Preparation and characterization of konjac glucomannan–poly(acrylic acid) IPN hydrogels for controlled release

In this paper, we reported the synthesis and properties of interpenetrating polymer network (IPN) hydrogel systems designed for colon targeted drug delivery. The gels were composed of konjac glucomannan (KGM) and cross-linked poly(acrylic acid) (PAA) by N,N-methylene-bis-(acrylamide) (MBAAm). It was possible to modulate the swelling degree of the gels. And the swelling ratio has sensitive respondence to the environmental pH value variation. The degradation tests show that the hydrogels retain the enzymatic degradation character of KGM. In vitro release of model drug VB₁₂ was studied in the presence of Cellulase E0240 in pH 7.4 phosphate buffer at 37 °C. The accumulative release percent of the model drug reached 85.6% after 48 h and the drug release was controlled by the swelling and the degradation of the hydrogels. The results indicated that the IPN hydrogels can be exploited as potential carriers for colon-specific drug delivery.     

New route for synthesizing poly(ethylene glycol)-acrylic acid hydrogels using γ-irradiation for drug delivery carriers

Biocompatible and pH-responsive poly(ethylene glycol) (PEG)-acrylic acid (AAc) hydrogels were prepared by new technique using γ-irradiation for controlled oral drug delivery. The gel fraction was over 80% and the equal amounts of PEG and AAc blended hydrogel had efficient insulin loading using equilibrium swelling. These hydrogels exhibited unique pH-responsive characteristics in which interpolymer complexes were formed in acidic media and dissociated in neutral or basic environments. The insulin release from the gel was significantly retarded in acidic media while rapid release occurred under neutral/basic conditions. At the high pH solution, the gels swelled rapidly and over 70% of the insulin loaded was released over a period of 10 h. Within 2 h of administration of the insulin-containing gels, significant blood glucose reduction effects were observed in diabetic rats. The blood glucose reduction lasted for up to 10 h following administration.     

Biodegradable thermoresponsive hydrogels for aqueous encapsulation and controlled release of hydrophilic model drugs

A series of hydrogels with both thermoresponsive and completely biodegradable properties was developed for aqueous encapsulation and controlled release of hydrophilic drugs in response to temperature change. The hydrogels were prepared in phosphate-buffered saline (pH 7.4) through free radical polymerization of N-isopropylacrylamide (NIPAAm) monomer and a dextran macromer containing multiple hydrolytically degradable oligolactate-2-hydroxyethyl methacrylate units (Dex-lactateHEMA). Swelling measurement results demonstrated that four gels with feeding weight ratios of NIPAAm:Dex-lactateHEMA = 7:2, 6:3, 5:4, and 4:5 (w/w) were thermoresponsive by showing a lower critical solution temperature at approximately 32 degrees C. The swelling and degradation of the hydrogels strongly depended on temperature and hydrogel composition. An empirical mathematical model was established to describe the fast water absorption at the early stage and deswelling at the late stage of the hydrogels at 37 degrees C. Two hydrophilic model drugs, methylene blue and bovine serum albumin, were loaded into the hydrogels during the synthesis process. The molecular size of the drugs, the hydrophilicity and degradation of the hydrogels, and temperature played important roles in controlling the drug release.     

Dextran and hyaluronan methacrylate based hydrogels as matrices for soft tissue reconstruction

Polysaccharide hydrogels have become increasingly studied as matrices in soft tissue engineering because of their known cytocompatibility. In this work cross-linkable dextran methacrylates and hyaluronan methacrylate were synthesized and their transformation into stable hydrogels was studied. The in vitro degradation behaviour of the formed hydrogels could be controlled by the polysaccharide structure and the cross-linking density. Under in vitro conditions, the formed gels had no cytotoxic effects against fibroblasts, but cells could adhere only inefficiently in long term experiments. The use of composite gels improved the adherence of cells. Different scaffold architectures were studied including porous structures and perforated gel layers. Selected hydrogels were examined in an in vivo pilot study using a rabbit model to evaluate their biocompatibility, stability and degradation. No signs of inflammation were seen and with prolonged duration the material was degraded and lacunas were formed by immigrating or ingrowing cells. Optimizing their mechanical properties, the formed hydrogels represent promising candidates as matrices for soft tissue reconstruction.     

