Ethyl Alanato Research Articles

Molecular Mechanism Study on Effect of Biodegradable Amino Acid Ester-Substituted Polyphosphazenes in Stimulating Osteogenic Differentiation.

Amino acid ester substituted polyphosphazenes are osteoactive benefiting from their phosphorus-containing chemical structure, which highlights interests in bone tissue engineering. To correlate their chemical structures with cell activities, in this study, poly[(ethyl alanato)0.3 (ethyl glycinato)0.7 phosphazene] (PAGP) and poly[(ethyl phenylalanato)0.3 (ethyl glycinato)0.7 phosphazene] (PPGP) are synthesized to carry out studies on cell osteogenic differentiation. In the non-contact culture manner, bone mesenchymal stromal cells (BMSCs) are cultured in transwell chambers containing PAGP or PPGP films, while the cells and the materials do not contact. In the contact culture manner, BMSCs are cultured on the PAGP or PPGP films. In the meantime, solutions containing PAGP or PPGP degradation products (i.e., phosphate, ammonium, and corresponding amino acids) are applied for cell culture using inorganic phosphate (Pi) ion as control. Thus, the influences from substrate surface and degradation products can be identified separately. The results reveal that both the phosphorus-containing surface of PAGP and PPGP films and their degradation products play significant roles in regulating cell behaviors. In comparison with PAGP, PPGP seems able to provide relatively stable phosphorus-containing surface to strengthen the cell-scaffold interaction because of its slower degradation rate and higher Young's modulus, leading to greater promotion in osteogenic differentiation via contact effect.

Polyphosphazene functionalized polyester fiber matrices for tendon tissue engineering: in vitro evaluation with human mesenchymal stem cells

Poly[(ethyl alanato)1(p-methyl phenoxy)1] phosphazene (PNEA-mPh) was used to modify the surface of electrospun poly(ε-caprolactone) (PCL) nanofiber matrices having an average fiber diameter of 3000 ± 1700 nm for the purpose of tendon tissue engineering and augmentation. This study reports the effect of polyphosphazene surface functionalization on human mesenchymal stem cell (hMSC) adhesion, cell-construct infiltration, proliferation and tendon differentiation, as well as long term cellular construct mechanical properties. PCL fiber matrices functionalized with PNEA-mPh acquired a rougher surface morphology and led to enhanced cell adhesion as well as superior cell-construct infiltration when compared to smooth PCL fiber matrices. Long-term in vitro hMSC cultures on both fiber matrices were able to produce clinically relevant moduli. Both fibrous constructs expressed scleraxis, an early tendon differentiation marker, and a bimodal peak in expression of the late tendon differentiation marker tenomodulin, a pattern that was not observed in PCL thin film controls. Functionalized matrices achieved a more prominent tenogenic differentiation, possessing greater tenomodulin expression and superior phenotypic maturity according to the ratio of collagen I to collagen III expression. These findings indicate that PNEA-mPh functionalization is an efficient method for improving cell interactions with electrospun PCL matrices for the purpose of tendon repair.

Design and Optimization of Polyphosphazene Functionalized Fiber Matrices for Soft Tissue Regeneration

Electrospun polycaprolactone nanofiber matrices surface functionalized with poly[(ethyl alanato), (p-methyl phenoxy),] phosphazene were fabricated for the purpose of soft skeletal tissue regeneration. This preliminary study reports the effect of fiber diameter and polyphosphazene surface functionalization on significant scaffold properties such as morphology, surface hydrophilicity, porosity, tensile properties, human mesenchymal stem cell adhesion and proliferation. Six fiber matrices comprised of average fiber diameters in the range of 400-500, 900-1000, 1400-1500, 1900-2000, 2900-3000 and 3900-4000 nm were considered for primary evaluation. After achieving the greatest proliferation while maintaining moderate tensile modulus, matrices in the diameter range of 2900-3000 nm were selected to examine the effect of coating with 1%, 2% and 3% (weight/volume) polyphosphazene solutions. Polyphosphazene functionalization resulted in rougher surfaces that correlated with coating solution concentration. Analytical techniques such as energy dispersive X-ray analysis, Fourier transform infrared spectroscopy, elemental analysis, differential scanning calorimetry, water contact angle goniometry and confocal microscopy confirmed the presence of polyphosphazene and its distribution on the functionalized fiber matrices. Functionalization achieved through 2% polymer solutions did not affect average pore diameter, tensile modulus, suture retention strength or cell proliferation compared to PCL controls. Surface polyphosphazene functionalization significantly improved the matrix hydrophilicity evidenced through decreased water contact angle of PCL matrices from 130 degrees to 97 degrees. Further, enhanced total protein synthesis by cells during in vitro culture was seen on 2% PPHOS functionalized matrices over controls. Improving PCL matrix hydrophilicity via proposed surface functionalization may be an efficient method to improve cell-PCL matrix interactions.

