release the cells, are currently unavailable. The objectives of this study were to: (1) develop oxidized alginate-fibrin microbeads encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs); (2) investigate microbead degradation, cell release, and osteogenic differentiation of the released cells for the first time. Three types of microbeads were fabricated to encapsulate hUCMSCs: (1) Alginate microbeads; (2) oxidized alginate microbeads; (3) oxidized alginate-fibrin microbeads. Microbeads with sizes of about 100-500 μm were fabricated with 1 × 10(6) hUCMSCs/mL of alginate. For the alginate group, there was little microbead degradation, with very few cells released at 21 d. For oxidized alginate, the microbeads started to slightly degrade at 14 d. In contrast, the oxidized alginate-fibrin microbeads started to degrade at 4 d and released the cells. At 7 d, the number of released cells greatly increased and showed a healthy polygonal morphology. At 21 d, the oxidized alginate-fibrin group had a live cell density that was 4-fold that of the oxidized alginate group, and 15-fold that of the alginate group. The released cells had osteodifferentiation, exhibiting highly elevated bone marker gene expressions of ALP, OC, collagen I, and Runx2. Alizarin staining confirmed the synthesis of bone minerals by hUCMSCs, with the mineral concentration at 21 d being 10-fold that at 7 d. In conclusion, fast-degradable alginate-fibrin microbeads with hUCMSC encapsulation were developed that could start to degrade and release the cells at 4 d. The released hUCMSCs had excellent proliferation, osteodifferentiation, and bone mineral synthesis. The alginate-fibrin microbeads are promising to deliver stem cells inside injectable scaffolds to promote tissue regeneration.

Anti-biofilm Dentin Primer with Quaternary Ammonium and Silver Nanoparticles

Antibacterial bonding agents could combat recurrent caries at the tooth-composite margins. The objectives of this study were to develop novel antibacterial dentin primers containing quaternary ammonium dimethacrylate (QADM) and nanoparticles of silver (NAg), and to investigate the effects on dentin bond strength and dental plaque microcosm biofilms for the first time. Scotchbond Multi-Purpose (“SBMP”) bonding agent was used. QADM and NAg were incorporated into SBMP primer, yielding 4 primers: SBMP primer (control), control + 10% QADM (mass), control + 0.05% NAg, and control + 10% QADM + 0.05% NAg. Human saliva was collected to grow microcosm biofilms. The NAg particle size (mean ± SD; n = 100) was 2.7 ± 0.6 nm. Dentin shear bond strengths (n = 10) with human third molars were approximately 30 MPa for all groups (p > 0.1). QADM-NAg-containing primer increased the bacteria inhibition zone by 9-fold, compared with control primer (p < 0.05). QADM-NAg-containing primer reduced lactic acid production and colony-forming units of total micro-organisms, total streptococci, and mutans streptococci by an order of magnitude. In conclusion, novel QADM-NAg-containing primers were strongly antibacterial without compromising dentin bond strength, and hence are promising to inhibit biofilms and secondary caries. The processing method of incorporating QADM and NAg together into the same primer produced the strongest antibacterial effect, which could have a wide applicability to other bonding systems.