pH响应型蔗渣木质素水凝胶的制备及其对蛋白质的控释性能

该研究以蔗渣木质素和甲基丙烯酸为原料合成了pH敏感型蔗渣木质素/聚甲基丙烯酸水凝胶,对其合成条件、pH敏感性、溶胀-退溶胀性能以及对牛血清蛋白的控释等性质进行研究,并采用红外光谱、扫描电镜等对凝胶进行表征。结果表明:(1)对凝胶溶胀比影响的因素由大到小依次为甲基丙烯酸用量、交联剂用量、催化剂用量、反应的温度、木质素用量。当甲基丙烯酸单体浓度为1.75 mol·L~(-1)、木质素浓度为25 g·L~(-1)、交联剂浓度为3.25×10~(-2)mol·L~(-1)、引发剂浓度为1.25×10~(-2)mol·L~(-1)、反应温度为65℃时,所得水凝胶在模拟肠液中的溶胀比最大(28.16 g·g~(-1))。与不加木质素的聚甲基丙烯酸水凝胶相比,蔗渣木质素/聚甲基丙烯酸水凝胶的溶胀比有所下降,但其敏感pH由4~5碱移至6~8。(2)蔗渣木质素/聚甲基丙烯酸水凝胶的溶胀—退溶胀可逆性受组成的影响较大,但相对于聚甲基丙烯酸水凝胶,蔗渣木质素/聚甲基丙烯酸水凝胶对pH值的敏感响应性更强、响应速率更快,同时能在更短时间内达到溶胀平衡。(3)加入木质素可以提高水凝胶对牛血清蛋白的负载量,所试验的蔗渣木质素/聚甲基丙烯酸水凝胶样品对牛血清蛋白的最大负载量可达577 mg·g~(-1)。(4)牛血清蛋白在12 h后基本可达释放平衡;在模拟胃液中,牛血清蛋白的释放率仅10%,而在模拟肠液中释放率达92%。pH响应型蔗渣木质素/聚甲基丙烯酸水凝胶可以作为口服型蛋白类药物的潜在载体。     

Rheology and synergy of κ-carrageenan/locust bean gum/konjac glucomannan gels

The rheology and melting of mixed polysaccharide gels containing konjac glucomannan (KGM), locust bean gum (LBG) and κ-carrageenan (KC) were studied. Synergy-type peaks in the Young's modulus at optimal mixing ratios were found for both KC/LBG and KC/KGM binary gels at a fixed total polysaccharide content (1:5.5 for LBG:KC and 1:7 for KGM:KC). The Young's modulus peak for KC/KGM was higher than for KC/LBG gels. The same stoichiometric mixing ratios were found when either LBG or KGM was added to KC at a fixed KC concentration, where the Young's modulus increased up to additions at the stoichiometric ratio, but leveled off at higher LBG or KGM additions. Addition of KGM or LBG to the 2-component gels beyond the stoichiometric (optimal) mixing ratio at a fixed total polysaccharide content led to a decrease in the Young's modulus and an increase in the rupture strain and stress in extension, and both trends were stronger for KGM than for LBG.     

pH-sensitive cationic guar gum/poly (acrylic acid) polyelectrolyte hydrogels: Swelling and in vitro drug release

Novel polyelectrolyte hydrogels (coded as GA) based on cationic guar gum (CGG) and acrylic acid monomer by photoinitiated free-radical polymerization were synthesized with various feed compositions. Fourier transform infrared spectra (FTIR), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) confirmed that the formation of the polyelectrolyte hydrogels was attributed to the strong electrostatic interaction between cationic groups in CGG and anionic groups in poly (acrylic acid) (PAA). Swelling experiments provided important information on drug diffusion properties, which indicated the GA hydrogels were highly sensitive to pH environments. Potential applications of the hydrogels matrices in controlled drug delivery were also examined. The ketoprofen-loaded CGG/PAA matrices were prepared by hydrogels and directly compressed tablets, respectively. Release behavior of ketoprofen relied on the preparative methods of matrices, ratios of CGG/AA and pH environments. The release mechanism was studied by fitting experimental data to a model equation and calculating the corresponding parameters. The result showed that the kinetics of drug release from the hydrogels in pH 7.4 buffer solution was mainly non-Fickian diffusion. However, for tablets, the drug release in pH 7.4 buffer solution was mainly affected by polymer erosion. The pH of the dissolution medium appeared to have a strong effect on the drug transport mechanism. At more basic pH values, Case II transport was observed, indicating a drug release mechanism highly influenced by macromolecular chain relaxation. The ketoprofen release is also tested in the conditions chosen to simulate gastrointestinal tract conditions. The results implied that the GA hydrogels can be exploited as potential carriers for colon-specific drug delivery.     

