Gelatin Matrices Research Articles

Ascorbate enhances elastin synthesis in 3D tissue-engineered pulmonary fibroblasts constructs

Extracellular matrix remodeling is a continuous process that is critical to maintaining tissue homeostasis, and alterations in this process have been implicated in chronic diseases such as atherosclerosis, lung fibrosis, and emphysema. Collagen and elastin are subject to ascorbate-dependent hydroxylation. While this post-translational modification in collagen is critical for function, the role of hydroxylation of elastin is not well understood. A number of studies have indicated that ascorbate leads to reduced elastin synthesis. However, these studies were limited to analysis of cells grown under traditional 2D tissue culture conditions. To investigate this process we evaluated elastin and collagen synthesis in primary rat neonatal pulmonary fibroblasts in response to ascorbate treatment in traditional 2D culture and within 3D cross-linked gelatin matrices (Gelfoam). We observed little change in elastin or collagen biosynthesis in standard 2D cultures treated with ascorbate, yet observed a dramatic increase in elastin protein and mRNA levels in response to ascorbate in 3D cell-Gelfoam constructs. These data suggest that the cell-ECM architecture dictates pulmonary cell response to ascorbate, and that approaches aimed toward stimulating ECM repair or engineering functional cell-derived matrices should consider all aspects of the cellular environment.

Development of a morphogenetically active scaffold for three-dimensional growth of bone cells: biosilica-alginate hydrogel for SaOS-2 cell cultivation

Polymeric silica is formed from ortho-silicate during a sol-gel formation process, while biosilica is the product of an enzymatically driven bio-polycondensation reaction. Both polymers have recently been described as a template that induces an increased expression of the genes encoding bone morphogenetic protein 2 (BMP-2) and osteoprotegerin in osteoblast-related SaOS-2 cells; simultaneously or subsequently the cells respond with enhanced hydroxyapatite formation. In order to assess whether the biocompatible polymeric silica/biosilica can serve as a morphogenetically active matrix suitable for three-dimensional (3D) cell growth, or even for 3D cell bioprinting, SaOS-2 cells were embedded into a Na-alginate-based hydrogel. Four different gelatinous hydrogel matrices were used for suspending SaOS-2 cells: (a) the hydrogel alone; (b) the hydrogel with 400 μM ortho-silicate; (c) the hydrogel supplemented with 400 μM ortho-silicate and recombinant silicatein to allow biosilica synthesis to occur; and (d) the hydrogel with ortho-silicate and BSA. The SaOS-2 cells showed an increased growth if silica/biosilica components were present in the hydrogel. Likewise intensified was the formation of hydroxyapatite nodules in the silica-containing hydrogels. After an incubation period of 2 weeks, cells present in silica-containing hydrogels showed a significantly higher expression of the genes encoding the cytokine BMP-2, the major fibrillar structural protein collagen 1 and likewise of carbonic anhydrase. It is concluded that silica, and to a larger extent biosilica, retains its morphogenetic/osteogenic potential after addition to Na-alginate-based hydrogels. This property might qualify silica hydrogels to be also used as a matrix for 3D cell printing.

The Computational Analysis of Spirochete Motility in Viscous Fluids: Mimicking Host Reservoir Micro-Environments

Borrelia burgdorferi, the causative agent of Lyme disease, exists in an enzootic life-cycle involving the transmission and acquisition between arthropod vectors, Ixodes scapularis, and mammalian reservoirs. While either escaping from or disseminating within the host, the spirochetes encounter both visco-elastic networks of complex polymers and diverse viscous fluids. This study aims to establish practical in vitro systems, which accurately replicate the physiological enzootic milieu, to observe and quantify the effects of environmental conditions on spirochete motility.Gelatin matrices ranging from 2% (wt/vol) to 5% in concentration are utilized to reproduce the biomechanical behavior of numerous visco-elastic environments such as: the extracellular matrix, dermis and organ soft tissues, joint ligaments and tendons. The in vitro behavior of Borrelia in gelatin matrices intrinsically resembles the pathogen's movements in the chronically infected mouse dermis, and is characterized by four distinct motility states: non-motile, wriggling, lunging and translocating. Bio-physical modeling is used to demonstrate the relationship of transient membrane adhesions on motility state dynamics. Solutions of 1% (wt/vol) to 30% Ficoll, a non-ionic synthetic sucrose polymer, are used to imitate assorted viscous fluids encountered during spirochete infection, like: mammalian blood circulation, tick hemolymph, synovial and cerebrospinal fluids. Examination of spirochetes swimming in a purely viscous fluid simplifies the analysis of flagellar motor energy dissipation, enabling us to probe the speed-torque relationship of borrelial flagellum and motors. Analysis of the Treponema pallidum spirochete is included to help elucidate how differing morphological features, such as number of flagellum (wavelength and amplitude), effect swimming characteristics. MATLAB tracking and analysis algorithms are used to extract motility parameter quantities.

