Preparation and optimization of an eggshell membrane-based biomaterial for GTR applications.

OBJECTIVES: Guided Tissue Regeneration (GTR) is a popular clinical procedure for periodontal tissue regeneration. However, its key component, the barrier membrane, is largely collagen-based and is still quite expensive, posing a financial burden to the patients as well as healthcare systems and negatively impacting the patient's decision-making. Thus, our aim is to prepare a novel biomimetic GTR membrane utilizing a natural biomaterial, soluble eggshell membrane protein (SEP), which is economical as it comes from an abundant industrial waste from food and poultry industries, unlike collagen. Additive polymer, poly (lactic-co-glycolic acid) (PLGA), and a bioceramic, nano-hydroxyapatite (HAp), were added to improve its mechanical and biological properties. METHODS: For this barrier membrane preparation, we initially screened the significant factors affecting its mechanical properties using Taguchi orthogonal array design and further optimized the significant factors using response surface methodology. Furthermore, this membrane was characterized using SEM, EDAX, and ATR-FTIR, and tested for proliferation activity of human periodontal ligament fibroblasts (HPLFs). RESULTS: Optimization using response surface methodology predicted that the maximal tensile strength of 3.1 MPa and modulus of 39.9 MPa could be obtained at membrane composition of 8.9 wt% PLGA, 7.2 wt% of SEP, and 2 wt% HAp. Optimized PLGA/SEP/HAp membrane specimens that were electrospun on a static collector showed higher proliferation activity of HPLFs compared to tissue culture polystyrene and a commercial collagen membrane. SIGNIFICANCE: From the results observed, we can conclude that SEP-based nanofibrous GTR membrane could be a promising, environment-friendly, and cost-effective alternative for commercial collagen-based GTR membrane products.

Suspension culture of hepatocyte-derived reporter cells in presence of albumin to form stable three-dimensional spheroids.

Several studies in the past have formed 3-dimensional (3D) spheroids of primary hepatocytes in suspension culture. Unfortunately, primary hepatocytes in a suspension environment tend to lose their differentiated function over time, generally due to damage from fluid shear stress and eventual spheroid settling. We have therefore created a novel suspension culture system, by seeding H35 rat hepatoma cells, a hepatocyte-derived cell line, in a 24-well tissue culture polystyrene (TCPS) plate placed atop an orbital shaker to create 3D spheroids. To provide stability to the formed spheroids, we used a long-chain polymer, bovine serum albumin (BSA), dissolved in the cell culture medium and/or coated on TCPS surfaces placed in suspension configurations. Our results demonstrate that BSA coating of culture surfaces resulted in uniform and well-defined spheroids with little spheroid settling or "flattening" of cell colonies in either static or suspension configurations. In BSA-coated suspension systems, spheroid size scaled with the amount of BSA dissolved in culture medium. In static uncoated cultures, the normalized rat albumin production levels were enhanced by addition of BSA within culture medium. Thus, both addition of BSA to culture medium and application of BSA as a surface coating appear to be meaningful avenues for tailoring spheroid morphology and function. This 24-well plate suspension culture system may be a valuable tool for high throughput investigations of liver cell behavior in a stable, uniform, 3D spheroid state.

Effect of processing temperature on the morphology and drug-release characteristics of elastin-like polypeptide-collagen composite coatings.

Elastin-like polypeptides (ELPs) exhibit an inverse phase transition temperature (Tt) in response to changes in their environment. We hypothesized that processing ELP-collagen composites at temperatures higher than the Tt of ELP (∼32 °C) will affect their microstructure and subsequently, achieve tunable release of model drugs. The composite coatings were prepared by formation of ELP-collagen hydrogels at 37 °C, incubation at 37, 45, or 55 °C, and finally air-drying at 37 °C. Scanning electron micrographs revealed that the fabrication process affected both the collagen and ELP microaggregate phases. A gradual time dependent bovine serum albumin (BSA) release that followed the power law and a burst antibiotic doxycycline release followed by a linear zero-order release were observed. Importantly, BSA and doxycycline releases were dependent on the ELP microaggregate size, which was governed by the processing temperatures. This study lays the foundation to achieve optimized composite microstructures by controlling processing conditions for drug delivery applications.

Preparation and characterization of elastin-like polypeptide scaffolds for local delivery of antibiotics and proteins.

