Biomaterials (v.30, #20)

Two poly(ɛ-caprolactone fumarate)s (PCLFs) with distinct physical properties have been employed to prepare nanocomposites with hydroxyapatite (HA) nanoparticles via photo-crosslinking. The two PCLFs are PCLF530 and PCLF2000, named after their precursor PCL diol with molecular weight of 530 and 2000 g mol−1, respectively. Crosslinked PCLF530 is amorphous while crosslinked PCLF2000 is semi-crystalline with a melting temperature (T m) of ∼40 °C and a crystallinity of 40%. Consequently, the rheological and mechanical properties of crosslinked PCLF2000 are significantly greater than those of crosslinked PCLF530. Structural characterizations and physical properties of both series of crosslinked PCLF/HA nanocomposites with HA compositions of 0%, 5%, 10%, 20%, and 30% have been investigated. By adding HA nanoparticles, crosslinked PCLF530/HA nanocomposites demonstrate enhanced rheological and mechanical properties while the enhancement in compressive modulus is less prominent in crosslinked PCLF2000/HA nanocomposites. In vitro cell attachment and proliferation have been performed using rat bone marrow stromal cells (BMSCs) and correlated with the material properties. Cell attachment and proliferation on crosslinked PCLF530/HA nanocomposite disks have been enhanced strongly with increasing the HA composition. However, surface morphology and surface chemistry such as composition, hydrophilicity, and the capability of adsorbing protein cannot be used to interpret the cell responses on different samples. Instead, the role of surface stiffness in regulating cell responses can be supported by the correlation between the change in compressive modulus and BMSC proliferation on these two series of crosslinked PCLFs and PCLF/HA nanocomposites.
Keywords: Polycaprolactone fumarate (PCLF); Hydroxyapatite (HA); Nanocomposite; Photo-crosslinking; Bone marrow stromal cell responses;

An injectable, in situ enzymatically gellable, gelatin derivative for drug delivery and tissue engineering by Shinji Sakai; Keisuke Hirose; Kenichi Taguchi; Yuko Ogushi; Koei Kawakami (3371-3377).
A phenolic hydroxyl group was incorporated into gelatin, using aqueous-phase carbodiimide activation chemistry, to obtain in situ gellable and injectable protein-based materials for drug delivery and tissue engineering applications. By this means, gelatin derivatives that were gellable via a peroxidase-catalyzed reaction were obtained. The enzymatically cross-linked gelatin gels did not melt at 37 °C and showed tunable proteolytic degradability. The time necessary for gelation decreased with increasing content of the phenolic hydroxyl (Ph) group, peroxidase concentration and decreasing H2O2 concentration. Resistance to gel compression also depended on the content of Ph groups, with the gel containing the lowest Ph group content showing the greatest resistance to compression. We encapsulated L929 fibroblast cells in gelatin gels under conditions that induced gelation in about 10 s. The encapsulated cells showed about 95% viability. In addition, L929 cells seeded on the gels showed the same growth profiles as those seeded on an unmodified gelatin-coated dish. Subcutaneous rodent injection experiments demonstrated successful in situ formation of gels at the injected site.
Keywords: Gelatin; Enzyme; Hydrogel; Degradation; Cross-linking;

N-acetyl cysteine (NAC)-mediated detoxification and functionalization of poly(methyl methacrylate) bone cement by Naoki Tsukimura; Masahiro Yamada; Hideki Aita; Norio Hori; Fumihiko Yoshino; Masaichi Chang-Il Lee; Katsuhiko Kimoto; Anahid Jewett; Takahiro Ogawa (3378-3389).
Currently used poly(methyl methacrylate) (PMMA)-based bone cement lacks osteoconductivity and induces osteolysis and implant loosening due to its cellular and tissue-toxicity. A high percentage of revision surgery following the use of bone cement has become a significant universal problem. This study determined whether incorporation of the amino acid derivative N-acetyl cysteine (NAC) in bone cement reduces its cytotoxicity and adds osteoconductivity to the material. Biocompatibility and bioactivity of PMMA-based bone cement with or without 25 mm NAC incorporation was examined using rat bone marrow-derived osteoblastic cells. Osteoconductive potential of NAC-incorporated bone cement was determined by μCT bone morphometry and implant biomechanical test in the rat model. Generation of free radicals within the polymerizing bone cement was examined using electron spin resonance spectroscopy. Severely compromised viability and completely suppressed phenotypes of osteoblasts on untreated bone cement were restored to the normal level by NAC incorporation. Bone volume formed around 25 mm NAC-incorporated bone cement was threefold greater than that around control bone cement. The strength of bone–bone cement integration was 2.2 times greater for NAC-incorporated bone cement. For NAC-incorporated bone cement, the spike of free radical generation ended within 12 h, whereas for control bone cement, a peak level lasted for 6 days and a level greater than half the level of the peak was sustained for 20 days. NAC also increased the level of antioxidant glutathione in osteoblasts. These results suggest that incorporation of NAC in PMMA bone cement detoxifies the material by immediate and effective in situ scavenging of free radicals and increasing intracellular antioxidant reserves, and consequently adds osteoconductivity to the material.
Keywords: Arthroplasty; Total hip replacement; Antioxidant; Free radical; Bone cement implantation syndrome (BCIS); Toxicity;

