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Titanium anodization has been shown to produce crystalline oxides exhibiting photocatalytic reactions that form reactive oxygen species (ROS) when exposed to UV light. The ROS subsequently attack bacteria cells, and thus reduce bacteria attachment on titanium implant surfaces. Polyaniline (PANI) is a conductive polymer that has shown antibacterial properties when electropolymerized onto titanium. Our research group hypothesized the addition of PANI to crystalline titanium oxide surfaces would increase the available free electrons and thus increase photocatalytic activity (PCA). This research led to the development of a novel single-step anodization approach for PANI doping crystalline titanium oxide layers. The objective of the present study was to determine the proper aniline electrolyte concentration needed to maximize the PCA and reduce bacterial attachment on the formed oxides. Aniline concentrations up to 1 M were added into a 1 M sulfuric acid electrolyte. The formed oxides exhibited increased PANI surface coverage but decreased anatase and rutile crystalline titanium oxide phase formation with increasing aniline electrolyte concentrations. Despite exhibiting the lowest levels of anatase and rutile formation, the 0.75 M and 1 M aniline oxides with the greatest PANI surface coverage also exhibited the highest PCA levels. 1 M aniline oxides showed significantly higher PCA under UVA irradiation compared to oxides formed from aniline concentrations up to 0.5 M (p 0.001). 0.75 M aniline oxides exhibited significant reductions in Staphylococcus aureus attachment with or without UVA irradiation compared to control oxides without PANI. MTT and live/dead assays confirmed cytocompatibility and nearly 100% cell viability for the PANI doped oxides.

Critical-sized cranial bone defects fail to re-ossify and require the surgical intervention of cranioplasty. To achieve superior bone healing in such cases, a hydrogel consisting of an interpenetrating network of collagen and elastin-like polypeptide to encapsulate bone morphogenetic protein-2 (BMP-2), doxycycline, and 45S5 Bioglass is developed. This hydrogel has an appropriate elastic modulus of 39 ± 2.2 kPa to allow proper handling during implantation. The hydrogel promotes human adipose-derived stem attachment, proliferation, and differentiation toward the osteogenic lineage, including the deposition of hydroxyapatite particles embedded within a collagenous fibrillar structure after 21 days of in vitro culture. After eight weeks of implantation of the acellular hydrogel in a critical-sized rat cranial defect model, only a small quantity of various pro-inflammatory (20 pg mg

As life expectancy and the age of the general population increases so does the need for improved implants. A major contributor to the failure of implants is poor osseointegration, which is typically described as the direct connection between bone and implant. This leads to unnecessary complications and an increased burden on the patient population. Modification of the implant surfaces through novel techniques, such as varying topography and/or applying coatings, has become a popular method to enhance the osseointegration capability of implants. Recent research has shown that particular surface features influence how bone cells interact with a material; however, it is unknown which exact features achieve optimal bone integration. In this review, current methods of modifying surfaces will be highlighted, and the resulting surface characteristics and biological responses are discussed. Review of the current strategies of surface modifications found that many coating types are more advantageous when used in combination; however, finding a surface modification that utilizes the mutual beneficial effects of important surface characteristics while still maintaining commercial viability is where future challenges exist.

This paper evaluates the aggregation behavior of a potential drug and gene delivery system that combines branched polyethyleneimine (PEI), a positively-charged polyelectrolyte, and elastin-like polypeptide (ELP), a recombinant polymer that exhibits lower critical solution temperature (LCST). The LCST behavior of ELP has been extensively studied, but there are no quantitative ways to control the size of aggregates formed after the phase transition. The aggregate size cannot be maintained when the temperature is lowered below the LCST, unless the system exhibits hysteresis and forms irreversible aggregates. This study shows that conjugation of ELP with PEI preserves the aggregation behavior that occurs above the LCST and achieves precise aggregate radii when the solution conditions of pH (3, 7, 10), polymer concentration (0.1, 0.15, 0.3 mg/mL), and salt concentration (none, 0.2, 1 M) are carefully controlled. K-means cluster analyses showed that salt concentration was the most critical factor controlling the hydrodynamic radius and LCST. Conjugating ELP to PEI allowed crosslinking the aggregates and achieved stable particles that maintained their size below LCST, even after removal of the harsh (high salt or pH) conditions used to create them. Taken together, the ability to control aggregate sizes and use of crosslinking to maintain stability holds excellent potential for use in biological delivery systems.

Titanium surface modification by anodization has been shown to promote bone formation. However, interactions between anodized layers and bone cells depend upon the anodized layer surface compositio...