Characterization and in vitro evaluation of starch based hydrogels as carriers for colon specific drug delivery systems

A series of starch/methacrylic acid (MAAc) copolymer hydrogels of different compositions were synthesized using γ-rays induced polymerization and crosslinking. The effects of the preparation conditions such as the feed solution concentration, feed solution composition and irradiation dose on the gelation process of the synthesized copolymer were investigated. The swelling behavior of the starch/methacrylic acid (MAAc) copolymer hydrogels was characterized by studying the effect of the hydrogel composition on the time- and pH-dependent swelling. Swelling kinetics showed that the synthesized hydrogels possessed Fickian diffusion behavior at pH 1 and non-Fickian diffusion at pH 7 which recommend them as good candidate for colon specific drug delivery systems. The synthesized hydrogels were loaded with ketoprofen as a model drug to investigate the release behavior of the synthesized hydrogels. The results showed the ability of the hydrogels to keep the loaded drug at pH 1 and release it at pH 7. The data also showed that the release rate can be controlled by controlling the preparation conditions such as comonomer concentration and composition and irradiation dose.     

Impact of metal oxide nanoparticles on oral release properties of pH-sensitive hydrogel nanocomposites

In this article, modified κ-carrageenan hydrogel nanocomposites were synthesized to increase the release ability of carrageenan hydrogels under gastrointestinal conditions. The effect of MgO nanoparticle loading in a model drug (methylene blue) release is investigated. Characterization of hydrogels were carried out using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Field Emission Scanning Electron Microscope (FESEM) and Differential Scanning Calorimetry (DSC). Genipin was used to increase the delivery performance in gastrointestinal tract delivery by decreasing release in simulated stomach conditions and increasing release in simulated intestine conditions. It is shown that the amount of methylene blue released from genipin-cross-linked nanocomposites can be 67.5% higher in intestine medium and 56% lower in the stomach compared to κ-carrageenan hydrogel. It was found that by changing the nanoparticle loading and genipin concentration in the composite, the amount of drug released can be monitored. Therefore, applying nanoparticles appears to be a potential strategy to develop controlled drug delivery especially in gastrointestinal tract studies.     

壳聚糖基温敏水凝胶的研究进展

壳聚糖是一种由甲壳素脱乙酰化得到的氨基多糖,具有生物相容性、低细胞毒性和可生物降解性等特点。壳聚糖/β-甘油磷酸钠溶液温敏水凝胶在组织工程、药物缓释等领域多有报道,其成胶性能取决于凝胶的组分和浓度。针对单纯壳聚糖水凝胶强度较低、降解较快、药物突释等缺陷,通常对壳聚糖进行改性或引入新材料共混,获得更符合实际需要的壳聚糖基温敏水凝胶。对近年来壳聚糖基水凝胶的研究进展进行综述,包括改性壳聚糖、共混体系等,概述了其在组织工程(软骨、血管、神经修复)、药物缓释(癌症药物缓释、糖尿病治疗)领域中研究和应用的新进展,以期为后续温敏水凝胶的进一步研究提供参考。     

Gelation behavior of native and acetylated konjac glucomannan

Gelation kinetics of native and acetylated konjac glucomannan (KGM) samples in the presence of alkali (sodium carbonate) was studied by dynamic viscoelastic measurements. Molecular weight and other molecular parameters of KGM were determined by static light scattering and viscosity measurements. It was found that KGM molecules were degraded during acetylation treatment, but the molecular weights of acetylated samples were almost independent of the degree of acetylation (DA) and were about a half of that of a native sample. At a fixed alkaline concentration, increasing concentration of KGM or temperature shortened the gelation time, but increasing DA delayed it. The deacetylation reaction and subsequent aggregation process of acetylated samples needed longer time than that of native sample, and acetylated samples formed finally more elastic gels. It implied that the presence of acetyl groups exerts a strong influence on gelation behavior of KGM. It was suggested that the gelation rate of acetylated KGM and native KGM, which depends on the alkaline concentration and temperature, is an important factor that determines the elastic modulus of gels. This was supported by the experimental finding that the saturated elastic modulus tends to the same value when the ratio of alkali concentration to acetylated groups was kept constant. In slower gelation processes, junction zones are more homogeneously distributed and more numerous, leading to the more elastic gels.     