Tissue engineering applications could benefit from simultaneous release of growth factors, signaling molecules, and antibiotics to obtain optimal healing of tissues. Elastin-like polypeptides (ELPs) are genetically engineered polymers that possess good biocompatibility, are biodegradable, and exhibit mechanical properties similar to natural elastin. In addition, ELPs exhibit a characteristic inverse phase transition temperature (T(t)). This T(t) behavior is widely exploited in hyperthermia mediated drug delivery. The objectives of this research were to prepare ELP hydrogel scaffolds using a novel ultrasonication method and to investigate the release of a model protein (bovine serum albumin, BSA) and a commonly used antibiotic in periodontal therapy (doxycycline) from the scaffolds at two different temperatures (25 °C T(t)). Both BSA and doxycycline showed a gradual time dependent release and showed a trend of higher release fractions with higher loading doses. Based on the comparison between the release profiles at the two selected temperatures, the release was higher at 37 °C compared to that at 25 °C for both the loading concentrations of doxycycline (0.05 and 0.1 % v/v) and only one of the loading concentrations of BSA (0.5 % v/v) studied, while the release was higher at 25 °C compared to that at 37 °C only for the other loading concentration of BSA (1 % v/v) studied. These results suggested that the drug molecular weight and loading concentration were significant factors that affected the release kinetics. The experiments in this study demonstrated that the ELP hydrogel scaffolds can successfully release proteins and antibiotics critical to tissue engineering.

Surface modifications to enhance osseointegration-Resulting material properties and biological responses.

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.

Manipulating the solution environment to control the surface roughness of elastin-based polymer coatings.

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.

Comparison of the formation, adipogenic maturation, and retention of human adipose-derived stem cell spheroids in scaffold-free culture techniques.

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 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.

Photofunctionalization of anodized titanium surfaces using UVA or UVC light and its effects against Streptococcus sanguinis.

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.

Grafting amine-terminated branched architectures from poly(L-lactide) film surfaces for improved cell attachment.

Poly(L-lactide) (PLL) has been used as a bioabsorbable material in the medical and pharmaceutical fields. The unmodified hydrophobic PLL surface generally has low cell affinity; thus, modification of PLL film surface properties is necessary to improve its use as a biomaterial. Our surface modification method involved the use of photografting and typical wet chemistry to create branched architectures containing amine functionalities on the periphery of the grafted layers. Amine (-NH2) groups were first introduced on the PLL film surface by photoinduced grafting of 4,4'-diaminobenzophenone and the grafted branched architectures were created by subsequent reactions with succinic acid and tris(2-aminoethyl) amine. The resulting film surface was analyzed using contact angle goniometry and X-ray photoelectron spectroscopy. MC3T3 fibroblasts were cultured on unmodified PLL film and PLL films grafted with the branched structures and the films were subsequently analyzed by optical microscopy. The contact angle goniometry results showed an initial decrease and subsequent plateau in the water contact angles for the PLL films with each successive generation of the branched architectures. The X-ray photoelectron spectroscopy data provided insight into the structure of the grafted layer and revealed an increase in the nitrogen content with each generation. Optical micrographs showed enhanced cell attachment and viability on the surface-modified PLL films.

Spheroid model for functional osteogenic evaluation of human adipose derived stem cells.

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.

Effect of amine content and chemistry on long-term, three-dimensional hepatocyte spheroid culture atop aminated elastin-like polypeptide coatings.

Culture conditions that induce hepatic spheroidal aggregates sustain liver cells with metabolism that mimics in vivo hepatocytes. Here we present an array of elastin-like polypeptide conjugate coating materials (Aminated-ELPs) that are biocompatible, have spheroid-forming capacity, can be coated atop traditional culture surfaces, and maintain structural integrity while ensuring adherence of spheroids over long culture period. The Aminated-ELPs were synthesized either by direct conjugation of ELP and various polyelectrolytes or by conjugating both ELP and various small electrolytes to the reactive polymer poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA). Spheroid morphology, cellular metabolic function, and liver-specific gene expression over the long-term, 20-day culture period were assessed through optical microscopy, measurement of total protein content and albumin and urea production, and quantitative real-time (qRT) PCR. We found that the amine content of the Aminated-ELP coatings dictated the initial hepatocyte attachment, but not the subsequent hepatocyte spheroid formation and their continued attachment. A lower amine content was generally found to sustain higher albumin production by the spheroids. Out of the 19 Aminated-ELP coatings tested, we found that the lysine-containing substrates comprising ELP-polylysine or ELP-PVDMA-butanediamine proved to consistently culture productive spheroidal hepatocytes. We suggest that the incorporation of lysine functional groups in Aminated-ELP rendered more biocompatible surfaces, increasing spheroid attachment and leading to increased liver-specific function. Taken together, the Aminated-ELP array presented here has the potential to create in vitro hepatocyte culture models that mimic in vivo liver functionality and thus, lead to better understanding of liver pathophysiology and superior screening methods for drug efficacy and toxicity. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 377-388, 2017.