Mechanical properties and osteocompatibility of novel biodegradable alanine based polyphosphazenes: Side group effects

The versatility of polymers for tissue regeneration lies in the feasibility to modulate the physical and biological properties by varying the side groups grafted to the polymers. Biodegradable polyphosphazenes are high-molecular-weight polymers with alternating nitrogen and phosphorus atoms in the backbone. This study is the first of its kind to systematically investigate the effect of side group structure on the compressive strength of novel biodegradable polyphosphazene based polymers as potential materials for tissue regeneration. The alanine polyphosphazene based polymers, poly(bis(ethyl alanato) phosphazene) (PNEA), poly((50% ethyl alanato) (50% methyl phenoxy) phosphazene) (PNEA 50mPh 50), poly((50% ethyl alanato) (50% phenyl phenoxy) phosphazene) (PNEA 50PhPh 50) were investigated to demonstrate their mechanical properties and osteocompatibility. Results of mechanical testing studies demonstrated that the nature and the ratio of the pendent groups attached to the polymer backbone play a significant role in determining the mechanical properties of the resulting polymer. The compressive strength of PNEA 50PhPh 50 was significantly higher than poly(lactide- co-glycolide) (85:15 PLAGA) ( p < 0.05). Additional studies evaluated the cellular response and gene expression of primary rat osteoblast cells on PNEA, PNEA 50mPh 50 and PNEA 50PhPh 50 films as candidates for bone tissue engineering applications. Results of the in vitro osteocompatibility evaluation demonstrated that cells adhere, proliferate, and maintain their phenotype when seeded directly on the surface of PNEA, PNEA 50mPh 50, and PNEA 50PhPh 50. Moreover, cells on the surface of the polymers expressed type I collagen, alkaline phosphatase, osteocalcin, osteopontin, and bone sialoprotein, which are characteristic genes for osteoblast maturation, differentiation, and mineralization.

The influence of side group modification in polyphosphazenes on hydrolysis and cell adhesion of blends with PLGA

Polyphosphazenes have been synthesized with tris(hydroxymethyl)amino methane (THAM) side groups and with co-substituents glycine ethyl ester and alanine ethyl ester. The THAM side group was linked to the polyphosphazene backbone via the amino function. The three pendent hydroxyl functions on each THAM side group were utilized for hydrogen bonding association with poly(glycolic–lactic acid) (PLGA). Co-substitution of the polyphosphazene with both THAM and glycine or alanine ethyl esters was employed to avoid the insolubility of the single-substituent THAM-substituted polyphosphazenes. Both poly[(tris(hydroxymethyl)aminomethane)(ethyl glycinato)phosphazene] and poly[(tris(hydroxymethyl)aminomethane)(ethyl alanato)phosphazene] (1:1 ratio of side groups) were blended with PLGA (50:50) or PLGA (85:15). DSC analysis indicated miscible blend formation, irrespective of the detailed molecular structure of the polyphosphazene or the composition of PLGA in the blend. Hydrolysis studies of the polyphosphazene:PLGA (50:50) blends indicated that the PLGA component hydrolyzed more rapidly than the polyphosphazene. Primary osteoblast cell studies showed good cell adhesion to the polymer blends during 14 days, but subsequent limited cell spreading due to increased surface roughness as the two polymers eroded at different rates.

Biodegradable Polyphosphazene-Nanohydroxyapatite Composite Nanofibers: Scaffolds for Bone Tissue Engineering

Bone is a natural composite comprised of hierarchically arranged collagen fibrils, hydroxyapatite and proteoglycans in the nanometer scale. This preliminary study reports the fabrication of biodegradable poly[bis(ethyl alanato)phosphazene]-nanohydroxyapatite (PNEA-nHAp) composite nanofiber matrices via electrospinning. Binary solvent compositions of THF and ethanol were used as a spinning solvent to attain better nanohydroxyapatite dispersibility in PNEA solution. These nanocomposites were characterized for morphology, nHAp distribution and content using spectroscopy and gravimetric estimations. Composite nanofibers fabricated in the diameter range of 100-310 nm could encapsulate 20-40 nm nHAp crystals. A better composite nanofiber yield was obtained for 50% (w/w) nHAp experimental loadings. Incremental experimental loading beyond 60% (w/w) hindered electrospinning due to polymer-nHAp phase separation. Composites nanofibers had a rougher surface and nodules along the length of the fibers suggesting nHAp encapsulation. Further, characterization via energy dispersive X-ray spectroscopy and X-ray mapping confirmed the nHAp encapsulation. Providing cells with a natural bone like environment with a fibrillar structure and natural hydroxyapatite can enhance bone tissue regeneration/repair.