Mechanisms regulating increased production of osteoprotegerin by osteoblasts cultured on microstructured titanium surfaces by Zvi Schwartz; Rene Olivares-Navarrete; Marco Wieland; David L. Cochran; Barbara D. Boyan (3390-3396).
Osteoblasts grown on microstructured Ti surfaces enhance osteointegration by producing local factors that regulate bone formation as well as bone remodeling, including the RANK ligand decoy receptor osteoprotegerin (OPG). The objective of this study was to explore the mechanism by which surface microstructure and surface energy mediate their stimulatory effects on OPG expression. Titanium disks were manufactured to present different surface morphologies: a smooth pretreatment surface (PT, Ra < 0.2 μm), microstructured sandblasted/acid etched surface (SLA, Ra = 3–4 μm), and a microstructured Ti plasma-sprayed surface (TPS, Ra = 4 μm). Human osteoblast-like MG63 cells were cultured on these substrates and the regulation of OPG production by TGF-β1, PKC, and α2β1 integrin signaling determined. Osteoblasts produced increased amounts of OPG as well as active and latent TGF-β1 and had increased PKC activity when grown on SLA and TPS. Exogenous TGF-β1 increased OPG production in a dose-dependent manner on all surfaces, and this was prevented by adding blocking antibody to the TGF-β type II receptor or by reducing TGF-β1 binding to the receptor by adding exogenous soluble type II receptor. The PKC inhibitor chelerythrine inhibited the production of OPG in a dose-dependent manner, but only in cultures on SLA and TPS. shRNA knockdown of α2 or a double knockdown of α2β1 also reduced OPG, as well as production of TGF-β1. These results indicate that substrate-dependent OPG production is regulated by TGF-β1, PKC, and α2β1 and suggest a mechanism by which α2β1 signaling increases PKC, resulting in TGF-β1 production and TGF-β1 then acts on its receptor to increase transcription of OPG.
Keywords: Osteoblast; TGF-β1; Osteoprotegerin; Titanium; Microtopography;

Positioning and guidance of neurons on gold surfaces by directed assembly of proteins using Atomic Force Microscopy by Cristian Staii; Chris Viesselmann; Jason Ballweg; Lifang Shi; Gang-yu Liu; Justin C. Williams; Erik W. Dent; Susan N. Coppersmith; Mark A. Eriksson (3397-3404).
We demonstrate that Atomic Force Microscopy nanolithography can be used to control effectively the adhesion, growth and interconnectivity of cortical neurons on Au surfaces. We demonstrate immobilization of neurons at well-defined locations on Au surfaces using two different types of patterned proteins: 1) poly-d-lysine (PDL), a positively charged polypeptide used extensively in tissue culture and 2) laminin, a component of the extracellular matrix. Our results show that both PDL and laminin patterns can be used to confine neuronal cells and to control their growth and interconnectivity on Au surfaces, a significant step towards the engineering of artificial neuronal assemblies with well-controlled neuron position and connections.
Keywords: AFM; Microfabrication; Protein patterning; Cortical neurons; Self-assembled monolayers; Neural network;

