Preparation and characterization of novel polymeric microcapsules for live cell encapsulation and therapy

This article describes the preparation and in vitro characterization of novel genipin cross-linked alginate-chitosan (GCAC) microcapsules that have potential for live cell therapy applications. This microcapsule system, consisting of an alginate core with a covalently cross-linked chitosan membrane, was formed via ionotropic gelation between calcium ions and alginate, followed by chitosan coating by polyelectrolyte complexation and covalent cross-linking of chitosan by naturally derived genipin. Results showed that, using this design concept and the three-step procedure, spherical GCAC microcapsules with improved membrane strength, suppressed capsular swelling, and suitable permeability can be prepared. The suitability of this novel membrane formulation for live cell encapsulation was evaluated, using bacterial Lactobacillus plantarum 80 (pCBH1) (LP80) and mammalian HepG2 as model cells. Results showed that capsular integrity and bacterial cell viability were sustained 6 mo postencapsulation, suggesting the feasibility of using this microcapsule formulation for live bacterial cell encapsulation. The metabolic activity of the encapsulated HepG2 was also investigated. Results suggested the potential capacity of this GCAC microcapsule in cell therapy and the control of cell signaling; however, further research is required.     

The effects of different crossing-linking conditions of genipin on type I collagen scaffolds: an in vitro evaluation

The purpose of this paper is to analyze the properties of fabricating rat tail type I collagen scaffolds cross-linked with genipin under different conditions. The porous genipin cross-linked scaffolds are obtained through a two step freeze-drying process. To find out the optimal cross-link condition, we used different genipin concentrations and various cross-linked temperatures to prepare the scaffolds in this study. The morphologies of the scaffolds were characterized by scanning electron microscope, and the mechanical properties of the scaffolds were evaluated under dynamic compression. Additionally, the cross-linking degree was assessed by ninhydrin assay. To investigate the swelling ratio and the in vitro degradation of the collagen scaffold, the tests were also carried out by immersion of the scaffolds in a PBS solution or digestion in a type I collagenase respectively. The morphologies of the non-cross-linked scaffolds presented a lattice-like structure while the cross-linked ones displayed a sheet-like framework. The morphology of the genipin cross-linked scaffolds could be significantly changed by either increasing genipin concentration or the temperature. The swelling ratio of each cross-linked scaffold was much lower than that of the control (non-cross-linked).The ninhydrin assay demonstrated that the higher temperature and genipin concentration could obviously increase the cross-linking efficiency. The in vitro degradation studies indicated that genipin cross-linking can effectively elevate the biostability of the scaffolds. The biocompatibility and cytotoxicity of the scaffolds was evaluated by culturing rat chondrocytes on the scaffold in vitro and by MTT. The results of MTT and the fact that the chondrocytes adhered well to the scaffolds demonstrated that genipin cross-linked scaffolds possessed an excellent biocompatibility and low cytotoxicity. Based on these results, 0.3 % genipin concentrations and 37 °C cross-linked temperatures are recommended.     

Preparation of Chitosan Cryostructurates with Controlled Porous Morphology and Their Use as 3D-Scaffolds for the Cultivation of Animal Cells

The influence of the conditions of the formation of genipin cross-linked chitosan cryostructurates on the porous morphology and physicochemical properties of these cryostructurates and on the possibility of their use as biopolymer 3D scaffolds for tissue engineering was studied. The chitosan cryostructurates were obtained by freeze-drying a chitosan acetate solution, treating the resulting sponge with an alcohol solution of ammonia to transform the polyaminosaccharide from a salt into a chitosan-base, and then cross-linking the polymer with genipin (the molar ratios of genipin to the number of chitosan amino groups were 0.05, 0.033, and 0.02, respectively). The pore sizes, water-holding capacity, and in vitro biodegradation rate of the cryostructurates were shown to depend on the aforementioned ratio. The properties of the prepared chitosan cryostructurates, the hydrogels formed by chitosan cross-linking with genipin at positive temperatures, and the films cast from genipin-containing chitosan solutions after solvent evaporation were studied and compared. The biocompatibility of the obtained macroporous sponge materials was demonstrated using L929 mouse fibroblasts. Confocal laser microscopy showed that the cells in all of the 3D scaffolds obtained were evenly distributed; they grew and proliferated when cultured in vitro for seven days.     

