Delivery of Bioactive Albumin from Multi-Functional Polyampholyte Hydrogels.

Tissue engineered scaffolds are currently being explored to aid in healing and regeneration of non-union fractures in bone. Additionally, albumin has been demonstrated to provide benefits to healing when applied to injury sites. This paper focuses on delivery of calcium modified, bioactive bovine serum albumin (BSA) from a multi-functional polyampholyte polymer scaffold. First, the inherent nonfouling and conjugation properties of the polyampholyte hydrogel were verified to determine the impact of calcium exposure. The polyampholyte hydrogel delivery platform was then assessed with calcium titrations and osteoblast-like cell (MC3T3-E1) adhesion, proliferation, and viability evaluations. Finally, integrin inhibitors were used to identify the binding mechanisms that mediate cell adhesion to the calcium-modified BSA-conjugated hydrogels. An increase in cell adhesion was observed following calcium exposure up to 0.075 M, although this and higher calcium concentrations affected hydrogel stability and cell growth. BSA exposed to 0.05 M calcium and delivered from polyampholyte hydrogels promoted the most promising viable cell adhesion over 7 days. Cell adhesion to the calcium-modified BSA-conjugated hydrogels appeared to be regulated by arginine-glycine-aspartic acid (RGD) and collagen specific integrins. These results demonstrate that the delivery of calcium modified BSA from an implantable polymer scaffold is promising for bone tissue engineering applications.

Paired Simulations and Experimental Investigations into the Calcium-Dependent Conformation of Albumin.

Serum albumin is the most abundant protein in blood plasma, and it is involved in multiple biological processes. Serum albumin has recently been adapted for improving biomaterial integration with bone tissue, and studies have shown the importance of this protein in bone repair and regeneration. However, the mechanism of action is not yet clear. In stark contrast, other studies have demonstrated that albumin blocks cell adhesion to surfaces, which is seen as a limitation to its bone healing role. These apparent contradictions suggest that the conformation of albumin facilitates its bioactivity, leading to enhanced bone repair. Serum albumin is known to play a major role in maintaining the calcium ion concentration in blood plasma. Due to the prevalence of calcium at bone repair and regeneration sites, it has been hypothesized that calcium binding to serum albumin triggers a conformational change, leading to bioactivity. In the current study, molecular modeling approaches including molecular docking, atomic molecular dynamics (MD) simulation, and coarse-grained MD simulation were used to test this hypothesis by investigating the conformational changes induced in bovine serum albumin by interaction with calcium ions. The computational results were qualitatively validated with experimental Fourier-transform infrared spectroscopy analysis. We find that free calcium ions in solution transiently bind with the three major loops in albumin, triggering a conformational change where N-terminal and C-terminal domains separate from each other in a partial unfolding process. The separation distance between these domains was found to correlate with the calcium ion concentration. The experimental data support the simulation results showing that albumin has enhanced conformational heterogeneity upon exposure to intermediate levels of calcium, without any significant secondary structure changes.

Enhancement and mechanisms of MC3T3-E1 osteoblast-like cell adhesion to albumin through calcium exposure.

Serum albumin is the most prominent protein in blood, and it aids in bone fracture healing, though the manner through which enhanced healing occurs is not well understood. This study investigates the influence of calcium on the bioactivity of albumin due to the prevalence of calcium at bone injury sites. Bovine serum albumin (BSA) was exposed to varying concentrations of calcium, adsorbed to tissue culture polystyrene, and the subsequent BSA-coated surfaces were evaluated with calcium titration, and cell adhesion, viability, and binding inhibition studies. Calcium-modified BSA improved overall MC3T3-E1 osteoblast-like cell adhesion, although high calcium concentrations induced cell death. Inhibiting specific integrins revealed that without calcium exposure, cell binding to BSA was primarily mediated by integrins that typically bind to the GFOGER sequence of collagen. As calcium exposure increases, the primary binding interaction transitioned to integrins known to bind RGD. However, cell binding to calcium-modified BSA was not completely eliminated during the inhibition studies indicating additional unidentified binding interactions occur. Overall, these results suggest that the exposure to calcium induces conformational changes that affect the cell-binding bioactivity of BSA, which may explain the beneficial impact of albumin in bone tissue.

Synthesis of a zwitterionic N-Ser-Ser-C dimethacrylate cross-linker and evaluation in polyampholyte hydrogels.

Polyampholyte hydrogels are attractive materials for tissue engineering scaffolds as they offer a wide variety of features including nonfouling, selective protein delivery, and tunable physical characteristics. However, to improve the potential performance of these materials for in vivo applications, there is a need for a higher diversity of zwitterionic cross-linker species to replace commonly used ethylene glycol (EG) based chemistries. Towards this end, the synthesis of a dipeptide based zwitterionic cross-linker, N-Ser-Ser-C dimethacrylate (S-S) from N-Boc-l-serine is presented. The strategy utilized a convergent coupling of methacrylated serine partners followed by careful global deprotection to yield the zwitterionic cross-linker with good overall yields. This novel cross-linker was incorporated into a polyampholyte hydrogel and its physical properties and biocompatibility were compared against a polyampholyte hydrogel synthesized with an EG-based cross-linker. The S-S cross-linked hydrogel demonstrated excellent nonfouling performance, while promoting enhanced cellular adhesion to fibrinogen delivered from the hydrogel. Therefore, the results suggest that the S-S cross-linker will demonstrate superior future performance for in vivo applications.

