Biomedical subjects

Carl G Simon

Publications and source records attributed to Carl G Simon.

15 recordsLinked to original sources

PubMed2006

Strong calcium phosphate cement-chitosan-mesh construct containing cell-encapsulating hydrogel beads for bone tissue engineering.

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.

Alginates↗

PubMed2006

Premixed calcium phosphate cements: synthesis, physical properties, and cell cytotoxicity.

OBJECTIVES: Calcium phosphate cement (CPC) is a promising material for dental, periodontal, and craniofacial repairs. However, its use requires on-site powder-liquid mixing that increases the surgical placement time and raises concerns of insufficient and inhomogeneous mixing. The objective of this study was to determine a formulation of premixed CPC (PCPC) with rapid setting, high strength, and good in vitro cell viability. METHODS: PCPCs were formulated from CPC powder+non-aqueous liquid+gelling agent+hardening accelerator. Five PCPCs were thus developed: PCPC-Tartaric, PCPC-Malonic, PCPC-Citric, PCPC-Glycolic, and PCPC-Malic. Formulations and controls were compared for setting time, diametral tensile strength, and osteoblast cell compatibility. RESULTS: Setting time (mean+/-S.D.; n=4) for PCPC-Tartaric was 8.2+/-0.8 min, significantly less than the 61.7+/-1.5 min for the Premixed Control developed previously (p<0.001). On 7th day immersion, the diametral tensile strength of PCPC-Tartaric reached 6.5+/-0.8 MPa, higher than 4.5+/-0.8 MPa of Premixed Control (p=0.036). Osteoblast cells displayed a polygonal morphology and attached to the nano-hydroxyapatite crystals in the PCPCs. All cements had similar live cell density values (p=0.126), indicating that the new PCPCs were as cell compatible as a non-premixed CPC control known to be biocompatible. Each of the new PCPCs had a cell viability that was not significantly different (p>0.1) from that of the non-premixed CPC control. SIGNIFICANCE: PCPCs will eliminate the powder-liquid mixing during surgery and may also improve the cement performance. The new PCPCs supported cell attachment and yielded a high cell density and viability. Their mechanical strengths approached the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. These nano-crystalline hydroxyapatite cements may be useful in dental, periodontal, and craniofacial repairs.

3T3 Cells↗

PubMed2005

Cytotoxicity of three-dimensionally ordered macroporous sol-gel bioactive glass (3DOM-BG).

In this study, 80% SiO(2)-20% CaO (mole fraction) three-dimensionally ordered macroporous sol-gel bioactive glass (3DOM-BG, average pore size: 345 nm) particles were prepared and characterized. Since the 3DOM-BGs have a novel microstructure and ion-releasing profile, the cytotoxicity of 3DOM-BG particles was tested. The cytotoxicity tests were performed using MC3T3-E1 osteoblast-like cells: (1) Wst-1 assay for cell viability after culture in extracts from 3DOM-BG particles; (2) phase contrast microscopy for cell morphology after culture with 3DOM-BG particles; and (3) fluorescence microscopy for imaging cells cultured directly on 3DOM-BG particles. The results showed that 3DOM-BG particles were not cytotoxic, and that cells attached, spread and proliferated on and around 3DOM-BG particles.

3T3 Cells↗

PubMed2005

Fast setting calcium phosphate-chitosan scaffold: mechanical properties and biocompatibility.