Characterization of internal structure of hydrated agar and gelatin matrices by cryo‐SEM

There has been a considerable interest in recent years in developing polymer gel matrices for many important applications such as 2DE for quantization and separation of a variety of proteins and drug delivery system to control the release of active agents. However, a well-defined knowledge of the ultrastructures of the gels has been elusive. In this study, we report the characterization of two different polymers used in 2DE: Gelatin, a naturally occurring polymer derived from collagen (protein) and agar, a polymer of polysaccharide (sugar) origin. Low-temperature SEM is used to examine the internal structure of these gels in their frozen natural hydrated states. Results of this study show that both polymers have an array of hollow cells that resembles honeycomb structures. While agar pores are almost circular, the corresponding Gaussian curve is very broad exhibiting a range of radii from nearly 370 to 700 nm. Gelatin pores are smaller and more homogeneous reflecting a narrower distribution from nearly 320 to 650 nm. Overall, these ultrastructural findings could be used to correlate with functions of the polymers.

A nanostructural investigation of glassy gelatin oligomers: molecular organization and interactions with low molecular weight diluents

The effects of low molecular weight diluents (namely water and glycerol) on the nanostructure and thermodynamic state of low water content gelatin matrices are explored systematically by combining positron annihilation lifetime spectroscopy (PALS) with calorimetric measurements. Bovine gelatin matrices with a variation in the glycerol content (0–10 wt.%) are equilibrated in a range of water activities (aw = 0.11–0.68, T = 298 K). Both water and glycerol reduce the glass transition temperature, Tg, and the temperature of dissociation of the ordered triple helical segments, Tm, while having no significant effect on the level of re-naturation of the gelatin matrices. Our PALS measurements show that over the concentration range studied, glycerol acts as a packing enhancer and in the glassy state it causes a nonlinear decrease in the average hole size, vh, of the gelatin matrices. Finally, we report complex changes in vh for the gelatin matrices as a function of the increasing level of hydration. At low water contents (Qw ∼ 0.01–0.10), water acts as a plasticizer, causing a systematic increase in vh. Conversely, for water contents higher than Qw ∼ 0.10, vh is found to decrease, as small clusters of water begin to form between the polypeptide chains.

The heterogeneous motility of the Lyme disease spirochete in gelatin mimics dissemination through tissue

The Lyme disease spirochete Borrelia burgdorferi exists in nature in an enzootic cycle that involves the arthropod vector Ixodes scapularis and mammalian reservoirs. To disseminate within and between these hosts, spirochetes must migrate through complex, polymeric environments such as the basement membrane of the tick midgut and the dermis of the mammal. To date, most research on the motility of B. burgdorferi has been done in media that do not resemble the tissue milieus that B. burgdorferi encounter in vivo. Here we show that the motility of Borrelia in gelatin matrices in vitro resembles the pathogen's movements in the chronically infected mouse dermis imaged by intravital microscopy. More specifically, B. burgdorferi motility in mouse dermis and gelatin is heterogeneous, with the bacteria transitioning between at least three different motility states that depend on transient adhesions to the matrix. We also show that B. burgdorferi is able to penetrate matrices with pore sizes much smaller than the diameter of the bacterium. We find a complex relationship between the swimming behavior of B. burgdorferi and the rheological properties of the gelatin, which cannot be accounted for by recent theoretical predictions for microorganism swimming in gels. Our results also emphasize the importance of considering borrelial adhesion as a dynamic rather than a static process.

MG63 osteoblast-like cells exhibit different behavior when grown on electrospun collagen matrix versus electrospun gelatin matrix.