Elastin-like polypeptides (ELP) have been used as a genetically-engineered, biocompatible substitute for elastin. Cell culture coatings prepared using ELP conjugated to low molecular weight polyethyleneimine (PEI) entices cells to form three-dimensional cellular aggregates that mimic their in vivo counterparts. This study seeks to control the deposition of the ELP and ELP-PEI molecules to control the roughness of the final coatings. The two polymers were coated onto three different substrates (glass, polystyrene, tissue-culture polystyrene) and the solution environment was altered by changing the polymer concentration (0.5, 1.0, 1.5 mg/mL) and/or salt concentration (None, 0.2 M phosphate buffered saline) for a total of 36 conditions. Atomic force microscopy (AFM) was used to measure the average roughness (Ra) of the samples and found that ELP coated samples had a higher Ra than their ELP-PEI counterparts. The coatings were tested for stability by performing cell culture media changes every three days for 11 days. AFM showed that the average roughness of the tested samples increased with each media change. To address this, the surfaces were crosslinked using hexamethyl diisocyanate, which minimized the change in surface roughness even when subjected to an intense sonication process. This study provides parameters to achieve elastin-based coatings with controlled roughness that can be used to support stable, long-term in vitro cell culture.

Thermosensitive chitosan hydrogels—renewable, biocompatible materials—have many applications as injectable biomaterials for localized drug delivery in the treatment of a variety of diseases. To combat infections such as Staphylococcus aureus osteomyelitis, localized antibiotic delivery would allow for higher doses at the site of infection without the risks associated with traditional antibiotic regimens. Fosfomycin, a small antibiotic in its own class, was loaded into a chitosan hydrogel system with varied beta-glycerol phosphate (β-GP) and fosfomycin (FOS) concentrations. The purpose of this study was to elucidate the interactions between FOS and chitosan hydrogel. The Kirby Bauer assay revealed an unexpected concentration-dependent inhibition of S. aureus, with reduced efficacy at the high FOS concentration but only at the low β-GP concentration. No effect of FOS concentration was observed for the planktonic assay. Rheological testing revealed that increasing β-GP concentration increased the storage modulus while decreasing gelation temperature. NMR showed that FOS was removed from the liquid portion of the hydrogel by reaction over 12 h. SEM and FTIR confirmed gels degraded and released organophosphates over 5 days. This work provides insight into the physicochemical interactions between fosfomycin and chitosan hydrogel systems and informs selection of biomaterial components for improving infection treatment.

Many structural variants of elastin-like polypeptides (ELPs), the genetically engineered equivalents of part of human elastin, currently are being investigated for drug delivery and tissue engineering. Here, we report preparation of six different aminated ELP conjugates via two strategies. In the first, a direct linking strategy was used to couple hydrophobic ELP with either polyethyleneimine, polylysine, or polyarginine. In the second, conjugates were made by attaching ELP onto the reactive polymer, poly(2-vinyl-4,4-dimethyl azlactone), and then exhaustively reacting residual azlactone groups with either ethylenediamine, 1,4-butanediamine, or arginine. Molecular size and chemistry of the resulting six aminated-ELP conjugates were confirmed through gel electrophoresis, FTIR spectroscopy, and mass spectrometry. Dynamic light scattering analysis showed that the conjugates prepared using the "direct reaction scheme" formed small aggregates as well as retained their inverse volume-phase transition temperature behavior. The conjugates prepared using the "reactive polymer linker scheme" also retained this transition temperature behavior.

This work demonstrates the use of canola oil as a biologically compatible solvent for a thiol–ene driven suspension polymerization that effectively creates micron-sized particles.

Objectives: Collagen scaffolds for guided bone regeneration have poor mechanical properties and fail due to lack of rigidity and rapid degradation by collagenases. The objectives of this study were to reinforce collagen with elastin-like polypeptide (ELP) and optimize composite mechanical properties using novel response surface methodology (RSM).Methods: 2-7mg/mL collagen and 0-24mg/mL ELP were used in 11 different proportions to form the composites (Fig.1). Tensile strength, elastic modulus and toughness of hydrated composites were determined by uniaxial tensile testing (n=6) and optimized by two factor (collagen/ELP concentration) analysis with RSM (DOE++ software). Composites (n=3) were characterized by swelling ratio, differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and FTIR spectroscopy.Results: Mechanical properties varied with composition (Fig.1a,b,c). H9 composite had the maximum strength (20.2±11.3kPa) and modulus (61.8±22.7kPa), but had lower toughness (0.20±0.04mJ/mm3) than many other composites. The efficient RSM technique performed equally weighted two-factor, three-objective optimization using just 5 compositions (versus the 11 we had to prepare for Fig.1a,b,c) and directed us to 6:12mg/mL collagen:ELP composition that may have high toughness without negatively impacting strength and modulus (Fig.1d,e). Physical characterization of freeze-dried H9 composite against its control (H7) by swelling ratio, DSC, and FTIR revealed that the addition of ELP in composites reduced the residual water content (swelling ratio for H7=53.8±3.7 versus H9=30.8±2.1). SEM images of both H7 (Fig.2a,c) and H9 (Fig.2b,d) composites showed porous fibrillar and afibrillar collagenous microstructure. Additionally, H9 composite showed dense microstructure with characteristic ELP aggregates.Conclusions: A biomaterial with suboptimal properties should be optimized by a compromise between the minimal requirements, target level, and relative importance of each property. RSM efficiently performed such optimization for ELP-collagen composites and predicted a new composition for future testing. Our study demonstrated that ELP-Collagen composites have the capability to form good quality, rigid porous structures required for guided bone regeneration.

