Regenerative medicine is a multidisciplinary field of research which involves the use of biomaterials, growth factors, and stem cells to repair, replace, or regenerate tissues and organs damaged by injury or disease and has definitely evolved in parallel with advances in the biotechnological field.
Tissue engineering enhances the tissues regeneration through the use of biodegradable scaffolds combined with in vitro cultured cells. Regarding the cellular component, stem cells are ideal candidates for regenerative medicine due to their ability to commit to multiple cell lineages and to self-renew.
Stem cells for regenerative medicine applications should meet the following criteria: i) abundance of cells (millions to billions); ii) minimally invasive procedure with minimal morbidity harvest; iii) differentiation potential along multiple cell lineages in a controllable and reproducible manner; iv) safe transplantation to either an autologous or allogeneic host; v) possibility of isolation in accordance with the current Good Manufacturing Practice guidelines
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Several sources of stem cells are likely to meet the criteria, yet the human adipose derived stem cells (hADSCs) have multiple benefits with the increased occurrence of obesity, SAT is accessible and thus hADSCs can be harvested in large quantities with minimal risk. In addition, AT yields manifold greater numbers of mesenchymal stem cells (MSCs) compared to bone marrow. Due to their secretory profile, hADSCs delivered into injured or diseased tissue, stimulate the recovery in a paracrine manner. These cells were shown to modulate the "stem cell niche" of the host by stimulating the recruitment of the endogenous stem cells to the site of injury where they promote the differentiation toward the required lineage pathway. In addition, hADSCs secrete almost all of the growth factors involved in normal wound healing. Since 2001, when the existence of stem cells within this tissue was reported, AT has acquired increased importance as a stem cell source for a wide range of potential applications in regenerative medicine strategies.
Traumas, resection of tumors as well as congenital abnormalities, often result in defects due to the loss of soft tissue, composed of SAT. Besides the reconstruction of the functional tissue, a modern requirement in regenerative medicine is also the aesthetic restoration of the resulting imperfections. Classical clinical strategies for AT engineering (ATE) include the use of autologous fat implants, which are considered to be the ideal filling material in terms of biocompatibility, immune response and avoidance of graft rejection. However, AT transplantation yields unpredictable results, due to varying degrees of graft resorption over time (40%-60% volume loss) and lack of sufficient revascularization.
The alternative use of synthetic surrogates (Teflon, silicone implants) or allogenic materials, like bovine collagen, have the advantage of endless supply, but clinical experiences revealed various deficiencies, such as rupture, capsular contracture, dislocation, suboptimal biocompatibility of the implants and allergic reactions.
Modern strategies in current ATE applications involve the design of 3D cell-scaffold bioconstructs obtained by preseeding the scaffold with undifferentiated cells. In order to achieve in situ functional de novo tissue, the embedded hADSCs are committed towards the adipogenic lineage by subjecting the bioconstructs to in vitro adipogenic conditions. Subsequently, the engineered tissue is expected to be structurally, mechanically and functionally integrated to the implantation site. Overall, the most important feature of this modern strategy is the achievement of a long-term and predictable clinical application result ensured by the control of the scaffold's composition, implanted cell number and the differentiation status and kinetics. After implantation, hADSCs remain viable at the wound site and continuously secrete growth factors, just as it occurs in the natural process of wound healing. Consequently, at the injury site, implanted cells that undergo differentiation generate not only an inert filling tissue, but they are also able to stimulate cell recruitment from stem cell niches in order to aesthetically restore the site from injury.
Hydrogels are crosslinked networks of hydrophilic polymers that have the capacity to retain large volumes of water. For certain tissue engineering applications, hydrogels have specific advantages over other scaffold materials, such as their capacity to be mixed with cells in liquid form, and subsequently injected to fill irregularly shaped tissue defects for in situ gelation. Hydrogels are also favorable as carriers for drugs, peptides, and proteins in controlled delivery applications. Numerous studies have demonstrated the importance of understanding structure-property relationships of hydrogels. With respect to hydrogels as tissue engineering scaffolds, this knowledge is crucial because material properties affect both scaffold performance and cell function.
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To successfully engineer an adipose construct into a complex 3D system, a scaffold is required to serve as a site for cellular attachment, proliferation and subsequent differentiation. It should also possess proper mechanical and chemical properties, designed in accordance with the final host tissue.