Novel chitosan-based films cross-linked by genipin with improved physical properties

Novel cross-linked chitosan-based films were prepared using the solution casting technique. A naturally occurring and nontoxic cross-linking agent, genipin, was used to form the chitosan and chitosan/poly(ethylene oxide) (PEO) blend networks, where two types of PEO were used, one with a molecular weight of 20 000 g/mol (HPEO) and the other of 600 g/mol (LPEO). Genipin is used in traditional Chinese medicine and extracted from gardenia fruit. Importantly, it overcomes the problem of physiological toxicity inherent in the use of some common synthetic chemicals as cross-linking agents. The mechanical properties and the stability in water of cross-linked and un-crosslinked chitosan and chitosan/PEO blend films were investigated. It was shown that, compared to the transparent yellow, un-cross-linked chitosan/PEO blend films, the genipin-cross-linked chitosan-based film, blue in color, was more elastic, was more stable, and had better mechanical properties. Genipin-cross-linking produced chitosan networks that were insoluble in acidic and alkaline solutions but were able to swell in these aqueous media. The swelling characteristics of the films exhibit sensitivity to the environmental pH and temperature. The surface properties of the films were also examined by contact angle measurements using water and mixtures of water/ethanol. The results showed that, with the one exception of cross-linked pure chitosan in 100% water, the cross-linked chitosan and chitosan/PEO blends were more hydrophobic than un-crosslinked ones.     

Rheological study of genipin cross-linked chitosan hydrogels

This paper reports the rheological behavior of chitosan solutions that have been cross-linked with different amounts of genipin, at body temperature and physiological pH. The effect of the cross-linker loading on the rheological properties of hydrogels has been evaluated. The oscillatory time sweep method was used to monitor the dynamic viscoelastic parameters during in situ (i.e., in the rheometer) gelation experiments, enabling the determination of the gelation time. The stress and frequency sweeps were employed to measure G' of the cured hydrogels. It was found that the solutions of chitosan cross-linked with genipin, under physiological conditions, could form relatively strong elastic gels when compared to those of pure chitosan. Moreover, the gelation time obtained from the crossover of G' and G' was in excellent agreement with the value obtained from the Winter-Chambon criterion. A significant reduction on this parameter was achieved even at low genipin concentrations. This behavior suggests that these formulations are able to be produced in situ and thus constitute promising matrices for cells and bioactive molecule encapsulations.     

Synthesis and characterization of a novel chitosan-gelatin bioconjugate with fluorescence emission

Polysaccharide-protein conjugations have generated increasing interests for biomedical applications in recent years. A naturally occurring cross-linking reagent, genipin, which has been used in herbal medicine, was employed to cross-link chitosan and gelatin for the preparation of a novel chitosan-gelatin conjugate. The primary amine groups on chitosan and gelatin were covalently linked with genipin, leading to the formation of a chitosan-gelatin conjugate with nitrogen-containing heterocycle units, the pyrindine-like derivatives. The FT-IR and UV-vis studies revealed that chitosan could react with genipin via a nucleophilic ring-opening reaction to construct more sufficient and extensive cross-link networks, as compared with its gelatin counterpart. The UV-vis absorption properties of the chitosan-gelatin conjugates were strongly related to the chitosan-to-gelatin weight ratio in the compositions. It is worth noting that the conjugation process endows the special emission properties of the chitosan-gelatin conjugates, which depends on the cross-linking reaction and the formation of hydrogen bonding involved chitosan-gelatin complex. Fluorescence quenching or enhancement was observed from the chitosan-gelatin conjugates upon coordinated with a wide variety of heavy metal ions (Ag+, Cu2+, Fe2+, and Co2+). This study also examined the possibility of covalent coupling the capture chelator (chitosan) with bioactive protein (e.g., albumin, alpha-globulin, and fibrinogen) to create fluorescence emission. These findings may provide a novel way to deliver therapeutic radionuclides for immuno-targeting purposes in the future.     