Entrapment and in vitro release of delta-sleep inducing peptide from polymer hydrogels based on modified polyvinyl alcohol

The aim of this study was to entrap delta-sleep inducing peptide (DSIP) in cross-linked poly(vinyl alcohol)-based hydrogels of different structures and to determine kinetics of the peptide release from these hydrogels using an in vitro model. Isotropic and macroporous hydrogels based on poly(vinyl alcohol) acrylic derivative (Acr-PVA) and also macroporous epoxy groups containing hydrogels synthesized by copolymerization of this macromer and glycidyl methacrylate, have been used in this study. Isotropic hydrogels were prepared at positive temperatures while macroporous ones were obtained by formation in cryo-conditions. The peptide was entrapped into macroporous PVA hydrogels by adding the peptide solution onto preformed matrices, while peptide immobilization on PVA-GMA hydrogels, containing free epoxy groups, was carried out by sorption of peptide from its aqueous solution. In the case of DSIP entrapment into isotropic PVA gel the peptide solution was added into the polymer mixture at hydrogel formation. The kinetics of peptide release from hydrogels was studied by incubating matrices in PBS solution (pH 7.4), in physiological solution (0.9% NaCl) and in water. DSIP concentration in supernatants was determined by reverse-phase HPLC. Incubation of macroporous PVA gels in PBS, 0.9% NaCl, and water for 30 min caused release of 74, 70, and 64% DSIP, respectively, and this processes completed within 3 h. From hydrogel containing epoxy groups the release of neither peptide nor its degradation products was observed even after incubation for 48 h. For freshly prepared isotropic hydrogel the release kinetics was as follows: 27 and 78% DSIP were released within first 30 min and 33 h, relatively. For the lyophilized hydrogel samples the peptide release was 63% after incubation for 30 min, while drying of samples at room temperature for 3 days caused significant peptide loss because of its structure damage.     

One-step synthesis of interpenetrating network hydrogels: Environment sensitivities and drug delivery properties

A novel interpenetrating network hydrogel for drug controlled release, composed of modified poly(aspartic acid) (KPAsp) and carboxymethyl chitosan (CMCTS), was prepared in aqueous system. The surface morphology and composition of hydrogels were characterized by SEM and FTIR. The swelling properties of KPAsp, KPAsp/CMCTS semi-IPN and KPAsp/CMCTS IPN hydrogels were investigated and the swelling dynamics of the hydrogels was analyzed based on the Fickian equation. The pH, temperature and salt sensitivities of hydrogels were further studied, and the prepared hydrogels showed extremely sensitive properties to pH, temperature, the ionic salts kinds and concentration. The results of controlled drug release behaviors of the hydrogels revealed that the introduction of IPN observably improved the drug release properties of hydrogels, the release rate of drug from hydrogels can be controlled by the structure of the hydrogels and pH value of the external environment, a relative large amount of drug released was preferred under simulated intestinal fluid. These results illustrated high potential of the KPAsp/CMCTS IPN hydrogels for application as drug carriers.     

Preparation and characterization of imogolite/DNA hybrid hydrogels

Imogolite is one of the clay minerals contained in volcanic ash soils. The novel hybrid hydrogels were prepared from imogolite nanofibers and DNA by utilizing strong interaction between the aluminol groups on imogolite surface and phosphate groups of DNA. The hybrid hydrogels of imogolite and DNA were prepared in various feed ratios, and their physicochemical properties and molecular aggregation states were investigated in both dispersion and gel states. The maximum DNA content in the hybrid gels was shown in equivalent molar ratio of imogolite and DNA. The physical properties of the hybrid gels were changed by varying DNA blend ratios. In the dispersion state, the hybrid gels showed a fibrous structure of imogolite, whereas a continuous network structure was observed in pure imogolite, indicating that the hybrid with DNA enhanced the dispersion of imogolite. In the gel state, DNA and imogolite nanofibers formed a 3D network structure.     