Electrospinning is a simple and efficient method of fabricating a non-woven polymeric nanofiber matrix. However, using fluorinated alcohols as a solvent for the electrospinning of proteins often results in protein denaturation. TEM and circular dichroism analysis indicated a massive loss of triple-helical collagen from an electrospun collagen (EC) matrix, and the random coils were similar to those found in gelatin. Nevertheless, from mechanical testing we found the Young's modulus and ultimate tensile stresses of EC matrices were significantly higher than electrospun gelatin (EG) matrices because matrix stiffness can affect many cell behaviors such as cell adhesion, proliferation and differentiation. We hypothesize that the difference of matrix stiffness between EC and EG will affect intracellular signaling through the mechano-transducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MG63 osteoblast-like cells. From the results, we found there was no significant difference between the EC and EG matrices with respect to either cell attachment or proliferation rate. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the EC than in those grown on the EG. In addition, the phosphorylation levels of Y397-FAK, ERK1/2, BSP, and OPN proteins, as well as ALP activity, were also higher on the EC than on the EG. We further inhibited ROCK activation with Y27632 during differentiation to investigate its effects on matrix-mediated osteogenic differentiation. Results showed the extent of mineralization was decreased with inhibition after induction. Moreover, there is no significant difference between EC and EG. From the results of the protein levels of phosphorylated Y397-FAK, ERK1/2, BSP and OPN, ALP activity and mineral deposition, we speculate that the mechanism that influences the osteogenic differentiation of MG63 osteoblast-like cells on EC and EG is matrix stiffness and via ROCK-FAK-ERK1/2.

The Heterogeneous Motility of the Lyme Disease Spirochete in Gelatin Mimics Dissemination through Tissue

The Lyme disease spirochete Borrelia burgdorferi exists in nature in an enzootic cycle that involves the arthropod vector Ixodes scapularis and mammalian reservoirs. To disseminate within and between these hosts, spirochetes must migrate through complex, polymeric environments such as the basement membrane of the tick midgut and the dermis of the mammal. To date, most research on the motility of B. burgdorferi has been done in media that do not resemble the tissue milieus that B. burgdorferi encounter in vivo. Here we show that the motility of Borrelia in gelatin matrices in vitro resembles the pathogen's movements in the chronically infected mouse dermis imaged by intra-vital microscopy. More specifically, B. burgdorferi motility in mouse dermis and gelatin is heterogeneous, with the bacteria transitioning between at least three different motility states that depend on transient adhesions to the matrix. We also show that B. burgdorferi is able to penetrate matrices with pore sizes much smaller than the diameter of the bacterium. We find a complex relationship between the swimming behavior of B. burgdorferi and the rheological properties of the gelatin, which cannot be accounted for by recent theoretical predictions for microorganism swimming in gels. Our results also emphasize the importance of considering borrelial adhesion as a dynamic rather than a static process.

Retention of selected aroma compounds by gelatine matrices

The retention of various aroma compounds (linalool, limonene, ethyl butyrate, butyl acetate, isobutyl acetate, pentyl acetate, isopentyl acetate, hexyl acetate) by gelatine gels (2.5, 5, 10 and 20wt%) was investigated by Static Headspace Gas Chromatography. Prior to analysis the samples were left to equilibrate at 37°C for 24h. The selected volatiles differed in their hydrophobicity and structure. One of the major conclusions was that increased hydrophobicity, in volatiles within the same homologous series, resulted in increased release. Branched volatiles gave greater partition coefficient values than their linear counterparts. When volatiles without the same functional group were compared, no correlation between hydrophobicity and retention was observed. The different mechanical properties of the matrix, as a result of different protein concentration, also affected the aroma retention. The effect of equilibration temperature on partition coefficients was also studied. Increased temperature resulted in higher partition coefficient values, probably due to the increased volatility of the aroma compound. Both positive and negative percentage of retention values were calculated suggesting varying protein-volatile interactions.