Three-dimensional spheroid cell model of in vitro adipocyte inflammation.

To improve treatment of obesity, a contributing factor to multiple systemic and metabolic diseases, a better understanding of metabolic state and environmental stress at the cellular level is essential. This work presents development of a three-dimensional (3D) in vitro model of adipose tissue displaying induced lipid accumulation as a function of fatty acid supplementation that, subsequently, investigates cellular responses to a pro-inflammatory stimulus, thereby recapitulating key stages of obesity progression. Three-dimensional spheroid organization of adipose cells was induced by culturing 3T3-L1 mouse preadipocytes on an elastin-like polypeptide-polyethyleneimine (ELP-PEI)-coated surface. Results indicate a more differentiated phenotype in 3D spheroid cultures relative to two-dimensional (2D) monolayer analogues based on triglyceride accumulation, CD36 and CD40 protein expression, and peroxisome proliferator-activated receptor-γ (PPAR-γ) and adiponectin mRNA expression. The 3T3-L1 adipocyte spheroid model was then used to test the effects of a pro-inflammatory microenvironment, namely maturation in the presence of elevated fatty acid levels followed by acute exposure to tumor necrosis factor alpha (TNF-α). Under these conditions, we demonstrate that metabolic function was reduced across all cultures exposed to TNF-α, especially so when pre-exposed to linoleic acid. Further, in response to TNF-α, enhanced lipolysis, monitored as increased extracellular glycerol and fatty acids levels, was observed in adipocytes cultured in the presence of exogenous fatty acids. Taken together, our 3D spheroid model showed enhanced adipogenic differentiation and presents a platform for elucidating the key phenotypic responses that occur in pro-inflammatory microenvironments that characterize obesogenic states.

Spheroid organization kinetics of H35 rat hepatoma model cell system on elastin-like polypeptide-polyethyleneimine copolymer substrates.

Though two-dimensional systems have yielded some success in deriving morphological and functional markers of hepatocyte culture, they largely fail to capture the three-dimensional organization, long-term viability, and functionality of the hepatic tissue. We have engineered a system for inducing self-assembly of model H35 rat hepatoma spheroids using a copolymer comprised of biocompatible elastin-like polypeptide (ELP) chemically conjugated to positively charged polyethyleneimine (PEI). We have achieved a conjugation ratio of 30 mol %, though our studies analyzing spheroid organization kinetics indicate conjugate ratios of 5 mol % and greater to be optimal for cell culture based on least variability in spheroid sizes and minimum incidence of overgrown aggregates. Furthermore, our ELP-PEI system indicated the potential for influencing ultimate spheroid dimensions, with spheroid size inversely related to polyelectrolyte conjugation. Overall, this study provides a good starting point to investigate functional correlations between spheroid size and functional markers and their future use as an in vitro diagnostic or tissue engineering tool.

Preparation and characterization of novel elastin-like polypeptide-collagen composites.

Collagen-based biomaterials suffer from poor mechanical properties and rapid degradation. Elastin-like polypeptides (ELPs) possess good biocompatibility and have unique solution properties that allow them to coacervate above a critical temperature. The objective of this research was to prepare a series of freeze dried ELP-collagen composite scaffolds as a proof of concept. Combination of ELP and collagen has the potential to produce composite structures with varying strengths. Four different composite structures were prepared by varying the ratio of ELP to collagen. Increased ELP content in the scaffolds appears to have reduced the residual water content based on Fourier transformed infrared spectroscopy and differential scanning calorimetry. Scanning electron microscopy images of ELP-collagen composites showed a three-dimensional, open porous structure with the formation of characteristic aggregates of ELP. The mechanical testing experiments showed that the elastic modulus, tensile strength, and toughness of ELP-collagen scaffolds were significantly greater than neat collagen scaffolds. The improved mechanical properties were attributed to a homogeneous network structure with additional reinforcement coming from the ELP aggregates. Our study confirms that ELP-collagen composites with superior physical and mechanical properties compared to collagen scaffolds can be produced. Further optimization of design parameters will allow producing ELP-collagen composites for specific biomedical applications.