Engineering retinal progenitor cell and scrollable poly(glycerol-sebacate) composites for expansion and subretinal transplantation by Stephen Redenti; William L. Neeley; Santiago Rompani; Sunita Saigal; Jing Yang; Henry Klassen; Robert Langer; Michael J. Young (3405-3414).
Retinal degenerations cause permanent visual loss and affect millions world-wide. Presently, a novel treatment highlights the potential of using biodegradable polymer scaffolds to induce differentiation and deliver retinal progenitor cells for cell replacement therapy. In this study, we engineered and analyzed a micro-fabricated polymer, poly(glycerol sebacate) (PGS) scaffold, whose useful properties include biocompatibility, elasticity, porosity, and a microtopology conducive to mouse retinal progenitor cell (mRPC) differentiation. In vitro proliferation assays revealed that PGS held up to 86,610 (±9993) mRPCs per square millimeter, which were retained through simulated transplantations. mRPCs adherent to PGS differentiated toward mature phenotypes as evidenced by changes in mRNA, protein levels, and enhanced sensitivity to glutamate. Transplanted composites demonstrated long-term mRPC survival and migrated cells exhibited mature marker expression in host retina. These results suggest that combining mRPCs with PGS scaffolds for subretinal transplantation is a practical strategy for advancing retinal tissue engineering as a restorative therapy.
Keywords: Biodegradation; Cell adhesion; Elastomer; Stem cell; Nerve tissue engineering; Ophthalmology;

The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering by Robert Pytlík; David Stehlík; Tomáš Soukup; Marie Kalbáčová; František Rypáček; Tomáš Trč; Katarína Mulinková; Petra Michnová; Linda Kideryová; Jan Živný; Pavel Klener; Romana Veselá; Marek Trněný; Pavel Klener (3415-3427).
Clinical application of human multipotent mesenchymal stromal cells (hMSCs) requires their expansion to be safe and rapid. We aimed to develop an expansion protocol which would avoid xenogeneic proteins, including fetal calf serum (FCS), and which would shorten the cultivation time and avoid multiple passaging. First, we have compared research-grade alpha-MEM medium with clinical grade CellGro™ for Hematopoietic Cells' Medium. When FCS was used for supplementation and non-adherent cells were discarded, both media were comparable. Both media were comparable also when pooled human serum (hS) was used instead of FCS, but the numbers of hMSCs were lower when non-adherent cells were discarded. However, significantly more hMSCs were obtained both in alpha-MEM and in CellGro™ supplemented with hS when the non-adherent cells were left in the culture. Furthermore, addition of recombinant cytokines and other supplements (EGF, PDGF-BB, M-CSF, FGF-2, dexamethasone, insulin and ascorbic acid) to the CellGro™ co-culture system with hS led to 40-fold increase of hMSCs' yield after two weeks of cultivation compared to alpha-MEM with FCS. The hMSCs expanded in the described co-culture system retain their osteogenic, adipogenic and chondrogenic differentiation potential in vitro and produce bone-like mineralized tissue when propagated on 3D polylactide scaffolds in immunodeficient mice. Our protocol thus allows for very effective one-step, xenogeneic protein-free expansion of hMSCs, which can be easily transferred into good manufacturing practice (GMP) conditions for large-scale, clinical-grade production of hMSCs for purposes of tissue engineering.
Keywords: Tissue engineering; Multipotent mesenchymal stromal cells; Human serum; Expansion; Differentiation; Orthopedic surgery;

The effect of ultra-nanocrystalline diamond films on the proliferation and differentiation of neural stem cells by Ying-Chieh Chen; Don-Ching Lee; Chao-Yang Hsiao; Yu-Fen Chung; Huang-Chin Chen; Joseph P. Thomas; Way-Faung Pong; Nyan-Hwa Tai; I.-Nan Lin; Ing-Ming Chiu (3428-3435).
The interaction of ultra-nanocrystalline diamond (UNCD) with neural stem cells (NSCs) has been studied along with its surface modification in order to improve its function as a biomaterial. Hydrogen- and oxygen-terminated UNCD films were compared with standard grade polystyrene in terms of their impact on the growth, expansion and differentiation of NSCs. When NSCs were cultured on these substrates in low serum and without any differentiating factors, hydrogen-terminated UNCD films spontaneously induced cell proliferation and neuronal differentiation. Oxygen-terminated UNCD films were also shown to further improve neural differentiation, with a preference to differentiate into oligodendrocytes. Hence, controlling the surface properties of UNCD could manipulate the differentiation of NSCs for different biomedical applications. These observations raise the potential for the use of UNCD as a biomaterial for central nervous system transplantation and tissue engineering.
Keywords: Neural stem cells; Neural differentiation; Ultra-nanocrystalline diamond; ECM;