Calcium phosphate cement (CPC) sets in situ to form hydroxyapatite and is highly promising for a wide range of clinical applications. However, its low strength limits its use to only non-stress applications, and its lack of macroporosity hinders cell infiltration, bone ingrowth and implant fixation. The aim of this study was to develop strong and macroporous CPC scaffolds by incorporating chitosan and water-soluble mannitol, and to examine the biocompatibility of the new graft with an osteoblast cell line and an enzymatic assay. Two-way ANOVA identified significant effects on mechanical properties from chitosan reinforcement and powder:liquid ratio (p<0.001). The flexural strength of CPC-chitosan composite at a powder:liquid ratio of 2 was (13.6+/-1.2) MPa, which was significantly higher than (3.2+/-0.6) MPa for CPC control without chitosan (Tukey's at 0.95). At a powder:liquid ratio of 3.5, CPC-chitosan had a strength of (25.3+/-2.9) MPa, which was significantly higher than (10.4+/-1.7) MPa for CPC control. The scaffolds possessed total pore volume fractions ranging from 42.0% to 80.0%, and macroporosity up to 65.5%. At total porosities of 52.2-75.2%, the scaffold had strength and elastic modulus values similar to those of sintered porous hydroxyapatite and cancellous bone. Osteoblast mouse cells (MC3T3-E1) were able to adhere, spread and proliferate on CPC-chitosan specimens. The cells, which ranged from about 20 to 50 microm including the cytoplasmic extensions, infiltrated into the 165-271 microm macropores of the scaffold. In summary, substantial reinforcement and macroporosity were imparted to a moldable, fast-setting, biocompatible, and resorbable hydroxyapatite graft. The highly porous scaffold may facilitate bone ingrowth and implant fixation in vivo. In addition, the two to three times increase in strength may help extend the use of CPC to larger repairs in moderately stress-bearing locations.

Absorbable Implants↗

PubMed2005

Premixed rapid-setting calcium phosphate composites for bone repair.

Although calcium phosphate cement (CPC) is promising for bone repair, its clinical use requires on site powder-liquid mixing. To shorten surgical time and improve graft properties, it is desirable to develop premixed CPC in which the paste remains stable during storage and hardens only after placement into the defect. The objective of this study was to develop premixed CPC with rapid setting when immersed in a physiological solution. Premixed CPCs were formulated using the following approach: Premixed CPC = CPC powder + nonaqueous liquid + gelling agent + hardening accelerator. Three premixed CPCs were developed: CPC-monocalcium phosphate monohydrate (MCPM), CPC-chitosan, and CPC-tartaric. Setting time for these new premixed CPCs ranged from 5.3 to 7.9 min, significantly faster than 61.7 min for a premixed control CPC reported previously (p < 0.05). SEM revealed the formation of nano-sized needle-like hydroxyapatite crystals after 1 d immersion and crystal growth after 7 d. Diametral tensile strength for premixed CPCs at 7 d ranged from 2.8 to 6.4 MPa, comparable to reported strengths for cancellous bone and sintered porous hydroxyapatite implants. Osteoblast cells attained a normal polygonal morphology on CPC-MCPM and CPC-chitosan with cytoplasmic extensions adhering to the nano-hydroxyapatite crystals. In summary, fast-setting premixed CPCs were developed to avoid the powder-liquid mixing in surgery. The pastes hardened rapidly once immersed in physiological solution and formed hydroxyapatite. The cements had strengths matching those of cancellous bone and sintered porous hydroxyapatite and non-cytotoxicity similar to conventional non-premixed CPC.

3T3 Cells↗

PubMed2005

Combinatorial screening of cell proliferation on poly(L-lactic acid)/poly(D,L-lactic acid) blends.

We have combined automated fluorescence microscopy with a combinatorial approach for creating polymer blend gradients to yield a rapid screening method for characterizing cell proliferation on polymer blends. A gradient in polymer blend composition of poly(L-lactic acid) (PLLA) and poly(D,L-lactic acid) (PDLLA) was created in the form of a strip-shaped film and was annealed to allow PLLA to crystallize. Fourier transform infrared (FTIR) microspectroscopy was used to determine the composition in the gradients and atomic force microscopy was used to characterize surface topography. Osteoblasts were cultured on the gradients and proliferation was assessed by automated counting of cells using fluorescence microscopy. Surface roughness varied with composition, was smooth on PDLLA-rich regions and was rough on the PLLA-rich regions. Cell adhesion was similar on all regions of the gradients while proliferation was faster on the smooth, PDLLA-rich end of the gradients than on the rough, PLLA-rich end of the gradients. These results demonstrate the feasibility of a new, combinatorial approach for evaluating cell proliferation on polymer blends.