The scaffolds must be biocompatible and biodegradable (resorbing over time, leaving nothing but living de novo tissue behind). The process of scaffold degradation is critical as it should not produce any toxic byproducts. In addition, the timing of this process must be accurately controlled and predicted. If the scaffold does not degrade quickly enough, fibrous tissue may reduce the effective area for preadipocyte expansion and differentiation and vascular ingrowth. If degradation occurs too quickly, the construct may lose its desired three-dimensional shape.
The microarchitecture of a scaffold is important for controlling cell behavior and tissue integration. The biomaterials used to create an engineered construct must provide an adequate pore size and shape for cellular attachment and growth and an appropriate pore density (porosity) to allow the diffusion of nutrients and vascular ingrowth.
Ideally, materials should provide a chemically reactive foundation that allows the attachment of biologically activity moieties (growth factors, adhesion receptors, peptides, etc) that might be necessary for the induction or sustenance of cellular growth.
In the following sections the attention is focused in several protein and polysaccharide-based polymers that are commonly used in research works for drug or cell delivery within the tissue engineering field.
4.1. Protein-based scaffolds
With respect to protein-based polymers, collagen and gelatin scaffolds will be described in more detail in the following sections. Protein-based polymers have the advantage of mimicking many features of ECM and thus have the potential to direct the migration, growth and organization of cells during tissue regeneration and wound healing and for stabilization of encapsulated and transplanted cells.
4.1.1. Collagen scaffolds
A great number of biomaterials have been used in the perspective of tissue reconstruction, but collagen-based scaffolds were proven to provide the best results [12 al nostru]. Collagen is regarded as an ideal scaffold or matrix for TE as it is the major protein component of the ECM, providing support to connective tissues such as skin, tendons, bones, cartilage, blood vessels, and ligaments. In its native environment, collagen interacts with cells in connective tissues and transduces essential signals for the regulation of cell anchorage, migration, proliferation, differentiation, and survival.
Twenty-seven types of collagens have been identified to date, but collagen type I is the most abundant and the most investigated for biomedical applications. Extracted as aqueous solution or gel, type I collagen can be processed in different forms such as: medical devices, artificial implants, or support for drug release and scaffolds for tissue regeneration that have an important role in medicine today.
In order to tailor the degradation of collagen in accordance with the target tissue's properties, crosslinking of the fibers is a must. In addition, one of the most important difficulties in the processability of collagen based 3D scaffolds is the sterilization, as nearly all the common methods produce some degree of alteration.
The addition of bioactive molecules of natural origin in the composition of the currently used collagen biomaterials could improve the biological performances of the resulting scaffold in terms of cellular adhesion, proliferation potential, extracellular matrix synthesis, intercellular signaling, modulation of stem cells differentiation, etc. An attractive source of natural polymers with great physico-chemical properties is the silk isolated from Bombyx mori cocoons. These fibers are composed primarily of two types of proteins: fibroin, the core filaments of silk, and sericin, the antigenic gum-like protein surrounding the fibers. Silk sericin (SS) is a granular protein with adhesive and gelatin-like characteristics, which was shown to be responsible for the proliferation and attachment of several mammalian cell lines, as well as for the activation of collagen production, both in vitro and in vivo.
Recently [al nostru] we demonstrated not only the biocompatibility of a new superporous collagen-sericin (Coll-SS) hydrogel, but also its potential to sustain the adipogenic differentiation of hADSCs embedded inside the scaffold and subjected to adipogenic conditions.
The biocompatibility of Coll-SS versus Coll was tested in terms of viability and proliferation, by double fluorescence Live/Dead staining and quantitative MTT assay. In addition, the cytotoxic potential of both matrices on hADSCs was evaluated using lactate dehydrogenase (LDH) spectrophotometric test.
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LIVE/DEAD staining of the hADSCs-3Dscaffold revealed that the ratio between the viable (green labeled) and the dead (red labeled) cells was constantly positive, whereas a higher cellular density was revealed on Coll-SS than on the control system. Consequently, hADSCs on the surface of Coll-SS reached a confluent monolayer faster than the cells on top of Coll scaffold, thus displaying a higher proliferative potential in the presence of sericin. In addition, in the context of these proliferative 3D cultures, the amount of dead cells observed was lower at 6 days, as compared to 2 and 4 days post-seeding in both bioconstructs, suggesting that hADSCs were able to adapt to the 3D microenvironment provided by the scaffolds. The fluorescence microscopy investigation also revealed the fibroblast-like morphology of the green-labeled living cells. However, cell density on Coll-SS was higher than on Coll system at 2 days post-seeding, probably due to the sticky properties of sericin. These observations are in accordance with previous findings, which stated that sericin enhances cell proliferation and attachment.