https://iadr.abstractarchives.com/abstract/47am-2840432/optimization-of-collagen-elp-bioglass-composite-scaffolds-for-guided-bone-regeneration

https://iadr.abstractarchives.com/abstract/18iags-2954828/stem-cell-differentiation-in-collagen-elp-bioglass-scaffolds-for-guided-bone-regeneration

This study aimed to probe the effect of formulation of scaffolds prepared using collagen and elastin-like polypeptide (ELP) and their resulting physico-chemical and mechanical properties on the adipogenic differentiation of human adipose derived stem cells (hASCs). Six different ELP-collagen scaffolds were prepared by varying the collagen concentration (2 and 6 mg/mL), ELP addition (6 mg/mL), or crosslinking of the scaffolds. FTIR spectroscopy indicated secondary bonding interactions between collagen and ELP, while scanning electron microscopy revealed a porous structure for all scaffolds. Increased collagen concentration, ELP addition, and presence of crosslinking decreased swelling ratio and increased elastic modulus and compressive strength of the scaffolds. The scaffold characteristics influenced cell morphology, wherein the hASCs seeded in the softer, non-crosslinked scaffolds displayed a spread morphology. We determined that stiffer and/or crosslinked elastin-collagen based scaffolds constricted the spreading of hASCs, leading to a spheroid morphology and yielded an enhanced adipogenic differentiation as indicated by Oil Red O staining. Overall, this study underscored the importance of spheroid morphology in adipogenic differentiation, which will allow researchers to create more physiologically-relevant three-dimensional, in vitro culture models.

Studies have shown ultraviolet-A (UVA) irradiation of crystalline titanium oxides leads to the production of reactive oxygen species (ROS) via a photocatalytic process. The ROS exhibit antimicrobial properties that may be of benefit in preventing bacterial attachment to implant devices. Recent studies have suggested a potential benefit of mixed anatase and rutile oxides and dopants on the photocatalytic properties of titanium oxides. The goal of this work was to compare the photocatalytic activity of different anodized commercially pure titanium grade 4 (CPTi4) surfaces. CPTi4 specimens were anodized in three mixed-acid electrolytes to create crystalline oxide surfaces that were either primarily anatase, primarily rutile, or a combination of anatase and rutile. Additionally, the primarily anatase and combination oxides incorporated some phosphorous from the phosphoric acid component in the electrolyte. The photocatalytic activity of the anodized specimens was measured using both methylene blue (MB) degradation assay and comparing the attachment of S. aureus under irradiation with UVA light of differing intensities (1 mW/cm2, 8 mW/cm2, and 23 mW/cm2). Primarily rutile oxides exhibited significantly higher levels of MB degradation after exposure to 1 mW/cm2 UVA lights. Primarily rutile specimens also had the largest reduction in bacterial attachment followed by the mixed phase specimens and the primarily anatase specimens at 1 mW/cm2 UVA lights. Phosphorous-doped, mixed phase oxides exhibited an accelerated MB degradation response during exposure to 8 mW/cm2 and 23 mW/cm2 UVA lights. All anodized and unanodized CPTi4 groups revealed similar S. aureus attachment at the two higher UVA intensities. Although MB degradation assay and the bacteria attachment assay both confirmed photocatalytic activity of the oxides formed in this study, the results of the MB degradation assay did not accurately predict the oxides performance against S. aureus.

While three-dimensional spheroids outperform traditional two-dimensional monolayer culture for human adipose-derived stem cells (hASCs), there is not a consensus on the most successful method for enhancing their adipogenic differentiation and minimizing the loss of physiologically relevant, fatty spheroids during culture. To this end, we compared three culture methods, namely, elastin-like polypeptide-polyethyleneimine (ELP-PEI) coated surfaces, ultra-low attachment static culture, and suspension culture for their ability to form and retain productive hASC spheroids. The ELP-PEI coatings used the ELP conjugated to two molecular weights of PEI (800 or 25,000 g/mol). FTIR spectroscopy, atomic force microscopy, and contact angle goniometry revealed that the ELP-PEI coatings had similar chemical structures, surface topography, and hydrophobicity. Time-lapse microscopy showed that increasing the PEI molecular weight resulted in smaller spheroids. Measurement of triglyceride content showed that the three methods induced comparable differentiation of hASCs toward the adipogenic lineage. DNA content and morphometric analysis revealed the merging of spheroids to form larger spheroids in the ultra-low attachment static culture and suspension culture methods. In contrast, the retention of hASC spheroid sizes and numbers with a regular spheroid size (~100 μm) were best atop the ELP-PEI800 coatings. Overall, this research shows that the spheroid culture atop the ELP-PEI coatings is a suitable cell culture model for future studies involving long-term, three-dimensional culture of mature adipocytes derived from hASCs.