Enhanced Biocompatibility of Polyampholyte Hydrogels.

Tissue-engineered scaffolds encounter many challenges including poor integration with native tissue. Nonspecific protein adsorption can trigger the foreign body response leading to encapsulation and isolation from the native injured tissue. This concern is mitigated with nonfouling polymer scaffolds. This study investigates the long-term biocompatibility of a nonfouling polyampholyte system composed of positively charged [2-(acryloyloxy)ethyl]trimethylammonium chloride monomers and negatively charged 2-carboxyethyl acrylate monomers, cross-linked with triethylene glycol dimethacrylate. This system has previously shown resistance to nonspecific protein adsorption and short-term cell attachment via conjugated proteins. However, longer-term cell survival has not been evaluated with this system. First, the environmental pH was monitored with varying amounts of counter ions present in the hydrogel synthesis buffer. The lowest level (3 M NaOH) and the level that resulted in pH values closest to physiological conditions (6.7 M NaOH) were chosen for further investigation. These two formulations were then compared in terms of their contact angle, qualitative protein adsorption and conjugation capacity, and quantitative cell adhesion, proliferation, and viability. The 3 M NaOH formulation showed higher initial protein conjugation and cell adhesion compared to the 6.7 M NaOH formulation. However, the 3 M NaOH hydrogels had low cell viability after 24 h due to the acidic component release into the culture environment. The 6.7 M NaOH formulation showed a lower initial conjugation and cell adhesion but overcame this limitation by providing a stable environment that maintained cell viability for over 5 days. The 6.7 M NaOH polyampholyte hydrogel formulation shows increased biocompatibility, while maintaining resistance to nonspecific protein adsorption, as demonstrated by the targeted cell adhesion and proliferation. Therefore, this polyampholyte formulation demonstrates strong potential as a tissue-engineered scaffold.

Evaluation of chlorine substituted hydroxyapatite (ClHAP)/polydopamine composite coatings on Ti64.

Titanium (Ti) and its alloys (especially Ti-6Al-4V or Ti64) are commonly used as load-bearing implants because of their biocompatibility and resistance to fatigue and corrosion. However, Ti/alloys are bio-inert metals and not prone to osseointegration. In order to further improve the bioactivity and osseointegration of Ti64, this study evaluated the modification of the Ti64 surface with a deposited chlorine substituted hydroxyapatite (ClHAP)/polydopamine (Pda) composite coating. Pda serves as an adhesion molecule and ClHAP releases slight acidity that stimulates osteoclastic activity. The composite coating with 10-30 % ClHAP particles is shown to promote bioactivity as evidenced by osteoblast proliferation. Therefore, this coating approach may enhance osseointegration in vivo.

Impacts of cross-linker chain length on the physical properties of polyampholyte hydrogels.

Polymeric tissue engineering scaffolds have shown promise to aid in regeneration and repair of damaged tissue. In particular, nonfouling polymers have been proposed for eliminating biomaterial-induced concerns such as infection, scarring, and rejection by the immune system. Polyampholyte polymers are one class of nonfouling polymers that are composed of an equimolar mixture of positively and negatively charged monomer subunits. They possess nonfouling properties, bioactive molecule conjugation capabilities, and tunable mechanical properties. In this study, the influence of the cross-linker species on the degradation behavior, mechanical strength, and nonfouling properties of polyampholytes composed of a 1:1 molar ratio of [2-(acryloyloxy)ethyl] trimethylammonium chloride (positively charged) and 2-carboxyethyl acrylate (negatively charged) monomers was investigated. Specifically, the impact of ethylene glycol repeat units on the overall material performance was evaluated by synthesizing and characterizing hydrogels containing di-, tri-, and tetra-ethylene glycol dimethacrylate cross-linker species. The degradation studies were conducted for over 100 days in Sorenson's buffer with pH values of 4.5, 7.4, and 9.0 by tracking the swelling behavior and weight change over time. The mechanical properties were assessed using compression and tensile testing to failure. The retention of the nonfouling and protein conjugation capabilities was demonstrated using fluorescently labeled bovine serum albumin. The results demonstrate the tunability of both degradation behavior and mechanical properties through the cross-linker selection, without impacting the underlying nonfouling and biomolecule delivery capabilities. Therefore, it is concluded that polyampholyte hydrogels represent a promising platform for tissue engineering.

Polyampholyte Hydrogels in Biomedical Applications.

Polyampholytes are a class of polymers made up of positively and negatively charged monomer subunits. Polyampholytes offer a unique tunable set of properties driven by the interactions between the charged monomer subunits. Some tunable properties of polyampholytes include mechanical properties, nonfouling characteristics, swelling due to changes in pH or salt concentration, and drug delivery capability. These characteristics lend themselves to multiple biomedical applications, and this review paper will summarize applications of polyampholyte polymers demonstrated over the last five years in tissue engineering, cryopreservation and drug delivery.