An embolic gelling solution based on acrylic copolymers in ethanol for the treatment of arteriovenous malformations by Aymeric Seron; Laurence Moine; Alexandre Laurent; Michel Wassef; Gérard Guiffant; Patrice Flaud; Denis Labarre (3436-3443).
We report the preparation of an embolic agent based on specific association of an acrylic copolymer with dedicated particles formulated in ethanol. The copolymers were synthesized by radical polymerization of tertiobutylacrylamide (tBA) and 2-hydroxypropyl methacrylate (HPMA). Influences of the monomers composition, molecular weight and copolymer concentration have been evaluated on an in vitro model. Introduction of tBA units improves significantly the occlusion properties but these properties are similar whatever the molecular weight of the copolymer. As observed by viscosity studies, it seems necessary to work with a relatively high polymer concentration (C > Ce) to form a cohesive embolus. Addition of solid particles composed by a crosslinked polymer of 2-hydroxyethyl methacrylate (HEMA) and N-trishydroxymethyl methacrylamide (TRIS) in the acrylic copolymer solution has allowed to obtain an embole having an enhanced cohesion and giving a more compact structure. An in vivo evaluation has been performed by injection of this embolic agent in intercostal arteries and renal artery of sheep. There was no fragmentation of the plug during and after injection and a complete arterial occlusion by a cohesive embole. The pathological examination confirmed that there was a complete arterial occlusion by the plug and that the dedicated particles were as expected embedded in the precipitate acrylic copolymer.
Keywords: Embolization; Gelling solution; Acrylic copolymer; Crosslinked particles;

The mechanical properties and bioactivity of poly(methyl methacrylate)/SiO2–CaO nanocomposite were investigated using dimethyldiethoxysilane (DMDES) and tetraethoxysilane (TEOS), which could produce two and four siloxane linkages, respectively, after a sol–gel reaction. Methyl methacrylate was co-polymerized with 3-(trimethoxysilyl)propyl methacrylate and then co-condensed with DMDES (specimen D) and TEOS (specimen T), respectively, with calcium nitrate tetrahydrate under acidic conditions. The fracture toughness of specimen D was much improved compared to that of specimen T, whereas its fracture strength, hardness, and apatite-forming ability in simulated body fluid (SBF) were slightly decreased. The improved fracture toughness of specimen D without losing apatite-forming ability was explained by the decrease of siloxane linkage numbers and the introduction of alkyl groups in silica structure because covalently bonded siloxane linkages produce hard and brittle fracture behavior in the nanocomposite while the alkyl groups help to make the silica as linear chain structure. The practical implication of these results is that this new nanocomposite can be applied to the filler materials for bone cement and dental composite resin because of its good bioactivity and improved mechanical properties.
Keywords: PMMA; SiO2–CaO; Siloxane linkage; Fracture toughness; Bioactivity;

Temperature-responsive monodisperse spheres are useful for various in vivo and in vitro applications. Size, response temperature and biocompatibility are particularly important consideration with in vivo applications. In this work, we constructed fusion proteins of low antigenic elastin-like peptide (ELP) and a polyaspartic acid chain, and studied the particles that had a favorable size and temperature of formation of particle. From DLS analysis, we confirmed that some of them formed particles with less than 100 nm in diameter around 37 °C, while the diameter of ELPs alone is larger than 1 μm in diameter. The (PGVGV)160D22, which is composed of a short aspartic acid chain and a long ELP region, had a tendency to form large particles. The temperature of formation and collapse of the protein particle were dependent on the length of the ELP and the polyaspartic acid chain, and the concentration of proteins. The direct observation with TEM indicated that the morphologies of the particles were spherical except when (PGVGV)160D22 was used. The intensities of the environment-sensitive hydrophobic fluorescence increased at 37 °C more than 1.5 times as much as at 25 °C both in free form and modified at the ELP region. These results indicated that the polarity of the environment surround the fluorescence decreased or the movement of fluorescence was limited, and thus, implied that the ELP formed a more hydrophobic or rigid region and could hold hydrophobic drugs. These results suggest that a temperature-responsive protein particle with favorable size and temperature of formation can be constructed that is suitable for any in vitro or in vivo application.
Keywords: Drug delivery; Elastin; Nanoparticle; Thermally responsive material;

