Amol V. Janorkar

In vitro evaluation of elastin-like polypeptide–collagen composite scaffold for bone tissue engineering

OBJECTIVES Collagen and elastin are two key structural proteins found in the extra-cellular matrices (ECMs) of most tissues, yet very little is known about the response of bone cells to elastin or its derivatives. Recently, we have designed and characterized a novel class of ECM-based composite scaffolds with collagen and a genetically engineered polymer, elastin-like polypeptide (ELP) and subsequently showed their superior mechanical properties and drug release characteristics compared to collagen scaffolds. The objective of this research was to evaluate osteoblast growth and expression on these composite scaffolds. METHODS A thorough biochemical and morphological characterization was performed on MC3T3-E1 pre-osteoblast cells cultured on collagen and ELP-collagen scaffolds. Cell viability was assessed using a live/dead assay. Total DNA content of all cells present on various surfaces was quantified. Pre-osteoblast differentiation was assessed by measuring the alkaline phosphatase and osteocalcin production. Mineral deposition by the cultured cells was visualized using the Von Kossa stain. RESULTS Our results showed that the ELP-collagen scaffolds were suitable substrates for cell culture that allowed MC3T3-E1 pre-osteoblast cell attachment, differentiation, and subsequent mineralization over a period of 3 weeks. The ELP-collagen scaffolds displayed equivalent biocompatibility and cell-interacting properties to those of the neat collagen scaffolds. SIGNIFICANCE The novel ELP-collagen composite material may have future implications as a scaffold material for bone tissue engineering applications, for example, the treatment of alveolar bone loss.

Use of three‐dimensional spheroids of hepatocyte‐derived reporter cells to study the effects of intracellular fat accumulation and subsequent cytokine exposure

Non‐alcoholic fatty liver disease (NAFLD) is a family of liver diseases associated with obesity. Initial stage of NAFLD is characterized by a fatty liver, referred to as steatosis, which progresses in some individuals to non‐alcoholic steatohepatitis (NASH) and liver failure. In order to study and treat the many liver diseases such as NAFLD, an improved in vitro cellular model is needed. Several studies in the past have attempted to elucidate these mechanisms using primary hepatocytes or relevant hepatoma cell lines in two‐dimensional (2D) monolayer in vitro cultures. These 2D planar culture systems, unfortunately, do not represent the complex architecture of hepatic tissue in vivo. Therefore, we have engineered an elastin‐like polypeptide (ELP)–polyethyleneimine (PEI) copolymer and shown that ELP–PEI coated surfaces influenced H35 rat hepatoma cell morphology to create 3D spheroids. Our reporter cell model recapitulates many cellular features of the human disease, including fatty acid uptake, intracellular triglyceride accumulation, decreased proliferation, decreased liver‐specific function, and increased reactive oxygen species accumulation. Finally, to demonstrate the utility of the reporter cells for studying transcriptional regulation, we compared the transcriptional dynamics of nuclear factor κB (NFκB) in response to its classical inducer (tumor necrosis factor‐α, TNF‐α) under lean and fatty conditions in both 2D and 3D culture configurations. We found that, in 3D spheroids, linoleic acid treatment activated NFκB at earlier time points during the development of steatosis, but suppressed the TNF‐mediated NFκB activation at later time points. These studies therefore provide a good starting point to evaluate such relationships observed during NAFLD in a 3D in vitro cell culture. Bioeng. 2011; 108:1171–1180.

Mechanical & Cell Culture Properties of Elastin-Like Polypeptide, Collagen, Bioglass, and Carbon Nanosphere Composites

