Dual Role of Mesenchymal Stem Cells Allows for Microvascularized Bone Tissue-Like Environments in PEG Hydrogels.

In vitro engineered tissues which recapitulate functional and morphological properties of bone marrow and bone tissue will be desirable to study bone regeneration under fully controlled conditions. Among the key players in the initial phase of bone regeneration are mesenchymal stem cells (MSCs) and endothelial cells (ECs) that are in close contact in many tissues. Additionally, the generation of tissue constructs for in vivo transplantations has included the use of ECs since insufficient vascularization is one of the bottlenecks in (bone) tissue engineering. Here, 3D cocultures of human bone marrow derived MSCs (hBM-MSCs) and human umbilical vein endothelial cells (HUVECs) in synthetic biomimetic poly(ethylene glycol) (PEG)-based matrices are directed toward vascularized bone mimicking tissue constructs. In this environment, bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2) promotes the formation of vascular networks. However, while osteogenic differentiation is achieved with BMP-2, the treatment with FGF-2 suppressed osteogenic differentiation. Thus, this study shows that cocultures of hBM-MSCs and HUVECs in biological inert PEG matrices can be directed toward bone and bone marrow-like 3D tissue constructs.

NMR imaging of the diffusion of water at 310 K into semi-IPNs of PEM and Poly(HEMA-co-THFMA)

Magnetic resonance imaging has been used to monitor the diffusion of water at 310 K into a series of semi-IPNs of poly(ethyl methacrylate), PEM, and copolymers of 2-hydroxyethyl methacrylate, HEMA, and tetrahydrofurfuryl methacrylate, THFMA. The diffusion was found to be well described by a Fickian kinetic model in the early stages of the water sorption process, and the diffusion coefficients were found to be slightly smaller than those for the copolymers of HEMA and THFMA, P(HEMA-co-THFMA), containing the same mole fraction of HEMA in the matrix. A second stage sorption process was identified in the later stage of water sorption by the PEM/PTHFMA semi-IPN and for the systems containing a P(HEMA-co-THFMA) component with a mole fraction HEMA of 0.6 or less. This was characterized by the presence of Water near the surface of the cylinders with a longer NMR T-2 relaxation time, which would be characteristic of mobile water, such as water present in large pores or surface fissures. The presence of the drug chlorhexidine in the polymer matrixes at a concentration of 5.625 wt % was found not to modify the properties significantly, but the diffusion coefficients for the water sorption were systematically smaller when the drug was present.

Investigation into the diffusion of water into HEMA-co-MOEP hydrogels

Cross-linked homopolymers and copolymers of 2-hydroxyethyl methacrylate, HEMA, and ethylene glycol methacrylate phosphate, MOEP, have been synthesized, and the diffusion of water into these systems has been investigated. Only polymers with 0-20 mot % MOEP exhibited ideal swelling behavior as extensive fracturing occurred in the systems with greater than 20 mot % MOEP as the polymers began to swell during water sorption. Gravimetric studies were used in conjunction with magnetic resonance imaging of the diffusion front to elucidate the diffusion mechanism for these systems. In the case of the cross-linked HEMA homopolymer gets, the water transport mechanism was determined to be concentration-independent Fickian diffusion. However, as the fraction of MOEP in the network increased, the transport mechanism became increasingly exponentially concentration-dependent but remained Fickian until the polymer consisted of 30 mot % MOEP where the water transport could no longer been described by Fickian diffusion.

Water diffusion in methacrylate based copolymer hydrogels of 2-hydroxyethyl methacrylate

The diffusion of water into cylinders of polyHEMA and copolymers of HEMA with THFMA, BMA and CHMA were studied over a range of copolymer compositions. The diffusion of water into the polymers was found to follow a Fickian, or case I mechanism. The diffusion coefficients of water were determined from mass measurements and NMR imaging studies. They were found to vary from 1.7 +/- 0.2 x 10(-11) m(2) s(-1) for polyHEMA at 37 degreesC to lower values for the copolymers. The mass of water absorbed at equilibrium relative to the mass of dry polymer varied from 52-58 wt% for polyHEMA to lower values for the copolymers.

Study of the calcification of PHEMA hydrogels using a two compartment permeation cell

To simulate the process of calcification in hydrogel implants, particularly calcification inside hydrogels, in vitro experiments using two compartment permeation cells have been performed. PHEMA hydrogel membranes were synthesized by free radical polymerization in bulk. The permeability and diffusion coefficient for Ca2+ ions at 37 ° C were determined using Fick's laws of diffusion. It was evident that Ca2+ ions either from CaCl2 or SBF solutions may diffuse through PHEMA hydrogel membranes. The fort-nation of calcium phosphate deposits inside the hydrogel was observed and attributed to a heterogeneous nucleation from diffusing calcium and phosphate ions. The morphology of the deposits both on the surface and inside the hydrogels was found to be similar, i.e. spherical aggregates with a diameter of less than one micron. © 2005 Elsevier B.V. All rights reserved.