In this study, scanning electron microscopy (SEM) was used to observe the interaction between de-solvated SynB1-elastin-like polypeptide (SynB1-ELP) and silica at a temperature above ELP's lower critical solution temperature (LCST). ELP was seen to initially wet the surface of the silica before rearranging to form narrowly distributed spherical particles. After formation, the ELP particles dynamically rearranged to increase and subsequently decrease in size until 24 h at which time they collapsed. SEM and Energy Dispersive X-ray Spectroscopy revealed that the formation of a thin layer of salt from the PBS solution preceded the initial wetting of ELP on silica, which was shown to play a role in the continuous rearrangement of ELP. FT-IR revealed that the salt, in combination with the hydrophilic silica, trapped water that provided a repulsive surface to the hydrophobic ELP and forced the ELP to continuously minimize its surface area until the water evaporated. This behavior shows that ELP's thermo-responsive nature coupled with its hydrophobicity can be used to create ELP particles and surfaces that can reorganize with minimal water present.

The design and development of multifunctional nanoparticles have attracted great interest in biomedical research. This study aims to prepare pH-responsive melanin-like nanoparticles for T1-weighted magnetic resonance imaging (MRI) and photothermal therapy. The new multifunctional nanoparticles (amino-Fe-PDANPs) are synthesized by copolymerization of dopamine and its derivative amino-N-[2-(diethylamino) ethyl]-3,4-dihydroxy-benzenepropanamide (N-Dopa) at room temperature. The size of nanoparticles can be controlled by NaOH concentration. The incorporation of N-Dopa is characterized by NMR and FT-IR. From transmission electron microscopy (TEM), the nanoparticles exhibit excellent dispersion stability in water and are spherical in shape. The MRI measurement has demonstrated that amino-Fe-PDANPs have a significant signal enhancement in responding to the acidic solution. Confirmed by the photothermal study, the nanoparticles exhibit a high photothermal conversion efficiency. The melanin-like multifunctional nanoparticles integrate both diagnosis and therapeutic functionalities, indicating the potential for theranostic application.

UV light preirradiation of anodized titanium oxide layers has recently been shown to produce a photocatalytic effect that may reduce early bacterial attachment on titanium surfaces. Streptococcus species have been identified as primary early colonizers and contribute to early biofilm formation on dental implant surfaces. Anodized layers with primarily amorphous, primarily anatase, primarily rutile, and mixtures of anatase and rutile phase oxides were preirradiated with UVA or UVC light for 10 min. Nanoscale surface roughness and pre- and post-UV-irradiated wettability were measured for each anodization group. Sample groups were subjected to streptococcus sanguinis for a period of 24 h. Bacterial attachment and killing efficacy were measured and compared to the corresponding non-UV control groups. UVA treatments showed trends of at least a 20% reduction in bacterial attachment regardless of the crystallinity, or combination of oxide phases present. Anodized layers consisting of primarily anatase phase on the outermost surface were shown to have a killing efficacy of at least 50% after preirradiation with UVA light. Anodized layers containing disperse mixtures of anatase and rutile phases at the outermost surface showed at least a 50% killing efficacy after pre-irradiation with either UVA or UVC light. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2284-2294, 2018.

Understanding the process of adipogenesis is critical if suitable therapeutics for obesity and related metabolic diseases are to be found. The current study presents proof of feasibility of creating a 3-D spheroid model using human adipose-derived stem cells (hASCs) and their subsequent adipogenic differentiation. hASC spheroids were formed atop an elastin-like polypeptide-polyethyleneimine (ELP-PEI) surface and differentiated using an adipogenic cocktail. Spheroids were matured in the presence of dietary fatty acids (linoleic or oleic acid) and evaluated based on functional markers including intracellular protein, CD36 expression, triglyceride accumulation, and PPAR-γ gene expression. Spheroid size was found to increase as the hASCs matured in the adipocyte maintenance medium, though the fatty acid treatment generally resulted in smaller spheroids compared to control. A stable protein content over the 10-day maturation period indicated contact-inhibited proliferation as well as minimal loss of spheroids during culture. Spheroids treated with fatty acids showed greater amounts of intracellular triglyceride content and greater expression of the key adipogenic gene, PPAR-γ. We also demonstrated that 3-D spheroids outperformed 2-D monolayer cultures in adipogenesis. We then compared the adipogenesis of hASC spheroids to that in 3T3-L1 spheroids and found that the triglyceride accumulation was less profound in hASC spheroids than that in 3T3-L1 adipocytes, correlated with smaller average spheroids, suggesting a relatively slower differentiation process. Taken together, we have shown the feasibility of adipogenic differentiation of patient-derived hASC spheroids, which with further development, may help elucidate key features in the adipogenesis process.