The impact of diamond nanocrystallinity on osteoblast functions by Lei Yang; Brian W. Sheldon; Thomas J. Webster (3458-3465).
Nanocrystalline diamond has been proposed as an anti-abrasive film on orthopedic implants. In this study, osteoblast (bone forming cells) functions including adhesion (up to 4 h), proliferation (up to 5 days) and differentiation (up to 21 days) on different diamond film topographies were systematically investigated. In order to exclude interferences from changes in surface chemistry and wettability (energy), diamond films with nanometer and micron scale topographies were fabricated through microwave plasma enhanced chemical-vapor-deposition and hydrogen plasma treatment. Scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy and water contact angle measurements verified the similar surface chemistry and wettability but varied topographies for all of the diamond films prepared on silicon in this study. Cytocompatibility assays demonstrated enhanced osteoblast functions (including adhesion, proliferation, intracellular protein synthesis, alkaline phosphatase activity and extracellular calcium deposition) on nanocrystalline diamond compared to submicron diamond grain size films for all time periods tested up to 21 days. An SEM study of osteoblast attachment helped to explain the topographical impact diamond had on osteoblast functions by showing altered filopodia extensions on the different diamond topographies. In summary, these results provided insights into understanding the role diamond nanotopography had on osteoblast interactions and more importantly, the application of diamond films to improve orthopedic implant lifetimes.
Keywords: Osteoblast; Adhesion; Proliferation; Differentiation; Nanocrystalline diamond;

Simultaneous delivery of doxorubicin and gemcitabine to tumors in vivo using prototypic polymeric drug carriers by Twan Lammers; Vladimir Subr; Karel Ulbrich; Peter Peschke; Peter E. Huber; Wim E. Hennink; Gert Storm (3466-3475).
Copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) are prototypic and well-characterized polymeric drug carriers that have been broadly implemented in the delivery of anticancer therapeutics. To demonstrate that polymers, as liposomes, can be used for simultaneously delivering multiple chemotherapeutic agents to tumors in vivo, we have synthesized and evaluated an HPMA-based polymer–drug conjugate carrying 6.4 wt% of gemcitabine, 5.7 wt% of doxorubicin and 1.0 mol% of tyrosinamide (to allow for radiolabeling). The resulting construct, i.e. poly(HPMA-co-MA-GFLG-gemcitabine-co-MA-GFLG-doxorubicin-co-MA-TyrNH2), was termed P-Gem-Dox, and was shown to effectively kill cancer cells in vitro, to circulate for prolonged period of time, to localize to tumors relatively selectively, and to inhibit tumor growth. As compared to control regimens, P-Gem-Dox increased the efficacy of the combination of gemcitabine and doxorubicin without increasing its toxicity, and it more strongly inhibited angiogenesis and induced apoptosis. These findings demonstrate that passively tumor-targeted polymeric drug carriers can be used for delivering two different chemotherapeutic agents to tumors simultaneously, and they thereby set the stage for more elaborate analyses on the potential of polymer-based multi-drug targeting.
Keywords: Chemotherapy; Copolymer; Drug delivery; Gamma irradiation; In vivo test;