Formation and properties of composites comprised of calcium-deficient hydroxyapatites and ethyl alanate polyphosphazenes

Composites comprised of calcium-deficient hydroxyapatite (HAp) and biodegradable polyphosphazenes were formed via cement-type reactions at physiologic temperature. The composite precursors were produced by blending particulate hydroxyapatite precursors with 10 wt% polymer using a solvent/non-solvent technique. HAp precursors having calcium-to-phosphate ratios of 1.5 (CDH) and 1.6 (CDS) were used. The polymeric constituents were poly[bis(ethyl alanato)phosphazene] (PNEA) and poly[(ethyl alanato)(1) (p-phenylphenoxy)(1) phosphazene] (PNEA(50)PhPh(50)). The effect of incorporating the phenyl phenoxy group was evaluated as a means of increasing the mechanical properties of the composites without retarding the rates of HAp formation. Reaction kinetics and mechanistic paths were characterized by pH determination, X-ray diffraction analyses, scanning electron microscopy, and infrared spectroscopy. The mechanical properties were analyzed by compression testing. These analyses indicated that the presence of the polymers slightly reduced the rate HAp formation. However, surface hydrolysis of polymer ester groups permitted the formation of calcium salt bridges that provide a mechanism for bonding with the HAp. The compressive strengths of the composites containing PNEA(50)PhPh(50) were superior to those containing PNEA, and were comparable to those of HAp produced in the absence of polymer. The current composites more closely match the structure of bone, and are thus strongly recommended to be used as bone cements where high loads are not expected.

Miscibility and in vitro osteocompatibility of biodegradable blends of poly[(ethyl alanato) ( p-phenyl phenoxy) phosphazene] and poly(lactic acid-glycolic acid)

Previously we demonstrated the ability of ethyl glycinato substituted polyphosphazenes to neutralize the acidic degradation products and control the degradation rate of poly(lactic acid-glycolic acid) (PLAGA) by blending. In this study, blends of high strength poly[(50% ethyl alanato) (50% p-phenyl phenoxy) phosphazene] (PNEA 50PhPh 50) and 85:15 PLAGA were prepared using a mutual solvent approach. Three different solvents, methylene chloride (MC), chloroform (CF) and tetrahydrofuran (THF) were studied to investigate solvent effects on blend miscibility. Three different blends were then fabricated at various weight ratios namely 25:75 (BLEND25), 50:50 (BLEND50), and 75:25 (BLEND75) using THF as the mutual solvent. The miscibility of the blends was evaluated by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). Among these, BLEND25 was miscible while BLEND50 and BLEND75 were partially miscible. Furthermore, BLEND25 formed apatite layers on its surface as evidenced in a biomimetic study performed. These novel blends showed cell adhesion and proliferation comparable to PLAGA. However, the PNEA 50PhPh 50 component in the blends was able to increase the phenotypic expression and mineralized matrix synthesis of the primary rat osteoblasts (PRO) in vitro. Blends of high strength PNEA 50PhPh 50 and 85:15 PLAGA are promising biomaterials for a variety of musculoskeletal applications.

In Vitro Evaluation of Poly[Bis(Ethyl Alanato)Phosphazene] as a Scaffold for Bone Tissue Engineering

Polyphosphazenes with amino acid ester as side groups are biocompatible polymers that could provide valid scaffolds for cell growth. In the present study we investigate the adhesion and growth of osteoblasts obtained from rat bone marrow on matrices composed of thin fibers of poly[bis(ethyl alanato)phosphazene] (PAlaP), poly(d,l-lactic acid) (PDLLA), or PAlaP/PDLLA blend. Our data show that scaffolds of PAlaP or PAlaP/PDLLA blend enhanced the cell adhesion and growth in comparison with that observed in cultures seeded on polystyrene tissue culture plates. Although collagenase-digestible protein synthesis remained unchanged, all scaffolds induced a decrease in alkaline phosphatase activity, suggesting that osteoblasts are in the proliferation phase. Both PAlaP and PAlaP blended with PDLLA may represent a new and interesting substrate for bone tissue engineering.