Characterization and scanning electron microscopic investigation of crosslinked freeze dried gelatin matrices for study of drug diffusivity and release kinetics

Drug delivery is a promising technique to enhance the therapeutic efficacy of the drug. However, properties of carrier materials require intense improvement for effective transport of drug molecules. In the current study, attempts have been made to develop freeze dried gelatin matrices cross linked with genipin at various temperatures (5 °C, 15 °C and 25 °C) prior to freeze-drying (−80 °C). The freeze dried matrices thus obtained at the said temperatures are characterized for crosslinking density, compression strength, swelling behaviors. The matrix crosslinked at 25 °C showed highest Flory–Rehner crosslinking density (467 ± 46) ( p < 0.05), highest compressive strength (12.36 ± 0.12) ( p < 0.05) and lowest equilibrium water content. In this context, scanning electron microscopy (SEM) was performed to study the surface morphology (size and shape of pores) of the crosslinked matrices. These images were further processed for quantitative analysis of morphological features, viz., areas, radius, ferret diameter, length of major and minor axis and eccentricity using MATLAB toolboxes. These quantitative analyses correlate transport and the release kinetics of model anti-inflammatory drug (indomethacin) from crosslinked matrices in vitro to tune as a controllable delivery system. The diffusional exponent ( n) for all constructs ranging from 0.61 to 0.69 ( p < 0.05) (0.45 < n < 0.89) indicated non-Fickian release kinetics.

GENIPIN CROSSLINKED DRUG–GELATIN COMPOSITE FOR DRUG TRANSPORT AND CYTOCOMPATIBILITY

Gelatin-based drug carrier matrices have emerged as very promising class of delivery system. The purpose of this investigation was to develop drug loaded gelatin-based gels (composites). Gelatin matrices were crosslinked with genipin, a naturally occurring crosslinker for the release of indomethacin. Indomethacin, a low molecular weight and moderately hydrophobic, anti-inflammatory agent was incorporated into the gelatin matrices to form drug loaded gel composites for the release study. The gels were subjected to temperature-dependent oscillatory rheology. The result showed pouring temperature in the range of ~31–34°C for the un-crosslinked gels while the crosslinked gels did not show crossover point. Gels were studied for surface morphology using scanning electron microscopy and a porous network structure was observed. The release of indomethacin from the gels indicated an initial increase in the release rate with the increase in drug concentrations. It was observed that drug composites with higher drug concentration exhibited higher drug transport. Swelling and crosslinking played a crucial role in regulating the drug transport. Further, viability assay suggested biocompatibility of these matrices in vitro. Gel in vitro cell compatibility using live dead assay evaluated with AH-927 cell line indicated normal cell proliferation without any harmful effect and thus suggesting appropriateness of crosslinked composites as potential drug carrier.

Controlled release of active DNA from uncrosslinked matrices

Gelatin is investigated as a matrix for temporarily encapsulating, then releasing active, short oligonucleotide stands. While most previous work has focused on using gelatin matrices with covalent crosslinks in which protease digestion triggers release of encapsulated agents, the current study involves uncrosslinked gelatin in order to preserve its relevant temperature sensitivity. First, 150 µL gelatin blocks loaded with fluorescently labeled DNA were investigated. Deposition of poly(allylamine)–poly(acrylic acid) bilayers on the gel typically hindered DNA release at room temperature, but promoted at least 5-fold greater release at 37 °C relative to the room temperature samples. Next, a polydisperse population of DNA-loaded gelatin microspheres was prepared and coated with the polyelectrolyte bilayer. The gelatin microsphere system exhibited a similar temperature dependence as the block system, however, the total amount of DNA encapsulated and released was greater due, mostly likely, to the higher surface area to volume ratio of the microspheres. In both systems, any active DNA released from the gelatin matrices was quantified through subsequent hybridization events with polystyrene particles functionalized with the complementary partner sequence. Overall, our studies indicate that these DNA-loaded, uncrosslinked gelatin carriers represent a promising system for triggered release of encapsulated oligonucleotides for a variety of bio-related applications.

Development of macroporous nanocomposite scaffolds of gelatin/bioactive glass prepared through layer solvent casting combined with lamination technique for bone tissue engineering

In this study, macroporous bioactive nanocomposite scaffolds were developed using cross-linked gelatin and bioactive glass (BaG) nanoparticles. First, BaG nanoparticles were synthesized via sol–gel method and characterized. Then, macroporous nanocomposites were prepared through layer solvent casting combined with freeze-drying and lamination techniques. This research has developed a new composition to produce a new bioactive nanocomposite which is porous with three-dimensional (3D) inter-connected microstructure, pore sizes are 200–500 μm, porosity are 72–86% and BaG nanoparticles are dispersed evenly among cross-linked gelatin matrices. It is mentionable that in this study, we have reported the formation of chemical bonds between BaG nanoparticles and gelatin for the first time. Finally, the in vitro cytocompatibility of the nanocomposite scaffolds was tested using SaOS-2 cell line.