Experimental Calcification of HEMA-Based Hydrogels in the Presence of Albumin and a Comparison to the in Vivo Calcification

The precipitation patterns and characteristics of calcium phosphate (CaP) phases deposited on HEMA-based hydrogels upon incubation in simulated body fluid (SBF-2) containing a protein (human serum albumin) have been investigated in relation to the calcification in an organic-free medium (SBF-1) and to that occurring after subcutaneous implantation in rats. In SBF-2, the deposits occurred exclusively as a peripheral layer on the surface of the hydrogels and consisted mainly of precipitated hydroxyapatite, a species deficient in calcium and hydroxyl ions, similarly to the deposits formed on the implanted hydrogels, where the deposited layer was thicker. In SBF-1, the deposits were mainly of brushite type. There was no evidence that albumin penetrated the interstices of hydrogels. As the X-ray diffraction patterns of the CaP deposits generated in SBF-2 showed a similar nature with those formed on the implanted hydrogel, it was concluded that the calcification in SBF-2 can mimic to a reliable extent the calcification process taking place in a biological environment.

In-vitro study of the spontaneous calcification of PHEMA-based hydrogels in simulated body fluid

In-vitro calcification of poly(2-hydroxyethyl methacrylate) (PHEMA)-based hydrogels in simulated body fluid (SBF) under a steady/batch system without agitation or stirring the solutions has been investigated. It was noted that the formation of calcium phosphate (CaP) deposits primarily proceeded through spontaneous precipitation. The CaP deposits were found both on the surface and inside the hydrogels. It appears that the effect of chemical structure or reducing the relative number of oxygen atoms in the copolymers on the degree of calcification was only important at the early stage of calcification. The morphology of the CaP deposits was observed to be spherical aggregates with a thickness of the CaP layer less than 0.5 mu m. Additionally, the CaP deposits were found to be poorly crystalline or to have nano-size crystals, or to exist mostly as an amorphous phase. Characterization of the CaP phases in the deposits revealed that the deposits were comprised mainly of whitlockite [Ca9MgH(PO4)(7)] type apatite and DCPD (CaHPO4 center dot 2H(2)O) as the precursors of hydroxyapatite [Ca-10(PO4)(6)(OH)(2)]. The presence of carbonate in the deposits was also detected during the calcification of PHEMA based hydrogels in SBF solution.

Zwitterionic and charge-balanced polyampholyte copolymer hydrogels

Zwitterionic copolymers were synthesised from N,N-dimethyl-N-(2- acryloylethyl)-N-(3-sulfopropyl) ammonium betaine (SPDA) and 2-hydroxyethyl methacrylate (HEMA) produce a series of polyzwitterion hydrogels. For the synthesis of the charge-balanced copolymer hydrogels, two cationic monomers were selected: 2-(diethylamino) ethyl methacrylate (DMAEMA) and 3-(dimethylamino) propyl methacrylamide (DMAPMA), and an anionic monomer; 2-acrylamido-2- methylpropane sulphonic acid (AMPS). Two series of charge-balanced copolymers were synthesized from stoichiometrically equivalent ratios of DMAEMA or DMAPMA and AMPS with HEMA as a termonomer. All synthesized copolymers produced clear and cohesive hydrogels. The zwitterionic and charge-balanced copolymers displayed similar equilibrium water contents together with similar mechanical and surface energy properties. The swelling of the zwitterionic and the charge-balanced copolymers shows some features of antipolyelectrolyte behavior.

Degradable poly(ethylene glycol)-based hydrogels:synthesis, physico-chemical properties and in vitro characterization

Designing degradable hydrogels is complicated by the structural and temporal complexities of the gel and evolving tissue. A major challenge is to create scaffolds with sufficient mechanical properties to restore initial function while simultaneously controlling temporal changes in the gel structure to facilitate tissue formation. Poly(ethylene glycol) was used in this work, to form biodegradable poly(ethylene glycol)-based hydrogels with hydrolyzable poly-l-lactide segments in the backbone. Non-degradable poly(ethylene glycol) was also introduced in the formulation to obtain control of the degradation profile that encompasses cell growth and new tissue formation. The dependence on polymer composition was observed by higher degradation profiles and decreased mechanical properties as the content of degradable segments was increased in the formulation. Based on in vitro tests, no toxicity of extracts or biomaterial in direct contact with human adipose tissue stem cells was observed, and the ultraviolet light treatment did not affect the proliferation capacity of the cells.

Soft hydrogels from nanotubes of poly(ethylene oxide)−tetraphenylalanine conjugates prepared by click chemistry

A new poly(ethylene oxide)-tetraphenylalanine polymer-peptide conjugate has been prepared via a “click” reaction between an alkyne-modified peptide and an azide-terminated PEO oligomer. Self-assembled nanotubes are formed after dialysis of a THF solution of this polymer-peptide conjugate against water. The structure of these nanotubes has been probed by circular dichroism, IR, TEM, and SAXS. From these data, it is apparent that self-assembly involves the formation of antiparallel ß-sheets and p-p-stacking. Nanotubes are formed at concentrations between 2 and 10 mg mL-1. Entanglement between adjacent nanotubes occurs at higher concentrations, resulting in the formation of soft hydrogels. Gel strength increases at higher polymer-peptide conjugate concentration, as expected.