Based on the evaluation of Coll and Coll-SS cytotoxic potential on hADSCs up to one week, the activity of LDH in the culture media was found to be increased at 2 days post seeding as compared to 4 and 6 days in both bioconstructs. Further on, LDH activity in Coll-SS system decreased dramatically in the first 4 days of culture and maintained this descending profile up to 6 days, but at a lower rate. Additionally, the cytotoxic effect of Coll showed an overall decreasing trend, displaying only one significant difference between 4 and 6 days of culture.
The behavior of the biomaterials in the adipogenic conditions used by us was revealed through SEM. Therefore, Coll biomatrix showed a slow and constant degradation rate, whereas Coll-SS strongly compacted, probably due to the release of sericin from the original scaffold into the medium. Considering wound healing applications, low levels of sericin released from the scaffold could be beneficial, as sericin is able to promote collagen production in wounds, but, at the same time, the matrix should also maintain its stability.
Contrast-phase microscopy images of Oil Red O stained bioconstructs revealed that the neutral lipid accumulation started at 7 days post adipogenic induction in cells loaded in both Coll and Coll-SS scaffolds. The number of lipid droplets, as well as their volume, increased during the adipogenic process in both bioconstructs, thus confirming the SEM observations regarding cellular morphology. No significant differences were observed in terms of intracytoplasmatic lipid droplets accumulation in cells undergoing adipogenesis in Coll and Coll-SS up to 28 days.
In order to evaluate the evolution of the differentiation process in our adipogenic conditions, the expression pattern of early and late adipogenic markers was investigated up to 28 days using the RealTime RT-PCR technique.
In our study, PPARÎ³2 transcript levels were detected, including in the samples harvested before inducing in vitro adipogenesis in both hADSCs-Coll and hADSCs-Coll-SS bioconstructs. This feature suggests that PPARÎ³2 gene is active at basal levels independent of the presence of pro-adipogenic conditions and confirms its potential as master activator and regulator of adipogenesis. Although our results show that PPARÎ³2 expression describes an ascendant trend post induction, statistical significant increases in gene expression were registered at 14 days, both for Coll and for Coll-SS systems, when compared to 7 days. Furthermore, we detected a significant upregulated PPARÎ³2 profile in the presence of sericin (hADSCs-Coll-SS bioconstruct) at 28 days, as compared to 21 days. This is in contrast with PPARÎ³2 mRNA levels obtained for control (hADSCs-Coll bioconstruct), since an increase was detected at 21 days versus 14 days, but no other significant upregulation was noticed until the end of the experiment. When comparing PPARÎ³2 expression pattern in the presence (hADSCs-Coll-SS bioconstruct) or absence of sericin (hADSCs-Coll construct), the statistical important differences occurred at 28 days post adipogenic induction.
Once activated, PPARÎ³2 induced the transcription of FAS, aP2 and perilipin, which act together in order to synthesize, transport and mediate triacylglycerol (TAG) metabolism, respectively. Thus, we first detected the activation of FAS gene, one of the downstream targets of PPARÎ³2 , at 7 days post-induction in both bioconstructs, but to a higher extent in the presence of sericin, as compared to the control system. Moreover, at 7 days of adipogenic induction, a significant change in FAS gene expression was reported only in hADSCs-Coll-SS bioconstruct as compared to 3 days post-induction. For this bioconstruct, a further significant increase was registered between 7 and 14 days, while for the hADSCs-Coll system, the first statistically significant increase was detected later, at 14 days, as compared to the previous time point. This upregulated profile registered a constant and statistically significant increase in both bioconstructs, during 14-21 days interval, suggesting the constant requirement of free fatty acids synthesis throughout the adipogenic differentiation process, independent of sericin influence. However, the FAS mRNA levels continued to increase up to 28 days post-induction in the presence of sericin, while the transcript levels corresponding to the control sample at 28 days were comparable to those at 21 days of adipogenesis. This difference registered between FAS transcript expression at 28 days in the presence and absence of sericin proved to have statistical significance, highlighting the possible influence of sericin on TAG synthesis during in vitro adipogenesis.