Effect of Side Group Chemistry on the Properties of Biodegradable l-Alanine Cosubstituted Polyphosphazenes

Biodegradable polyphosphazenes have been investigated for a variety of applications, such as controlled drug delivery matrixes, tissue-engineering scaffolds, membranes, and bone-type composites. In this study we have evaluated the effect of side group chemistry on the properties of biodegradable phosphazene polymers that contain ethyl alanato side groups together with ethyl glycinato, p-methylphenoxy, or p-phenylphenoxy side groups. The polymers were synthesized by a macromolecular substitution route. The molecular weights of aryloxy/amino acid ester cosubstituted polymers were much higher than the amino acid ester substituted polyphosphazenes described earlier. Polymer properties, such as glass transition temperature, hydrolytic degradation, surface wettability, tensile strength, and modulus of elasticity varied over a wide range following changes to the type of co-substituents on the polymer backbone. The glass transition temperatures varied from -10 to 35 degrees C and increased with the bulkiness of the side groups. Polymer films in phosphate buffer saline solution showed molecular weight declines ranging from 58% to >80% and mass loss ranging from 4% to 90% over a period of 7 weeks. Water contact angles for polymer films varied from 63 degrees to 107 degrees , with the highest angles for the alanine ethyl ester and p-phenylphenoxy cosubstituted polyphosphazene. The tensile strengths were in the range of 2.4-7.6 MPa and the modulus of elasticity was in the range of 31.4-455.9 MPa. Thus, in this study we have demonstrated the tunability of biodegradable polyphosphazenes to suit a range of biomedical applications.

Synthesis, characterization, and osteocompatibility evaluation of novel alanine‐based polyphosphazenes

This study deals with the synthesis and in vitro osteocompatibility evaluation of two novel alanine-containing biodegradable ester polyphosphazenes as candidates to form self-setting composites with hydroxyapatite (HAp) precursors. The two novel biodegradable polyphosphazenes synthesized were poly[(ethyl alanato)1.0(ethyl oxybenzoate)1.0 phosphazene] (PN-EA/EOB) and poly[(ethyl alanato)1.0(propyl oxybenzoate)1.0 phosphazene] (PN-EA/POB). The polymers were characterized by multinuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and gel permeation chromatography (GPC). Biodegradability and percentage water absorption of the polymers were evaluated by following the mass change in phosphate buffer (pH 7.4) at 37 degrees C. PN-EA/POB underwent faster degradation and showed higher water absorption compared to PN-EA/EOB. Both polymers became insoluble in common organic solvents following hydrolysis presumably due to crosslinking reactions accompanying the degradation process. Osteoblast cell adhesion and proliferation on PN-EA/EOB and PN-EA/POB was followed by scanning electron microscopy (SEM) and by using a biochemical assay. Both PN-EA/EOB and PN-EA/POB supported the adhesion and proliferation of primary rat osteoblast cells in vitro. Furthermore, the enzymatic activity of the osteoblast cells cultured on the polymers was confirmed by the alkaline phosphatase activity. Thus, these biodegradable amino-acid-based polyphosphazenes are promising new materials for forming self-setting bone cements.

Development of Novel Biodegradable Amino Acid Ester Based Polyphosphazene– Hydroxyapatite Composites for Bone Tissue Engineering

ASTRACT:Hydroxyapatite formed from low temperature setting calcium phosphate cements (CPC) are currently been used for various orthopaedic applications. CPCs are attractive candidates for the development of scaffolds for bone tissue engineering, since they are moldable, resorbable, set at physiological temperature without the use of toxic chemicals, and can be processed in an operating room setting. However they may have mechanical disadvantages which seriously limit them to non-load bearing orthopaedic applications. The aim of the present study was to develop composites from polyphosphazenes and calcium deficient hydroxyapatite precursors to form poorly crystalline hydroxyapatite-polymer composites. Composites were formed from calcium deficient hydroxyapatite precursors (Ca/P – 1.5, 1.6) and biodegradable polyphosphazenes, poly[bis(ethyl alanato)phosphazene] (PNEA) and poly[(50%ethyl alanato) (50%methyl phenoxy)phosphazene] (PNEA50mPh50) at physiological temperature. The results demonstrated that poorly crystalline hydroxyapatite that resembled the mineral component of bone was formed in the presence of biodegradable polyphosphazenes. The surface morphology of all the four composites was identical with a porous microstructure. The composites supported the adhesion and proliferation of osteoblast like MC3T3-E1 cells making them potential candidates for bone tissue engineering.