Unusual degradation behavior of citric acid-crosslinked gelatin in vitro and in vivo

The aim of this study was to evaluate the biodegradability and biocompatibility of gelatin matrices designed for drug-eluting stents (DESs). The matrices were prepared by crosslinking alkali-treated gelatin (AlGelatin) with a citric acid-based crosslinker, trisuccinimidyl citrate (TSC), to form AlGelatin–TSC. The biodegradation behavior of the matrices was evaluated in vitro and in vivo. An in vitro enzymatic degradation test showed that AlGelatin–TSC prepared at a TSC concentration of 20 mM is the most stable in collagenase solution compared to AlGelatin–TSC prepared at TSC concentrations higher or lower than 20 mM. Then, AlGelatin–TSC were implanted subcutaneously in rats to evaluate their biodegradability and tissue reaction in vivo. Similar to the in vitro degradation behavior, AlGelatin–TSC with TSC concentration of 20 mM exhibited the lowest biodegradable rate in vivo among all AlGelatin–TSC. In addition, strong inflammation and calcification were not observed for AlGelatin–TSC at any TSC concentration. From an analysis of the crosslinking density of the resulting AlGelatin–TSC, the lowest biodegradability of AlGelatin–TSC with TSC concentration of 20 mM was due to the highest crosslinking density of the matrix. These results suggest that AlGelatin–TSC is suitable for use as matrices in DESs because of its excellent biocompatibility and biodegradability.

Retention of trans-anethole by gelatine and starch matrices

The retention of trans-anethole by gelatine gels (2.5, 5, 10 and 20 wt.%) and starch solutions (10 and 20 wt.%) was investigated by Static Headspace Gas Chromatography. Prior to analysis the samples were left to equilibrate for 24 h at 5 and 37 °C, respectively. Higher partition coefficients were obtained at the higher temperature, probably due to the increased volatility of the aroma compound. The effect of freeze drying of the matrices in the presence of trans-anethole was also evaluated. No significant effect on the partition coefficient values was observed. However, the changes in the nature and the concentration of the matrix were not of great importance on the retention of trans-anethole.

Evaluation of cross-linked gelatin membranes as delivery carriers for retinal sheets

The delivery of intact sheet transplants to the subretinal space can prevent cell loss that is generally associated with the injection of cell suspensions or cell aggregates. The aim of this study was to develop chemically modified gelatin matrices that enhance the delivery efficiency and analyze whether the gelatin membranes cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) can be considered as potential carriers for retinal sheets. The characteristics of EDC cross-linked gelatin membranes were determined by mechanical and in vitro degradation tests, melting point measurements, cell proliferation assays, cytokine expression analyses, and tissue delivery studies. Gelatin membranes without cross-linking and glutaraldehyde cross-linked gelatin samples were used for comparison. Results of this study indicated that introduction of cross-links is capable of rendering the gelatin network more stable against mechanical stresses and deformations as well as rapid hydrolysis during intraocular delivery of delicate tissue sheets. In comparison with the glutaraldehyde treated samples, the EDC cross-linked gelatin membranes showed a better degradation profile and a relatively higher cytocompatibility. In addition, after EDC cross-linking, the gelatin matrices having an acceptable melting point could be used for the fabrication of a sandwich-like carrier with a high transfer and encapsulation efficiency. These findings suggest that the cross-linking agent type gives an influence on delivery functionality of gelatin membranes. In summary, the EDC cross-linked gelatin is an ideal candidate for use as a carrier material in retinal sheet delivery applications.

Monte Carlo simulation for an assessment of standard validity and quantitative X-ray microanalysis in plants

Protein matrices with known concentration of elements are used as standards in X-ray microanalysis of biological materials. The aim of the study was to assess if protein standards (gelatine) could be applied for quantitative X-ray microanalysis of carbohydrate material (cellulose) of plant cell walls by means of Monte Carlo simulations. Plant roots grown in 133Cs contaminated soil and gelatine with known elemental concentration were prepared according to freeze-drying protocols. The atomic fractions of gelatine and cellulose were used to simulate spectra and than compared to experimental ones. There was a good agreement in bremsstrahlung generation between gelatine and cellulose matrices. The experimental and simulated spectra of gelatine revealed a difference in background shape at low energies but corrected if specimen was tilted to -10° or the matrix density increased to 0.95 g/cm3. Minimum detection limit (MDL) simulated for 133Cs La in cellulose was also in agreement with that experimentally calculated for gelatine standard. Protein standard resembles carbohydrate materials and can be used for quantitative analysis of plant cell walls. However, using Monte Carlo simulations to predict analytical conditions for biological materials and elemental MDL, differences in topography and/or gelatine densities should be taken into consideration.