Fatty acid binding protein aP2, required for the transport of TAG across internal membranes, was detected at low levels starting with day 7 post-adipogenic induction. Important statistical differences in aP2 transcript levels were noticed between hADSCs-Coll-SS and hADSCs-Coll bioconstructs at 14, 21 and 28 days after adipogenic induction in our culture conditions, thus confirming that aP2 is a late adipogenic marker and raising the hypothesis that sericin is able to influence its expression in 3D culture systems.
Regarding the lipid droplet associated protein (perilipin), our results suggest that its expression pattern is highly influenced by the presence of sericin in 3D systems undergoing adipogenesis, since significant statistical differences appeared between the samples recovered simultaneously from Coll and Coll-SS biomatrices at 14, 21 and 28 days post adipogenic induction.
Perilipin protein expression was qualitatively analyzed by fluorescence and confocal microscopy and quantitatively evaluated by flow cytometric detection. Overall, both evaluation techniques revealed that perilipin expression was higher for cells that differentiated in the presence of sericin (Col-SS biomatrix) than for those undergoing adipogenesis in pure collagen bioconstructs (Coll biomatrix).
Briefly, our data showed that the addition of the sticky protein sericin enhanced the proliferation rate of the seeded cells, thus improving the biocompatibility of the Coll-SS scaffold. Furthermore, the study brought new valuable information on the in vitro adipogenic differentiation conducted in collagen-based biomatrices, in the presence or absence of sericin and the influence of the scaffold on the evolution of the process. Sericin stimulated an overexpression of PPARÎ³2, triggering a subsequent upregulated transcription of FAS, aP2 and perilipin markers. Moreover, based on the expression patterns obtained for these adipogenic markers in both constructs, a higher efficiency of adipogenesis could potentially be correlated with the presence of sericin in the 3D cellular environment.
Gelatin is a natural polymer that is derived from collagen, and is commonly used for pharmaceutical and medical applications because of its biodegradability and biocompatibility in physiological environments. Moreover, gelatin has relatively low antigenicity because of being denatured in contrast to collagen which is known to have antigenicity due to its animal origin. Gelatin is obtained by the acid and the alkaline processing of collagen. As a result, two different types of gelatin can be produced depending on the method in which collagen is pre-treated, prior to the extraction process. The biodegradable hydrogel matrices are prepared by chemical crosslinking of acidic or basic gelatin and are enzymatically degraded in the body, with time. Under specific conditions, such as temperature, solvent or pH, gelatin macromolecules present sufficient flexibility to realize a variety of conformations. Structural diversity of gelatin chain units determines the specific features of gelatin properties.
Despite of their advantages, these materials often display too low mechanical properties which reduce the potential use in TE. Interpenetrating hydrogels based on gelatin were developed in the aim of improving different properties to better match specific requirements. Interpenetrating polymer networks (IPNs) are described as "a class of stimuli-responsive materials consisting in polymer blends in network form". Remarkably, there are evidences on the fact that IPNs hydrogels would present improved mechanical properties with respect to their individual crosslinked networks.
Our results show that the new developed porous gelatin-alginate-polyacrilamide (G-CA-PAA) scaffold with IPN structure displayed a better biocompatibility when compared to a gelatin-alginate scaffold, probably due to its excellent stability in physiological environment. Although PAA is a highly cytotoxic synthetic polymer, its limited addition in the composition of a TE designed scaffold did not determine a decrease of the biocompatibility, on the contrary. SEM micrographs showed a uniform pose size and distribution in G-CA-PAA as compared to G-CA which subsequently determined a better cellular proliferation of the embedded hADSCs as revealed by fluorescence microscopy and spectrophotometric MTT assay.
4.2. Polysaccharidic polymers
Polysaccharides are a class of biopolymers constituted by simple sugar monomers. The monomers (monosaccharides) are linked together by O-glycosidic bonds that can be made to any of the hydroxyl groups of a monosaccharide, conferring polysaccharides the ability to form both linear and branched polymers. Differences in the monosaccharide composition, chain shapes and molecular weight dictate their physical properties including solubility, gelation and surface properties. These biological polymers can be obtained from different sources: microbial, animal and vegetal. Several advantages can be derived from the use of these macromolecules. They are non-toxic, show interaction with living cells and, with few exceptions, have low costs in comparison with others biopolymers such as collagen. These polysaccharidic polymers have been widely proposed as scaffold materials in TE applications as described in more detail in the following sections.