Poly(ethylene glycol) methacrylate/dimethacrylate hydrogels for controlled release of hydrophobic drugs

Hydrogels have been successfully used to entrap hydrophilic drugs and release them in a controlled fashion; however, the entrapment and release of hydrophobic drugs has not been well studied. We report on the release characteristics of a model hydrophobic drug, the steroid hormone estradiol, entrapped in low (MW 360/MW 550) and high (MW 526/MW 1000) molecular weight poly(ethylene glycol) methacrylate (PEG-MA)/dimethacrylate (PEG-DMA) hydrogels. The cross-linking ratio, temperature, and pH ranged from 10:1 to 10:3, from 33 to 41 degrees C, and from 2 to 12, respectively. The gelation of the PEG-MA/PEG-DMA hydrogel was initiated with UV irradiation. The absence of poly(glutamic acid) in the hydrogel formulation resulted in a loss of pH sensitivity in the acidic range, which was displayed by the hydrogels' similarities in swelling ratios in the pH buffers of pH 2, 4, and 7. Use of high molecular weight polymers resulted in a higher hydrogel swelling (300%) in comparison to the low molecular weight polymers. Drug size was found to be a significant factor. In comparison to 100% estradiol (MW 272) release, the fractional release of insulin (MW 5733) was 12 and 24% in low and high molecular weight gels at pH 2, respectively, and 17% in low molecular weight gels at pH 7. On the release kinetics of the estradiol drug, the hydrogels displayed a non-Fickian diffusion mechanism, which indicated that the media penetration rate is in the same range as the drug diffusion. The synthesis, entrapment, and release of estradiol by the PEG-MA/PEG-DMA hydrogels proved to be successful, but the use of ethanol in the buffers to promote the hydrophobic release of the estradiol in the in vitro environment caused complications, attributed to the process of transesterification.     

Physicochemical characterization of konjac glucomannan

Four commercial konjac glucomannan (KGM) samples and a glucomannan derived from yeast were characterized by aqueous gel permeation chromatography coupled with multi angle laser light scattering (GPC-MALLS). Disaggregation of aqueous glucomannan solutions through controlled use of a microwave bomb facilitated reproducible molar mass distribution determination alleviating the need for derivatization of the polymer or the use of aggressive solvents. Further characterization was undertaken by use of capillary viscometry and photon correlation spectroscopy (PCS). The weight average molecular masses (M(w)) determined were in the region of 9.0 +/- 1.0 x 10(5) g mol(-1) for KGM samples and 1.3 +/- 0.4 x 10(5) g mol(-1) for the yeast glucomannan. The values determined for KGM in aqueous solution are in agreement with those reported for KGM in aqueous cadoxen. The degradation of samples observed upon autoclaving has been quantified by GPC-MALLS and intrinsic viscosity determination, allowing comparison with reported Mark-Houwink parameters. Shear flow experiments were undertaken for a range of KGM solutions of concentration 0.05 to 2.0% using a combination of controlled stress and controlled strain rheometers. The concentration dependence of the zero shear specific viscosity was determined by analysis of the data using the Ellis model. The dependence of the zero shear specific viscosity on the coil overlap parameter was defined and interpretation discussed in terms of the Martin and Tuinier equations.     

In situ cross-linking of elastin-like polypeptide block copolymers for tissue repair

Rapid cross-linking of elastin-like polypeptides (ELPs) with hydroxymethylphosphines (HMPs) in aqueous solution is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. In order to examine the independent effect of the location and number of reactive sites on the chemical cross-linking kinetics of ELPs and the mechanical properties of the resulting hydrogels, we have designed ELP block copolymers comprised of cross-linkable, hydrophobic ELP blocks with periodic Lys residues (A block) and aliphatic, hydrophilic ELP blocks with no cross-linking sites (B block); three different block architectures, A, ABA, and BABA were synthesized in this study. All ELP block copolymers were rapidly cross-linked with HMPs within several minutes under physiological conditions. The inclusion of the un-cross-linked hydrophilic block, its length relative to the cross-linkable hydrophobic block, and the block copolymer architecture all had a significant effect on swelling ratios of the cross-linked hydrogels, their microstructure, and mechanical properties. Fibroblasts embedded in the ELP hydrogels survived the cross-linking process and remained viable for at least 3 days in vitro when the gels were formed from an equimolar ratio of HMPs and Lys residues of ELPs. DNA quantification of the embedded cells indicated that the cell viability within triblock ELP hydrogels was statistically greater than that in the monoblock gels at day 3. These results suggest that the mechanical properties of ELP hydrogels and the microenvironment that they present to cells can be tuned by the design of the block copolymer architecture.     