Fabrication of chemically cross-linked porous gelatin matrices.

The aim of this study was to chemically cross-link gelatin, by reacting its free amino groups with an aliphatic diisocyanate. To produce hydrogels with controllable properties, the number of reacting amino groups was carefully determined. Porosity was introduced into the gelatin-based hydrogels through the lyophilization process. Porous and non-porous matrices were characterized with respect to their chemical structure, morphology, water uptake and mechanical properties. The physical, chemical and mechanical properties of the porous matrices are related to the extent of their cross-linking, showing that they can be controlled by varying the reaction parameters. Water uptake values (24 hours) vary between 160% and 200% as the degree of cross-linking increases. The flexibility of the samples also decreases by changing the extent of cross-linking. Young's modulus shows values between 0.188 KPa, for the highest degree, and 0.142 KPa for the lowest degree. The matrices are potential candidates for use as tissue-engineering scaffolds by modulating their physical chemical properties according to the specific application.

Delivery Site of Perivascular Endothelial Cell Matrices Determines Control of Stenosis in a Porcine Femoral Stent Model

Endothelial cells, grown within gelatin matrices and implanted onto the adventitia of injured vessels, inhibit stenosis in experimental models. To determine if this technology could be adapted for minimally invasive procedures, the authors compared the effects of cells in an implantable sponge to that of an injectable formulation and investigated the importance of delivery site in a stent model. Stents were implanted in the femoral arteries of 30 pigs. This was followed by perivascular implantation of sponges or injection of particles containing allogeneic endothelial cells. Controls received acellular matrices or nothing. The effects of delivery site were assessed by injecting cellular matrices into or adjacent to the perivascular tissue or into the neighboring muscle. Animals were sacrificed after 28 days. Pre-sacrifice angiograms and tissue sections were evaluated for stenosis. Arteries treated with cellular matrices had a 55%-63% decrease in angiographic stenosis (P < .05) and a 38%-43% reduction in histologic stenoses (P < .05) compared to controls. Intimal area was greatest when cellular matrices were delivered into the muscle (6.35 mm(2) +/- 0.95) rather than into or adjacent to the perivascular tissue (4.05 mm(2) +/- 0.56 and 4.73 mm(2) +/- 0.53, respectively; P < .05). Perivascular endothelial cell matrices reduced stenosis after stent-induced injury. The effects were not dependent on the formulation but appeared to be dependent on delivery site. Minimally invasive injections of endothelial cell matrices to the adventitia of arteries following peripheral interventions may decrease restenosis rates.

Phase separation, pore structure, and properties of nanofibrous gelatin scaffolds

The development of three-dimensional (3D) biomimetic scaffolds which provide an optimal environment for cells adhesion, proliferation and differentiation, and guide new tissue formation has been one of the major goals in tissue engineering. In this work, a processing technique has been developed to create 3D nanofibrous gelatin (NF-gelatin) scaffolds, which mimic both the physical architecture and the chemical composition of natural collagen. Gelatin matrices with nanofibrous architecture were first created by using a thermally induced phase separation (TIPS) technique. Macroporous NF-gelatin scaffolds were fabricated by combining the TIPS technique with a porogen-leaching process. The processing parameters were systematically investigated in relation to the fiber diameter, fiber length, surface area, porosity, pore size, interpore connectivity, pore wall architecture, and mechanical properties of the NF-gelatin scaffolds. The resulting NF-gelatin scaffolds possess high surface areas (>32 m 2/g), high porosities (>96%), well-connected macropores, and nanofibrous pore wall structures. The technique advantageously controls macropore shape and size by paraffin spheres, interpore connectivity by assembly conditions (time and temperature of heat treatment), pore wall morphology by phase separation and post-treatment parameters, and mechanical properties by polymer concentration and crosslinking density. Compared to commercial gelatin foam (Gelfoam ®), the NF-gelatin scaffold showed much better dimensional stability in a tissue culture environment. The NF-gelatin scaffolds, therefore, are excellent scaffolds for tissue engineering.