Alginate is one of the most studied and applied polysaccharidic polymers in tissue engineering and drug delivery field. They are abundant in nature and are found as structural components of marine brown algae and as capsular polysaccharides in some soil bacteria. Commercial alginates are extracted from three species of brown algae. These include Laminaria hyperborean, Ascophyllum nodosum, and Macrocystis pyrifera in which alginate comprises up to 40% of the dry weight. Bacterial alginates have also been isolated from Azotobacter vinelandii and several Pseudomonas species.
Alginates are naturally derived polysaccharide block copolymers composed of regions of sequential Î²-D-mannuronic acid monomers (M-blocks), regions of Î±-L-guluronic acid (G blocks), and regions of interspersed M and G units. The length of the M- and G-blocks and sequential distribution along the polymer chain varies depending on the source of the alginate.
Alginates undergo reversible gelation in aqueous solution under mild conditions through interaction with divalent-cations such as Ca2+ that can cooperatively bind between the G-blocks of adjacent alginate chains creating ionic inter-chain bridges. This gentle property has led to their wide use as cell transplantation vehicles to grow new tissues and as wound dressings. Moreover, alginate as an anionic polymer with carboxyl end groups is a good mucoadhesive agent. However, alginate hydrogels used in these applications have uncontrollable degradation kinetics and gels dissolve in an uncontrollable manner following the loss of divalent-cations releasing high and low molecular weight alginate units. Attempts have been made to covalently crosslink sodium alginate with gelatin and sodium tetraborate or with albumin. The hydrogel is formed because blocks of guluronic residues bind to cations resulting in a three dimensional network of alginate fibers held together with ionic interactions. The model that best describes this network is the "egg-box model". The resultant network is a function of the frequency and length of contiguous guluronic acid residues as well as the concentration and type of the cation. The changes in frequency and length of adjacent guluronic acid units, as well as, changes in cation concentration can alter the number of alginate fibers held together changing the overall strength of the network. In a few words, alginates possessing a high guluronic acid content develop stiffer, more porous gels which maintain their integrity for longer periods of time.
Alginate-based materials are pH-sensitive. Biomolecules release from alginate-based materials in low pH solutions is significantly reduced which could be advantageous in the development of a delivery system. This pH-dependent behavior of alginate is exploited to tailor release profiles and in the development of 'smart' systems. However, at higher pH alginate undergoes a rapid dissolution which may result in burst release of protein drugs and subsequently their denaturation by proteolytic enzymes. Therefore, many modifications in the physicochemical properties are needed for the prolonged controlled release of protein drugs.
Alginate beads/hydrogels can be prepared by extruding/maintaining a solution of sodium alginate containing the desired protein or cells, as droplets/blocks, in to a divalent crosslinking solution such as Ca2+, Sr2+, or Ba2+. Monovalent cations and Mg2+ ions do not induce gelation. Although alginate beads/hydrogels can be prepared by simple and mild procedures, this method has a major limitation that is the drug loss during bead/hydrogel preparation, by leaching through the pores in the beads/hydrogels.
In the context of a small number of reports concerning AT regeneration using alginate 3D systems, we evaluated the biological performance of a novel layer-shaped alginate matrix that incorporates hADSCs. Therefore, the biological performances of two alginate hydrogel matrices, as temporary physical support for hADSCs, were compared in order to identify an appropriate environment for cell proliferation and adipogenic differentiation. These hydrogels were designed as thin layer disks and prepared by the controlled diffusion of two different cross-linking agents (calcium chloride and calcium gluconate) in cell-loaded alginate solution. The behavior of hADSCs cultured under 3D conditions within alginate hydrogels was analyzed in terms of viability, proliferation, morphology, and adipogenic differentiation. We found that both calcium gluconate and calcium chloride alginate hydrogels successfully support survival and adipogenic differentiation of hADSCs. Moreover, an enhancement of biological performance was detected in the case of calcium gluconate matrix, suggesting its promising application in soft tissue engineering.