3D culture systems have the ability to mimic the natural microenvironment by allowing better cell-cell interactions. We have prepared an in vitro 3D osteogenic cell culture model using human adipose derived stem cells (hASCs) cultured atop recombinant elastin-like polypeptide (ELP) conjugated to a charged polyelectrolyte, polyethyleneimine (PEI). We demonstrate that hASCs cultured atop the ELP-PEI coated tissue culture polystyrene (TCPS) formed 3D spheroids and exhibited superior differentiation toward osteogenic lineage compared to the traditional two dimensional (2D) monolayer formed atop uncoated TCPS. Live/dead viability assay confirmed >90% live cells at the end of the 3-week culture period. Over the same culture period, higher protein content was observed in 2D monolayer than 3D spheroids, as the 2D environment allowed continued proliferation, while 3D spheroids underwent contact-inhibited growth arrest. The normalized alkaline phosphatase (ALP) activity, which is an indicator for early osteogenic differentiation was higher for 3D spheroids. The normalized osteocalcin (OCN) production, which is an indicator for osteogenic maturation was also higher for 3D spheroids while 2D monolayer had no noticeable OCN production. On day 22, increased Alizarin red uptake by 3D spheroids showed greater mineralization activity than 2D monolayer. Taken together, these results indicate a superior osteogenic differentiation of hASCs in 3D spheroid culture atop ELP-PEI coated TCPS surfaces than the 2D monolayer formed on uncoated TCPS surfaces. Such enhanced osteogenesis in 3D spheroid stem cell culture may serve as an alternative to 2D culture by providing a better microenvironment for the enhanced cellular functions and interactions in bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1230-1236, 2017.

Previously, we found that elastin-like polypeptide (ELP), when dried above the lower critical solution temperature on top of a hydrophilic fused silica disk, exhibited a dynamic coalescence behavior. The ELP initially wet the silica, but over the next 12 h, dewett the surface and formed aggregates of precise sizes and shapes. Using Fourier-transform infrared (FT-IR) spectroscopy, the present study explores the role of secondary structures present in ELP during this progressive desiccation and their effect on aggregate size. The amide I peak (1600–1700 cm–1) in the ELP’s FT-IR spectrum was deconvoluted using the second derivative method into eight subpeaks (1616, 1624, 1635, 1647, 1657, 1666, 1680, 1695 cm–1). These peaks were identified to represent extended strands, β-turns, 3(10)-helix, polyproline I, and polyproline II using previous studies on ELP and molecules similar in peptide composition. Positive correlations were established between the various subpeaks, water content, and aggregate size to understand the contributions of the secondary structures in particle formation. The positive correlations suggest that type II β-turns, independent of the water content, contributed to the growth of the aggregates at earlier time points (1–3.5 h). At later time points (6–12 h), the aggregate growth was attributed to the formation of 3(10)-helices that relied on a decrease in water content. Understanding these relationships gives greater control in creating precisely sized aggregates and surface coatings with varying roughness.

As the most abundant extracellular matrix protein in the human body, collagen has been a popular biomaterial to prepare tissue engineering scaffolds. Unfortunately, collagen-only scaffolds do not possess adequate mechanical properties (modulus, strength) for in vitro handling and long-term cell culture. This paper reviews the improvements in the mechanical properties of collagen-based scaffolds using the gelation process and crosslinking agents. Effects of both chemical and physical crosslinking methods on scaffold mechanical properties are discussed along with the effects of wet versus dry testing conditions. Approaches involving the addition of polymer and ceramic additives in the collagen-based scaffolds forming composites to enhance their mechanical properties are also summarized.

It is well recognized that one of the major drawbacks of using traditional two dimensional cultures to model the living systems is inaccurately reflecting the physiological manner in which modulators, nutrients, oxygen, and metabolites are applied and removed. Moreover, the two dimensional culture system poorly reflects how different cell types interact with each other in the same microenvironment. Since the first global development of three dimensional (3D) cell culture techniques in the late 1960s, this last decade has seen an explosion of studies to promote 3D models in the fields of regenerative medicine and cancer. The recent surge of interest in 3D cell culture in cancer research is attributable to the interest in developing closer to real life models. The ability to include various cell types and extracellular components reflect more the physiological conditions of tumor microenvironment. In this short review, we will discuss different approaches of 3D culture system models and techniques with a focus on the 3D interactions of cancer cells with stromal cells in the goal to reevaluate old and develop new therapeutics.