3T3 Cells↗

PubMed2004

Self-hardening calcium phosphate cement-mesh composite: reinforcement, macropores, and cell response.

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.

Biocompatible Materials↗

PubMed2004

Quantification of inflammatory cellular responses using real-time polymerase chain reaction.

The introduction of tissue engineering strategies for the repair and replacement of human body components extends the application and importance of biomaterials. Implanted biomaterials frequently evoke inflammatory responses that are complex and not well understood at present. The goals of this work were to develop improved measurement methods for the quantification of cellular inflammatory responses to biomaterials and obtain data that lead to an enhanced understanding of the ways in which the body responds to the introduction of biomaterials. To evaluate the biocompatibility of materials, we established a system that allows for the analysis and quantitation of cellular inflammatory responses in vitro. In this study, the inflammatory responses of murine macrophages (RAW 264.7) were analyzed. The cells were incubated with polymethylmethacrylate (PMMA) microspheres in the presence and absence of lipopolysaccharide (LPS) at 8 and 18 h. The analysis of the genetic material obtained from the cells was quantitated using real-time reverse transcription polymerase chain reaction (RT-PCR). The cell populations treated with LPS or PMMA microspheres singly resulted in an elevation of cytokine levels compared to the untreated control. LPS resulted in a 258-fold increase, while PMMA resulted in an 87.9-fold increase at 8 h. RAW 264.7 cells incubated with LPS and PMMA particles demonstrated a synergistic effect by producing a marked increase in the level of cytokine expression, 336-fold greater than that of the untreated control at 8 h. Fluorescence microscopy studies that assessed cellular viability were also performed and are consistent with the RT-PCR results.

Animals↗

PubMed2004

Cell seeding into calcium phosphate cement.

To improve the effectiveness of calcium phosphate cement (CPC), we have developed a method to seed osteoblasts into the cement. CPC powder is mixed with water to form a paste that can be shaped to fit a bone defect in situ. The paste hardens in 30 min, reacts to form hydroxyapatite, and is replaced with new bone. Reacted CPC is biocompatible but unreacted CPC paste was found to have toxic effects when placed on cell monolayers (MC3T3-E1 cells). In contrast, when cells were indirectly exposed to CPC paste using a porous membrane or by placing a coverslip containing adherent cells onto a bed of CPC paste, the unreacted CPC was nontoxic. These results suggested that gel encapsulation of the cells might protect them from the CPC paste. Thus, cells were encapsulated in alginate beads (3.6-mm diameter), mixed with CPC paste, and incubated overnight. Both vital staining (calcein-AM and ethidium homodimer-1) and the Wst-1 assay (measures dehydrogenase activity) showed that cell survival in alginate beads that were mixed with CPC was similar to survival in untreated control beads. These results suggest that gel encapsulation could be used as a mechanism to protect cells for seeding into CPC.

Alginates↗

PubMed2004

Strong and bioactive composites containing nano-silica-fused whiskers for bone repair.

Self-hardening calcium phosphate cement (CPC) sets to form hydroxyapatite with high osteoconductivity, but its brittleness and low strength limit its use to only non-stress bearing locations. Previous studies developed bioactive composites containing hydroxyapatite fillers in Bis-GMA-based composites for bone repair applications, and they possessed higher strength values. However, these strengths were still lower than the strength of cortical bone. The aim of this study was to develop strong and bioactive composites by combining CPC fillers with nano-silica-fused whiskers in a resin matrix, and to characterize the mechanical properties and cell response. Silica particles were fused to silicon carbide whiskers to roughen the whisker surfaces for enhanced retention in the matrix. Mass ratios of whisker:CPC of 1:2, 1:1 and 2:1 were incorporated into a Bis-GMA-based resin and hardened by two-part chemical curing. Composite with only CPC fillers without whiskers served as a control. The specimens were tested using three-point flexure and nano-indentation. Composites with whisker:CPC ratios of 2:1 and 1:1 had flexural strengths (mean+/-SD; n=9) of (164+/-14) MPa and (139+/-22) MPa, respectively, nearly 3 times higher than (54+/-5) MPa of the control containing only CPC fillers (p<0.05). The strength of the new whisker-CPC composites was 3 times higher than the strength achieved in previous studies for conventional bioactive composites containing hydroxyapatite particles in Bis-GMA-based resins. The mechanical properties of the CPC-whisker composites nearly matched those of cortical bone and trabecular bone. Osteoblast-like cell adhesion, proliferation and viability were equivalent on the non-whisker control containing only CPC fillers, on the whisker composite at whisker:CPC of 1:1, and on the tissue culture polystyrene control, suggesting that the new CPC-whisker composite was non-cytotoxic.