In order to examine cell survival during culture, the viability the encapsulated hADSCs in the alginate hydrogels were evaluated after 2 and 7 days of culture by a flow cytometry-based LIVE/DEAD assay. Most of the entrapped cells (over 80%) successfully survived within the reference hydrogel and the calcium gluconate hydrogel. Furthermore, the percentage of viable cells was significantly higher within calcium gluconate hydrogel. These results are in keeping with the observation that the alginate matrices present a structure formed by interconnected pores, which is suitable to accommodate the hADSCs. Furthermore, these matrices successfully supported their viability, nutrient and protein transport. To get a more complete image on the cell survival and proliferation of hADSC embedded in the reference hydrogel and the calcium gluconate hydrogel, microscopic and spectrophotometric MTT based assays were issued at the same time intervals. These analyses provided evidence that both alginate-based hydrogels stimulated cell proliferation, the number of hADSCs within hydrogels increasing with the length of the incubation period. This finding is contrary to other studies showing that alginate hydrogels do not allow or inhibit proliferation and growth of several different types of cells when they are either grown on their surface as 2D monolayer culture or incorporated into the matrix of the gel . Interestingly, in our study, a higher number of metabolically active cells was found within the calcium gluconate hydrogel rather than in the reference hydrogel. Therefore, the growth and metabolic activity of hADSCs seemed to be influenced by the alginate crosslinking agent. The better cell survival and growth supporting activities of the calcium gluconate hydrogel could be explained by the larger pore sizes than in the case of reference hydrogel. Due to this particular structure, a more efficient transport of oxygen and nutrients may take place in the hydrogel matrix.
To evaluate the effect of calcium gluconate as alginate reticulating agent on adipogenesis, hADSCs encapsulated in alginate hydrogels were cultured with an adipogenic medium up to 21 days. Typical markers of lipid biosynthesis were analyzed. Thus, results of the Oil Red O staining showed that hADSCs embedded in calcium gluconate hydrogelstarted the process of neutral lipid accumulation at 7 days post-adipogenic induction, whereas in the cells embedded
in the reference hydrogel, a positive Oil Red O staining was observed after 15 days of adipogenic induction. This result demonstrated that in calcium gluconate hydrogels greater number of cells have undergone adipogenesis within 21 days of induction, as compared to cells embedded in reference hydrogel.
Flow cytometric detection of perilipin expression also confirmed a more rapid induction of adipogenesis in calcium gluconate hydrogelcompared with reference hydrogel. The delay in the adipogenic induction of hADSC embedded in the Reference hydrogel could be explained by the complexity of the effects that Ca2+ exerts on the adipogenesis process [58-60]. Thus, extracellular Ca2+ concentration ([Ca2+]e) can modulate many aspects of cellular behavior, such as: proliferation, differentiation, survival, and death. It has been shown that the levels of [Ca2+]e are important in regulating adipocyte lipid accumulation. Furthermore, increasing intracellular Ca ions ([Ca2+]i) in early stages of differentiation suppressed human adipocyte differentiation. Accordingly, the present study demonstrates a faster expression of adipogenic markers (intracellular lipid droplets accumulation and perilipin expression) in calcium gluconate hydrogel compared with the reference hydrogel, probably due to a slower release of the calcium ions. In addition, we cannot rule out a possible influence of [Ca2+] on the cell sensitivity to insulin, an adipogenic inductor contained by the adipogenic medium, as it is largely accepted that increased [Ca2+] contributes to insulin resistance . Since perillipin is not expressed before adipogenic differentiation , its high expression in the hADSCs embedded in calcium gluconate hydrogel suggests that calcium gluconate is effective for the adipogenic differentiation of hADSCs in this 3D culture system.
A hADSC-laden alginate hydrogel shaped as thin layer disk was developed by diffusion of a new compound, calcium gluconate, within the alginate gel matrix. In addition, we investigated whether calcium gluconate had a positive effect as alginate cross-linking agent on cell viability, proliferation and adipogenic differentiation in comparison with a reference matrix. Our results clearly demonstrate that both alginate microenvironments support hADSCs viability and proliferation. These matrices do not alter the cell morphology and create conditions that are favorable for adipogenic differentiation. Furthermore, an enhancement of all these cellular parameters was found out in the case of alginate hydrogel obtained by using calcium gluconate as reticulating agent, suggesting its promising application in soft tissue engineering.