Elastin-like polypeptides (ELP) belong to a family of recombinant polymers that shows great promise as biocompatible drug delivery and tissue engineering materials. ELPs aggregate above a characteristic transition temperature (T(t)). We have previously shown that the T(t) and size of the resulting aggregates can be controlled by changing the ELP’s solution environment (polymer concentration, salt concentration, and pH). When coupled to a synthetic polyelectrolyte, polyethyleneimine (PEI), ELP retains its T(t) behavior and gains the ability to be crosslinked into defined particle sizes. This paper explores several machine learning models to predict the transition temperature (T(t)) and hydrodynamic radius (R(h)) of ELP and two ELP-PEI polymers in varying solution conditions. An exhaustive design of experiments matrix consisting of 81 conditions of interest with varying salt concentration (0, 0.2, 1 M NaCl), pH (3, 7, 10), polymer concentration (0.1, 0.17, 0.3 mg/mL), and polymer type (ELP, ELP-PEI800, ELP-PEI10K) was investigated. The five models used in this study were multiple linear regression, elastic-net, support vector regression, multi-layer perceptron, and random forest. A multi-layer perceptron model was found to have the highest accuracy, with an R(2) score of 0.97 for both R(h) and T(t). This was followed closely by the random forest model, with an R(2) of 0.94 for R(h) and 0.95 for T(t). Feature importance was determined using the random forest and linear regression models. Both models showed that salt concentration and polymer type were the two most influential factors that determined R(h), while salt concentration was the dominant factor for T(t).

We have developed a multicomponent hydrogel scaffold that can mimic the bone extracellular matrix by incorporating collagen, elastin-like polypeptide (ELP), and Bioglass. We examined the effects of Bioglass addition to collagen-ELP scaffolds on mechanical properties, physical characteristics, and in vitro osteogenic differentiation, by varying the Bioglass amount and particle size. Response surface methodology with a central composite design predicted 5 mg (6.6 mg/mL) Bioglass with a particle size of 142 ± 5 µm as the optimal amount and particle size to be mixed with 6 mg/mL collagen and 18 mg/mL ELP to obtain a combination of maximized compressive properties. Swelling ratio and FTIR spectroscopy indicated lower hydrophilicity and the presence of hydrophobic and secondary interactions between collagen, ELP, and Bioglass. Scanning electron microscopy showed a nanofibrous morphology of intermingled collagen-ELP-Bioglass network. In vitro osteogenic characterization using human adipose-derived stem cells revealed increased cell attachment and proliferation with increased ALP activity, osteocalcin content, and mineralized deposit formation during a three-week culture. Numerous mineralized deposits composed of calcium and phosphorous were shown by energy dispersive spectroscopy. Overall, our results show that the collagen-ELP-Bioglass multicomponent composites have enhanced mechanical properties with adequate physical features and cell culture properties for bone tissue engineering.

Human adipose-derived stem cells (hASCs) have been shown to differentiate down many lineages including endothelial lineage. We hypothesized that hASCs would more efficiently differentiate toward the endothelial lineage when formed as three-dimensional (3D) spheroids and with the addition of vascular endothelial growth factor (VEGF). Three conditions were tested: uncoated tissue culture polystyrene (TCPS) surfaces that induced a 2D monolayer formation, elastin-like polypeptide (ELP)-collagen composite hydrogel scaffolds that induced encapsulated 3D spheroid culture, and ELP-polyethyleneimine-coated TCPS surfaces that induced 3D spheroid formation in scaffold-free condition. Cells were exposed to endothelial differentiation medium containing no additional VEGF or 20 and 50 ng/mL of VEGF for 7 days and assayed for viability and endothelial differentiation markers. While endothelial differentiation media supported endothelial differentiation of hASCs, our 3D spheroid cultures augmented this differentiation and produced more von Willebrand factor than 2D cultures. Likewise, 3D cultures were able to uptake LDL, whereas the 2D cultures were not. Higher concentrations of VEGF further enhanced differentiation. Establishing angiogenesis is a key factor in regenerative medicine. Future studies aim to elucidate how to produce physiological changes such as neoangiogenesis and sprouting of vessels which may enhance the survival of regenerated tissues.

Obesity is one of the most invaliding and preventable diseases in the United States. Growing evidence suggests that there are sex differences in obesity in human and experimental animals. However, the specific mechanisms of this disease are unknown. Consequently, there is any particular treatment according to the sex/gender at this time. During the last decade, we observe a rise in the study of adipocyte and the possible mechanisms involved in the different roles of the fat. Furthermore, the effect of sex steroids on the adipocyte is one of the fields that need elucidation. Supporting evidence suggests that sex steroids play an essential role not only in the fat distribution, but also, in its metabolism, proliferation, and function. Thus, using in vitro and in vivo studies will contribute to our fight against this critical health public problem encompassing both sexes. In the present review, we discuss some of the recent advances in the adipocytes and the effect of the sex steroids on the adipose tissue. Also, we propose a new alternative to study the role of sex steroids on adipocyte biology through human adipose-derived stem cells.