3T3 Cells↗

PubMed2004

Self-hardening calcium phosphate composite scaffold for bone tissue engineering.

Calcium phosphate cement (CPC) sets 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 craniofacial and orthopaedic repairs. However, its low strength and lack of macroporosity limit its use. This study investigated CPC reinforcement with absorbable fibers, the effects of fiber volume fraction on mechanical properties and macroporosity, and the cytotoxicity of CPC-fiber composite. The rationale was that large-diameter absorbable fibers would initially strengthen the CPC graft, then dissolve to form long cylindrical macropores for colonization by osteoblasts. Flexural strength, work-of-fracture (toughness), and elastic modulus were measured vs. fiber volume fraction from 0% (CPC Control without fibers) to 60%. Cell culture was performed with osteoblast-like cells, and cell viability was quantified using an enzymatic assay. Flexural strength (mean+/-SD; n=6) of CPC with 60% fibers was 13.5+/-4.4 MPa, three times higher than 3.9+/-0.5 MPa of CPC Control. Work-of-fracture was increased by 182 times. Long cylindrical macropores 293+/-46 microm in diameter were created in CPC after fiber dissolution, and the CPC-fiber scaffold reached a macroporosity of 55% and a total porosity of 81%. The new CPC-fiber formulation supported cell adhesion, proliferation and viability. The method of using large-diameter absorbable fibers in bone graft for mechanical properties and formation of long cylindrical macropores for bone ingrowth may be applicable to other tissue engineering materials.

Animals↗

PubMed2002

Co-extrusion of biocompatible polymers for scaffolds with co-continuous morphology.

A methodology for the preparation of porous scaffolds for tissue engineering using co-extrusion is presented. Poly(epsilon-caprolactone) is blended with poly(ethylene oxide) in a twinscrew extruder to form a two-phase material with micron-sized domains. Selective dissolution of the poly(ethylene oxide) with water results in a porous material. A range of blend volume fractions results in co-continuous networks of polymer and void spaces. Annealing studies demonstrate that the characteristic pore size may be increased to larger than 100 microm. The mechanical properties of the scaffolds are characterized by a compressive modulus on the order of 1 MPa at low strains but displaying a marked strain-dependence. The results of osteoblast seeding suggest it is possible to use co-extrusion to prepare polymer scaffolds without the introduction of toxic contaminants. Polymer co-extrusion is amenable to both laboratory- and industrial-scale production of scaffolds for tissue engineering and only requires rheological characterization of the blend components. This method leads to scaffolds that have continuous void space and controlled characteristic length scales without the use of potentially toxic organic solvents.

Algorithms↗

PubMed2002

Preliminary report on the biocompatibility of a moldable, resorbable, composite bone graft consisting of calcium phosphate cement and poly(lactide-co-glycolide) microspheres.