The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation by Ching-Li Tseng; Wen-Yun Su; Ko-Chung Yen; Kai-Chiang Yang; Feng-Huei Lin (3476-3485).
To develop a polymer-anticancer drug conjugate, we employed gelatin nanoparticles (GPs) as carriers of cisplatin (CDDP) with anticipated improved therapeutic effect and reduced side effects. The anticancer activities of CDDP-incorporated in GPs (GP–Pt) with biotinylated-EGF (bEGF) modification (GP–Pt–bEGF) were studied. GP–Pt–bEGF with EGFR affinity produced much higher Pt concentrations in A549 cells (high EGFR expression) than in HFL1 cells (low EGFR expression). An in vitro anticancer study showed that GP–Pt–bEGF was more potent than free CDDP or GP–Pt because of its rapid effect on the cell cycle as well as a lower IC50 (1.2 μg/ml) that inhibits A549 cell growth. PI staining showed that cells treated with GP–Pt-bEGF for only 4 h had the highest sub-G1 population.The CDDP formulations – free CDDP, GP–Pt, and GP–Pt–bEGF – were given by intratumorous injections to SCID mice in a subcutaneous model. This treatment showed that GP–Pt–bEGF had stronger anti-tumor activity and was less toxic than free CDDP in vivo. Mice treated with GP–Pt–bEGF showed slight body weight loss, whereas free CDDP treatment at the same dose caused a body weight loss of 20–30%. Furthermore, these formulations were given to mice with lung cancer via aerosol delivery. This treatment showed that inhaled GP–Pt–bEGF could target EGFR-overexpressing cells to achieve high cisplatin dosage in cancerous lungs.To summarize, gelatin nanoparticles loaded with CDDP and decorated with EGF tumor-specific ligand were successfully developed. Their in vitro and in vivo targeting ability and anticancer effect were confirmed. The aerosol delivery of the nanodrug carrier was demonstrated. Simple aerosol delivery of targeted drug carriers may prove useful for the clinical treatment of lung cancer patients.
Keywords: Gelatin nanoparticle (GPs); Cisplatin (CDDP); Lung cancer; Anticancer activity; Epidermal growth factor (EGF); Inhalation;

A key tenet of tissue engineering is the principle that the scaffold can perform the dual roles of biomechanical and biochemical support through presentation of the appropriate mediators to surrounding tissue. While growth factors have been incorporated into scaffolds to achieve sustained release, there are a limited number of studies investigating release of biologically active molecules from reactive two-component polymers, which have potential application as injectable delivery systems. In this study, we report the sustained release of platelet-derived growth factor (PDGF) from a reactive two-component polyurethane. The release of PDGF was bi-phasic, characterized by an initial burst followed by a period of sustained release for up to 21 days. Despite the potential for amine and hydroxyl groups in the protein to react with the isocyanate groups in the reactive polyurethane, the in vitro bioactivity of the released PDGF was largely preserved when added as a lyophilized powder. PUR/PDGF scaffolds implanted in rat skin excisional wounds accelerated wound healing relative to the blank PUR control, resulting in almost complete healing with reepithelization at day 14. The presence of PDGF attracted both fibroblasts and mononuclear cells, significantly accelerating degradation of the polymer and enhancing formation of new granulation tissue as early as day 3. The ability of reactive two-component PUR scaffolds to promote new tissue formation in vivo through local delivery of PDGF may present compelling opportunities for the development of novel injectable therapeutics.
Keywords: Polyurethane; Scaffold; Growth factors; Wound healing; Drug delivery; In vivo test;

Cryopreservation based on freezing protocols for the long-term storage of microencapsulated myoblasts by Ainhoa Murua; Gorka Orive; Rosa Ma. Hernández; José Luis Pedraz (3495-3501).
One important challenge in biomedicine is the ability to cryogenically preserve not only cells, but also tissue-engineered constructs. In the present paper, alginate-poly-l-lysine-alginate (APA) microcapsules containing erythropoietin (Epo)-secreting C2C12 myoblasts were elaborated, characterized and tested both in vitro and in vivo. Dimethylsulfoxide (DMSO) was selected as cryoprotectant to evaluate the maintenance of physiological activity of cryopreserved microencapsulated myoblasts employing procedures based on freezing protocols up to a 45-day cryopreservation period. High chemical resistance of the cryopreserved microcapsules was observed using 10% DMSO as cryoprotectant following a standard slow-cooling procedure. Although a 42% reduction in Epo release from the microencapsulated cells was observed in comparison with the non-cryopreserved group, the in vivo biocompatibility and functionality of the encapsulated cells subcutaneously implanted in Balb/c mice was corroborated by high and sustained hematocrit levels over 194 days and lacking immunosuppressive protocols. No major host reaction was observed. Based on the results obtained in our study, a slow-cooling protocol using 10% DMSO as cryoprotectant (confirmed for cryopreservation periods up to 45 days) might be considered a suitable therapeutic strategy if the long-term storage of microencapsulated cells, such as C2C12 myoblasts is pretended.
Keywords: Alginate; Cell encapsulation; Cryopreservation; Dimethylsulfoxide; Erythropoietin; Myoblast;