The ability of a tissue-engineered scaffold to regenerate functional tissues depends on its mechanical and biochemical properties. Though the commonly used collagen scaffolds have good biochemical properties, they fail due to their poor mechanical and physical properties. We have reinforced the collagen matrix with elastin-like polypeptide (ELP) to improve the mechanical and physical properties and optimized the composite composition using a novel statistical method of response surface methodology (RSM). RSM used a central composite design to correlate the 2 input factor variables (collagen and ELP concentrations) and 3 output objectives (tensile strength, elastic modulus, and toughness) using a second order polynomial equation. Upon uniaxial tensile testing and subsequent RSM optimization, a composite prepared using 6 mg/mL collagen and 18 mg/mL ELP was identified as having an optimal combination of all the three tensile properties. Physical properties of the 6:18 mg/mL composite versus the 6:0 mg/mL collagen-only hydrogel characterized by swelling ratio, differential scanning calorimetry, and FTIR spectroscopy revealed that the addition of ELP reduced the residual water content in the composites and provided evidence of the presence of collagen-ELP interactions. Scanning electron microscopy images of the collagen-only hydrogel showed porous fibrillar and dense afibrillar collagenous microstructure, but the collagen-ELP composite showed a dense collagenous microstructure with characteristic ELP aggregates. We surmise that because of the low water content and dense microstructure, the 6:18 mg/mL collagen-ELP composite had improved mechanical properties. Taken together, the composites prepared in this research can form good quality, rigid porous structures required for tissue engineering applications.

Titanium is one of the most commonly utilized metals for the manufacture of orthopedic and dental implants. The purpose of the present research was to investigate the effect of anodization duty cycle (2%–100%) using a pulsed potentiostatic waveform on the formation of crystalline titanium dioxide (anatase and rutile), surface features, and corrosion resistance while maintaining a homogenous oxide color. Based on the results of a previous study, specimens with gold and dark green colors were chosen as evaluation points in the present study. While both the gold and dark green anodized surfaces produced with the pulsed potentiostatic waveform contained anatase, significantly greater anatase levels were shown to be present on the dark green colored oxide specimens. Furthermore, rutile formation was shown only for the dark green specimens. Both the gold and dark green specimens were found to have nano-rough surfaces. The results of this study suggest that the dark green specimens produced with a lower duty cycle exhibited a thicker oxide, a higher degree of crystallinity, a homogenous nano-pore size and distribution, and a more corrosion resistant passive oxide layer.

This study highlights functional differences between 2-D monolayer and 3-D spheroid 3T3-L1 adipocyte culture models and explores the underlying genomic mechanisms responsible for the different phenotypes present. The spheroids showed higher triglyceride accumulation than the monolayer culture and further increase with larger spheroid size. Whole transcriptome analysis indicated significant differential expression of genes related to adipogenesis, including adipocytokine signaling, fatty acid metabolism, and PPAR-γ signaling. Spheroids also showed downregulation of matrix metalloproteinases (MMPs), integrin, actin-cytoskeleton associated genes, and Rho/GTPase3 expression relative to 2-D monolayer, indicating suppression of the Rho-ROCK pathway and thereby promoting adipogenic differentiation. When exposed to linoleic acid (500 μM) and TNF-α (125 ng/mL) to promote chronic adiposity, linoleic acid treatment resulted in increased intracellular triglycerides and subsequent TNF-α treatment resulted in significantly altered adipocytokine signaling, fatty acid metabolism, and PPAR signaling, in addition to upregulation of multiple MMPs in spheroids versus monolayer. Overall, 3-D spheroids showed enhanced adipogenic phenotype as indicated by triglyceride synthesis and transcriptome changes while retaining sensitivity to a pro-inflammatory stimulus. The 3-D spheroid culture thus may provide a simple, convenient, and sensitive in vitro model to study adipocyte response to metabolic stresses relevant to clinical pathologies.

UV light preirradiation of anodized titanium oxide layers has recently been shown to produce a photocatalytic effect that may reduce early bacterial attachment on titanium surfaces. Streptococcus species have been identified as primary early colonizers and contribute to early biofilm formation on dental implant surfaces. Anodized layers with primarily amorphous, primarily anatase, primarily rutile, and mixtures of anatase and rutile phase oxides were preirradiated with UVA or UVC light for 10 min. Nanoscale surface roughness and pre- and post-UV-irradiated wettability were measured for each anodization group. Sample groups were subjected to streptococcus sanguinis for a period of 24 h. Bacterial attachment and killing efficacy were measured and compared to the corresponding non-UV control groups. UVA treatments showed trends of at least a 20% reduction in bacterial attachment regardless of the crystallinity, or combination of oxide phases present. Anodized layers consisting of primarily anatase phase on the outermost surface were shown to have a killing efficacy of at least 50% after preirradiation with UVA light. Anodized layers containing disperse mixtures of anatase and rutile phases at the outermost surface showed at least a 50% killing efficacy after pre-irradiation with either UVA or UVC light. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2284-2294, 2018.