We have assessed the biocompatibility of a new composite bone graft consisting of calcium phosphate cement (CPC) and poly(lactide-co-glycolide) (PLGA) microspheres (approximate diameter of 0.18-0.36 mm) using cell culture techniques. CPC powder is mixed with PLGA microspheres and water to yield a workable paste that could be sculpted to fit the contours of a wound. The cement then hardens into a matrix of hydroxyapatite microcrystals containing PLGA microspheres. The rationale for this design is that the microspheres will initially stabilize the graft but can then degrade to leave behind macropores for colonization by osteoblasts. The CPC matrix could then be resorbed and replaced with new bone. In the present study, osteoblast-like cells (MC3T3-E1 cells) were seeded onto graft specimens and evaluated with fluorescence microscopy, environmental scanning electron microscopy and the Wst-1 assay (an enzymatic assay for mitochondrial dehydrogenase activity). Cells were able to adhere, attain a normal morphology, proliferate and remain viable when cultured on the new composite graft (CPC-PLGA) or on a control graft (CPC alone). These results suggest that our new cement consisting of CPC and PLGA microspheres is biocompatible. This is the first time that a 'polymer-in-mineral' (PLGA microspheres dispersed in a CPC matrix) cement has been formulated that is moldable, resorbable and that can form macropores after the cement has set.

Animals↗

PubMedDate not supplied

High-throughput investigation of osteoblast response to polymer crystallinity: influence of nanometer-scale roughness on proliferation.

A high-throughput method for analyzing cellular response to crystallinity in a polymer material is presented. Variations in crystallinity lead to changes in surface roughness on nanometer length scales, and it is shown that cells are exquisitely sensitive to these changes. Gradients of polymer crystallinity were fabricated on films of poly(L-lactic acid) using a gradient in annealing temperature. The resultant morphologies were characterized using an atomic force microscope. Root-mean-square (rms) roughness values ranging from 0.5 to 13 nm were created on a single sample. MC3T3-E1 osteoblastic cells were cultured for 1, 3 and 5 d, and the number of cells was measured using automated fluorescence microscopy. It is shown that the rate of proliferation on the smooth regions of the films is much greater than that on the rough regions, and a monotonic variation in rate is observed as a function of roughness. The critical rms roughness, above which a statistically significant reduction in rate of proliferation occurs, was approximately 1.1 nm. Fluorescence microscopy measurements on immunostained cells indicate there is no significant change in cell area, the number or type of adhesions formed, or the degree of actin polymerization. Results from enzyme-linked immunofluorescence assays indicated that there was no detectable change in adhesion protein accessibility, suggesting the cells directly respond to substrate topography. The use of the gradient library approach yielded the functional dependence of cell proliferation on nanometer-scale roughness and gave a sensitive estimate of the critical roughness for which a decrease in proliferation is observed.

3T3 Cells↗

PubMedDate not supplied

In situ formation of blends by photopolymerization of poly(ethylene glycol) dimethacrylate and polylactide.

Blends of cross-linked poly(ethylene glycol) dimethacrylate (PEGDMA) and poly(d,l-lactide) (PLA) were prepared by mixing photoactive PEGDMA (molecular mass: 875 g/mol) and PLA, and subsequently photopolymerizing the mixture with visible light. The effects of PLA molecular mass and mass fraction on the rheological properties of the PEGDMA/PLA mixtures, and on the degree of methacrylate vinyl conversion (DC), as well as blend miscibility, microstructure, mechanical properties, in vitro swelling behavior, and cell responses were studied. PLA-2K (molecular mass: 2096 g/mol) and PLA-63K (molecular mass: 63 000 g/mol) formed miscible and partially miscible blends with cross-linked PEGDMA, respectively. The addition of the PLA-2K did not affect the immediate or post-cure (>24 h) DC of the PEGDMA upon photopolymerization. However, the addition of PLA-63K decreased the immediate DC of the PEGDMA, which can be increased through extending the curing time or post-curing period. Compared to the cross-linked neat PEGDMA and PLA-2K/PEGDMA blends, PLA-63K/PEGDMA blends were significantly stronger, stiffer, and tougher. Both types of blends and the cross-linked PEGDMA swelled when soaked in a phosphate buffered saline (PBS) solution. The attachment and spreading of MCT3-E1 cells increased with increasing PLA-63K content in the blends. The facile and rapid formation of PEGDMA/PLA blends by photopolymerization represents a simple and efficient approach to a class of biomaterials with a broad spectrum of properties.

Acrylates↗