Characterization of protein release from hydrolytically degradable poly(ethylene glycol) hydrogels

We present a novel fully hydrophilic, hydrolytically degradable poly(ethylene glycol) (PEG) hydrogel suitable for soft tissue engineering and delivery of protein drugs. The gels were designed to overcome drawbacks associated with current PEG hydrogels (i.e., reaction mechanisms or degradation products that compromise protein stability): the highly selective and mild cross‐linking reaction allowed for encapsulating proteins prior to gelation without altering their secondary structure as shown by circular dichroism experiments. Further, hydrogel degradation and structure, represented by mesh size, were correlated to protein release. It was determined that polymer density had the most profound effect on protein diffusivity, followed by the polymer molecular weight, and finally by the specific chemical structure of the cross‐linker. By examining the diffusion of several model proteins, we confirmed that the protein diffusivity was dependent on protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., Ig). Furthermore, we demonstrated that the protein physical state was preserved upon encapsulation and subsequent release from the PEG hydrogels and contained negligible aggregation or protein–polymer adducts. These initial studies indicate that the developed PEG hydrogels are suitable for release of stable proteins in drug delivery and tissue engineering applications. Biotechnol. Bioeng. 2011; 108:197–206. © 2010 Wiley Periodicals, Inc.     

Release of 5-fluorouracil from poly(acrylamide-co-monopropyl itaconate) hydrogels

The aim of this work was to test the application of copolymeric poly(acrylamide-co-monopropyl itaconate) (A-MPI) hydrogels on the release of 5-fluorouracil (5-FU). The equilibrium degree of swelling in saline solution was 83 ± 2%. 5-FU, as the sodium salt, was trapped in gels by placing it in the polymerization feed mixture. The diffusion coefficients for both swelling of the gels and the release of 5-FU were determined, in addition to the activation energies for both processes. To determine the applicability of these copolymers, A-MPI (75:25) gel was subcutaneously implanted in rats and the drug plasma concentration was determined by HPLC.     

Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium

Growth and differentiation of keratinocytes in a serum-free medium (keratinocyte growth medium or KGM) was studied and compared to that under conditions in which serum and feeder cell layers were used. Cells were grown in KGM containing 0.1 mM calcium (KGM/low calcium), KGM containing 1.2 mM calcium (KGM/normal calcium), or Dulbecco's modified Eagles medium containing 5% fetal calf serum and 1.8 mM calcium in presence of mitomycin treated 3T3 M cells (DMEM/5% FCS). Plating efficiency and rate of growth were similar in the three media till confluence. In postconfluent cultures, protein and DNA content of cells attached to the plate in KGM/low-calcium dishes decreased as an increased number of cells were shed into the medium. Cell shedding was much less evident in the presence of normal calcium. Cells grown in KGM/low calcium had a higher rate of cell proliferation (3H-thymidine incorporation into cellular DNA) than cells grown in normal calcium. Transglutaminase activity, involucrin content, and cornified envelope formation were greatest in cells grown in KGM/normal calcium, intermediate in cells grown in DMEM/5% FCS, and least in cells grown in KGM/low calcium. Keratin profiles from cells grown in KGM/low calcium showed a lower percentage of high molecular weight bands compared to the keratin profiles from cells grown in the presence of normal calcium. Keratinocytes in KGM/low calcium grew as a monolayer of cuboidal cells with few features of differentiation, whereas cells grown in KGM/normal calcium stratified into multilayered islands (3-5 layers) surmounted by 2-4 layers of enucleated cells with thickened cornified envelopes. Cells grown in KGM/normal calcium also contained tonofilaments and lamellar bodies unlike cells grown in KGM/low calcium. Cells grown in DMEM/5% FCS also formed stratified layers comparable to cells grown in KGM/normal calcium but lacked cornified cells, keratohyalin granules, tonofilament bundles, and lamellar bodies. These studies indicate the usefulness of serum-free conditions for the culture of human keratinocytes and confirm the importance of extracellular calcium in keratinocyte differentiation.