Biodegradable Poly[bis(ethyl alanato)phosphazene] - Poly(lactide-co-glycolide) Blends: Miscibility and Osteocompatibility Evaluations

ABSTRACTWe have previously demonstrated that blending biodegradable glycine co-substituted polyphosphazenes with poly(lactide-co-glycolide) (PLAGA) results in novel biomaterials with versatile properties. The study showed that the degradation rate of polyphosphazene/PLAGA blends can be effectively controlled by varying the blend composition while at the same time the degradation products of polyphosphazenes effectively neutralized the acidic degradation products of PLAGA. In the present study, novel blends of hydrophobic, biodegradable polyphosphazene, poly[bis(ethyl alanato) phosphazene] (PNEA) and PLAGA (LA: GA; 85:15) were developed as candidates for bone tissue engineering applications. Two different blend compositions were developed by blending PNEA and PLAGA having weight ratios of 25:75 (Blend-1) and 50:50 (Blend-2) by the mutual solvent technique using dichloromethane as the solvent. The miscibility of the blends was determined using differential scanning calorimetry (DSC), fourier transform-infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). Surface analysis of the blends by SEM revealed a smooth uniform surface for Blend-1, whereas Blend-2 showed evidence of phase separation. PNEA is not completely miscible with PLAGA, as evidenced from DSC and FT-IR measurements. The osteocompatibilities of Blend-1 and Blend-2 were compared to those of parent polymers by following the adhesion and proliferation of primary rat osteoblast cells on two dimensional (2-D) polymer and blend films over a 21 day period in culture. Blend films showed significantly higher cell numbers on the surface compared to PLAGA and PNEA films.

Development of Biodegradable Polyphosphazene- Nanohydroxyapatite Composite Nanofibers Via Electrospinning

ABSTRACTBiodegradable polymeric nanofibers are of great interest as scaffolds for tissue engineering and drug delivery due to their extremely high surface area, high aspect ratio and similarity in structure to the extracellular matrix (ECM). Polyphosphazenes due to their synthetic flexibility, wide range of physico-chemical properties, non-toxic and neutral degradation products and excellent biocompatibility are suitable candidates for biomedical applications. The objective of the present study was to develop and evaluate composite nanofibers of a biodegradable polyphosphazene, poly[bis(ethyl alanato)phosphazene] (PNEA) and nanocrystals of hydroxyapatite (nHAp) via electrospinning. A suspension of nHAp in dimethyl formamide (DMF) sonicated with PNEA solution in tetrahydrofuran (THF) was used to develop composite nanofiber matrices via electrospinning at ambient conditions. In the present study the theoretical loading of nHAp was varied from 50%-90% (w/w) to PNEA. The nHAp content (actual loading of nHAp) of the composite nanofibers was determined by gravimetric estimation. The composite nanofibers were characterized by transmission electron microscopy (TEM), gravimetry and energy dispersive X-ray mapping. This study demonstrated the feasibility of developing novel composite nanofibers of biodegradable polyphosphazenes with more than 50% (w/w) loading of nHAp on and within the nanofibers.

Poly[(amino acid ester)phosphazenes] as substrates for the controlled release of small molecules

Three different poly[(amino acid ester) phosphazenes] have been examined in order to investigate their possible use as drug delivery vehicles. The three polymers are poly[di(ethyl glycinato)phosphazene], poly[di(ethyl alanato)phosphazene] and poly[di(benzyl alanato)phosphazene]. These macromolecules either share the same amino acid residue or the same ester group, and this facilitated comparisons of the hydrolytic decomposition and the small molecule release profiles of the polymers. The polymers were synthesized by treatment of poly(dichlorophosphazene) with an excess of the appropriate amino acid ester. Tetrahydrofuran solutions of each polymer were then thoroughly mixed with ethacrynic acid, a diuretic, or Biebrich Scarlet, an azo dye. Films cast from these solutions were immersed in aqueous media (pH 7) at 25°C and at 37°C for approximately 1400 h. During these experiments, the release of the small molecules was monitored by UV/visible spectroscopy. The molecular weight decline and the mass loss of the polyphosphazene films were measured.