Elastin-Collagen Based Hydrogels as Model Scaffolds to Induce Three-Dimensional Adipocyte Culture from Adipose Derived Stem Cells.

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.

OPTIMIZATION OF COLLAGEN-ELASTIN-LIKE POLYPEPTIDE-BIOGLASS SCAFFOLD COMPOSITION FOR OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS.

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.

Collagen-Elastin-Like Polypeptide-Bioglass Scaffolds for Guided Bone Regeneration.

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.

Optimization of collagen-elastin-like polypeptide composite tissue engineering scaffolds using response surface methodology.

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.

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.

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.

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.

Assessment of 2D:4D in subjects with anteroposterior mandibular dysplasia.

OBJECTIVES: To compare 2D:4D ratio which is determined by testosterone levels with patients having orthognathic, retrognathic and prognathic mandibles. MATERIALS AND METHODS: The study was performed at Chennai, on 320 subjects of which, 60 subjects (32 males and 28 females) had retrognathic mandible; 55 subjects (25 males and 30 females) had prognathic mandible and 205 subjects (98 males and 107 females) had normal mandible. All the subjects had a normal maxilla and were in the age group of 18 to 25 years. 2D:4D ratio was determined using the photocopies of the ventral surface of right hand made with vernier calipers of 0.01 mm accuracy. Statistical analysis was undertaken using Student's t- test, ANOVA test and TukeyHSD test. RESULTS: (i) Low 2D:4D is seen in subjects with mandibular prognathism, (ii) Among females, low 2D:4D is seen only in prognathic mandible. CONCLUSION: These findings highlight the fact that testosterone plays an important role in mandibular growth. Thus 2D:4D, a least invasive and reproducible procedure can be used as an early marker for mandibular progathism, and as a diagnostic tool in correlating the mandibular growth with causal relations between hormones and craniofacial development.