Collagen, the most commonly used extra-cellular matrix protein for tissue engineering applications, displays poor mechanical properties. Here, we report on the preparation and characterization of novel multi-component composite systems that incorporate a genetically engineered, biocompatible polymer (elastin-like polypeptide, ELP), biodegradable ceramic (45S5 bioglass), carbon nanosphere chains (CNSC), and minimal amount (~25% w/w) of collagen. We hypothesized that incorporation of bioglass and CNSC would improve mechanical properties of the composites. Our results showed that the tensile strength and elastic modulus nearly doubled after addition of the bioglass and CNSC compared to the control ELP–collagen hydrogels. Further, MC3T3-E1 pre-osteoblasts were cultured within the composite hydrogels and a thorough biochemical and morphological characterization was performed. Live/dead assay confirmed high cell viability (>95%) for all hydrogels by day 21 of culture. Alkaline phosphatase (ALP) activity and osteocalcin (OCN) production assessed the pre-osteoblast differentiation. Normalized ALP activity was highest for the cells cultured within ELP–bioglass–collagen hydrogels, while normalized OCN production was equivalent for all hydrogels. Alizarin red staining confirmed the mineral deposition by the cells within all hydrogels. Thus, the multi-component composite hydrogels displayed improved mechanical and cell culture properties and may be suitable scaffold materials for bone tissue engineering.

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.

A Surface-Tethered Spheroid Model for Functional Evaluation of 3T3-L1 Adipocytes

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.

Composition of elastin like polypeptide-collagen composite scaffold influences in vitro osteogenic activity of human adipose derived stem cells.

OBJECTIVE Collagen-based scaffolds for guided bone regeneration (GBR) are continuously improved to overcome the mechanical weaknesses of collagen. We have previously demonstrated superior mechanical characteristics of the elastin-like polypeptide (ELP) reinforced collagen composites. The objectives of this research were to evaluate the efficacy of ELP-collagen composites to culture human adipose-derived stem cells (hASCs) and allow them to undergo osteogenic differentiation. We hypothesized that hASCs would show a superior osteogenic differentiation in stiffer ELP-collagen composites compared to the neat collagen hydrogels. METHODS Composite specimens were made by varying ELP (0-18mg/mL) and collagen (2-6mg/mL) in a 3:1 ratio. Tensile strength, elastic modulus, and toughness were determined by uniaxial tensile testing. hASCs cultured within the composites were characterized by biochemical assays to measure cell viability, protein content, and osteogenic differentiation (alkaline phosphatase activity, osteocalcin, and Alizarin red staining). Scanning electron microscopy and energy dispersive spectroscopy were used for morphological characterization of composites. RESULTS All composites were suitable for hASCs culture with viable cells over the 22-day culture period. The ELP-collagen composite with 18mg/mL of ELP and 6mg/mL of collagen had greater tensile strength and elastic modulus combined with higher osteogenic activity in terms of differentiation and subsequent mineralization over a period of 3 weeks compared to other compositions. The extra-cellular matrix deposits composed of calcium and phosphorous were specifically seen in the 18:6mg/mL ELP-collagen composite. SIGNIFICANCE The success of the 18:6mg/mL ELP-collagen composite to achieve long-term, 3-dimensional culture and osteogenic differentiation indicates its potential as a GBR scaffold.

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.

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.

Characterization of Erucamide Profiles in LLDPE Films: Depth-Profiling Attempts Using FTIR Photoacoustic Spectroscopy and Raman Microspectroscopy

This research focuses on mapping the concentration profile of erucamide in LLDPE film. Attenuated Total Reflectance (ATR) FTIR and FTIR microspectroscopy (FTIR-mS) have both been used previously to map concentration profiles in polymer films. However, ATR-FTIR spectroscopy is restricted to the near-surface region of a film while FTIR-mS works well in the film bulk but has deficiencies near film surfaces. Two other techniques, Raman microspectroscopy (R-mS) and FTIR photoacoustic spectroscopy (FTIR-PAS), reportedly work well for depth-profiling in polymers with micron or submicron resolution. Experiments were conducted with R-mS and FTIR-PAS to attempt to quantify the spatial distribution of erucamide in LLDPE film. The amide I carbonyl peak was identified from neat erucamide powder for both R-mS and FTIR-PAS. That peak was not observed with R-mS for film containing erucamide, even for films with a relatively high erucamide loading (1 wt.%). However, FTIR-PAS could detect statistically significant differences in erucamide concentration as a function of penetration depth, indicating that FTIR-PAS may be used as an effective depth-profiling tool for the erucamide-LLDPE system.

Adipogenic differentiation of human adipose-derived stem cells grown as spheroids

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.