The goals of this study are to evaluate the ability of the multicomponent collagen-elastin-like polypeptide (ELP)-Bioglass scaffolds to support osteogenesis of rat mesenchymal stem cells (rMSCs), demonstrate in vivo biocompatibility by subcutaneous implantation in Sprague-Dawley rats, monitor degradation noninvasively, and finally assess the scaffold's ability in healing critical-sized cranial bone defects. The collagen-ELP-Bioglass scaffold supports the in vitro osteogenic differentiation of rMSCs over a 3 week culture period. The cellular (rMSC-containing) or acellular scaffolds implanted in the subcutaneous pockets of rats do not cause any local or systemic toxic effects or tumors. The real-time monitoring of the fluorescently labeled scaffolds by IVIS reveals that the scaffolds remain at the site of implantation for up to three weeks, during which they degrade gradually. Micro-CT analysis shows that the bilateral cranial critical-sized defects created in rats lead to greater bone regeneration when filled with cellular scaffolds. Bone mineral density and bone microarchitectural parameters are comparable among different scaffold groups, but the histological analysis reveals increased formation of high-quality mature bone in the cellular group, while the acellular group has immature bone and organized connective tissue. These results suggest that the rMSC-seeded collagen-ELP-Bioglass composite scaffolds can aid in better bone healing process.

Preparation of hydrogels that possess an effective antibiotic release profile and better mechanical properties compared to the traditionally used collagen hydrogels has the potential to minimize post-surgical infections and support wound healing. Toward this goal, we prepared elastin-like polypeptide (ELP)-collagen composite hydrogels that displayed a significantly higher elastic modulus compared to the collagen hydrogels. We then characterized the release behavior of the collagen and ELP-collagen hydrogels loaded with varying dosages (1-5% w/w) of a commonly used broad spectrum antibiotic, doxycycline hyclate. Both collagen and ELP-collagen hydrogels showed a gradual time dependent doxycycline release over a period of 5 days. The ELP-collagen hydrogels, in general, showed a slower release of the doxycycline compared to the collagen hydrogels. The released doxycycline was found to be effective against four bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Streptococcus sanguinis, and methicillin-resistant Staphylococcus aureus) in a dose dependent manner. Combined with their improved mechanical properties, the gradual and effective drug release from the biocompatible ELP-collagen hydrogels shown here may be beneficial for drug delivery and tissue engineering applications.

Emphasizing the role of hydrogel stiffness and cellular differentiation, this study develops collagen and elastin-like polypeptide (ELP)-based bone regenerative hydrogels loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) and doxycycline with mechanical properties suitable for osteogenesis. The drug-incorporated collagen-ELP hydrogels has significantly higher modulus of 35 ± 5 kPa compared to collagen-only hydrogels. Doxycycline shows a bi-phasic release with an initial burst release followed by a gradual release, while rhBMP-2 exhibits a nearly linear release profile for all hydrogels. The released doxycycline shows anti-microbial activity against Pseudomonas aeruginosa, Streptococcus sanguinis, and Escherichia coli. Microscopic observation of the hydrogels reveals their interconnected, macroporous, 3D open architecture with pore diameters between 160 and 400 µm. This architecture supports human adipose-derived stem cell attachment and proliferation from initial days of cell seeding, forming a thick cellular sheath by day 21. Interestingly, in collagen and collagen-ELP hydrogels, cell morphology is elongated with stretched slender lamellipodial formation, while cells assemble as spheroidal aggregates in crosslinked as well as drug-loaded hydrogels. Osteogenic markers, alkaline phosphatase and osteocalcin, are expressed maximally for drug-loaded hydrogels compared to those without drugs. The drug-loaded collagen-ELP hydrogels are thus promising for combating bacterial infection and promoting guided bone regeneration.

In order to effectively treat obesity, it must be better understood at the cellular level with respect to metabolic state and environmental stress. However, current two-dimensional (2D) in vitro cell culture methods do not represent the in vivo adipose tissue appropriately due to the absence of complex architecture and cellular signaling. Conversely, 3D in vitro cultures have been reported to have optimal results mimicking the adipose tissue in vivo. The main aim of this study was to examine the efficacy of a novel conjugate of a genetically engineered polymer, elastin-like polypeptide (ELP) and a synthetic polymer, polyethyleneimine (PEI), toward creating a 3D preadipocyte culture system. We then used this 3D culture model to study the preadipocyte differentiation and adipocyte maintenance processes when subjected to various dosages of nutritionally relevant free fatty acids with respect to total DNA and protein content, cell viability, and intracellular triglyceride accumulation. Our results showed that 3T3-L1 preadipocytes cultured on the ELP-PEI surface formed 3D spheroids within 72 h, whereas the cells cultured on unmodified tissue culture polystyrene surfaces remained in monolayer configuration. Significant statistical differences were discovered between the 3D spheroid and 2D monolayer culture with respect to the DNA and protein content, fatty acid consumption, and triglyceride accumulation, indicating differences in cellular response. Results indicated that the 3D culture may be a more sensitive modeling technique for in vitro adipocyte culture and provides a platform for future evaluation of 3D in vitro adipocyte function. Biotechnol. Bioeng. 2014;111: 174–183. © 2013 Wiley Periodicals, Inc.