Preparation and characterization of nano-hydroxyapatite/chitosan cross-linking composite membrane intended for tissue engineering

In this paper, a series of nano-hydroxyapatite(n-HA)/chitosan cross-linking composite membranes (n-HA; 0, 5, 10, 15, 20 and 30 wt%) were successfully developed by a simple casting/solvent evaporation method. n-HA with size about 20 nm in vertical diameter and about 100 nm in horizontal diameter was successfully synthesized by a hydro-thermal precipitation method, and then dispersed into chitosan/genipin solution with the aid of continuous ultrasound to develop n-HA/chitosan cross-linking composite membranes. The detailed characterizations including Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), water adsorption and tensile test were performed. With the analysis of FTIR spectra and TGA spectra, it suggested that there was existence of possible interactions between polymer and n-HA. Meanwhile, the n-HA content was greatly effected on the morphology as well as the tensile property of composite membrane. In vitro cytotoxicity test suggested that the developed n-HA/chitosan cross-linking composite membrane was non-cytotoxicity against L929 cells after 24 h's incubation might be suitable for further in vivo application.     

Chitosan nanogels by template chemical cross-linking in polyion complex micelle nanoreactors

Chitosan covalent nanogels cross-linked with genipin were prepared by template chemical cross-linking of chitosan in polyion complex micelle (PIC) nanoreactors. By using this method, we were able to prepare chitosan nanogels using only biocompatible materials without organic solvents. PIC were prepared by interaction between chitosan (X(n) = 23, 44, and 130) and block copolymer poly(ethylene oxide)-block-poly[sodium 2-(acrylamido)-2-methylpropanesulfonate] (PEO-b-PAMPS) synthesized by single-electron transfer-living radical polymerization (SET-LRP). PIC with small size (diameter about 50 nm) and low polydispersity were obtained up to 5 mg/mL. After cross-linking of chitosan with genipin, the nanoreactors were dissociated by adding NaCl. The dissociation of the nanoreactors and the formation of the nanogels were confirmed by (1)H NMR, DLS, and TEM. The size of the smallest nanogels was about 50 nm in the swollen state and 20 nm in the dry state. The amount of genipin used during reticulation was an important parameter to modulate the size of the nanogels in solution.     

Preparation and evaluation of decellularized porcine carotid arteries cross-linked by genipin: the preliminary results

Decellularized arteries have been considered as promising scaffolds for small-diameter vascular substitutes. However, weakened mechanical properties, immunological rejection and rapid degradation after transplantation still exist after decellularization. Previous studies indicated that genipin cross-linking can solve these problems. Therefore, genipin was selected as the cross-linking agent for the pre-treatment of decellularized arteries in our study. Histological analysis, scanning electron microscopy, mechanical properties analysis and subcutaneous embedding experiment were adopted to investigate the efficiency of decellularization and the effect of genipin cross-linking on improving mechanical, structural and biological properties of decellularized arteries. Decellularization protocols based on three trypsin concentrations were used to prepare decellularized arteries, after decellularization, arteries were cross-linked with genipin. Results showed that 0.5% trypsin was the most efficient concentration to remove cellular components and preserve ECM. However, mechanical properties of 0.5% trypsin decellularized arteries weakened significantly, while genipin cross-linking improved mechanical properties of decellularized arteries to the same level as fresh arteries. After 4 weeks subcutaneous embedding, cross-linked arteries caused the mildest inflammatory response. In conclusion, genipin could be employed as an ideal cross-linking agent to strengthen mechanical properties, enhance the resistance to degradation and reduce the antigenicity of decellularized arteries for small-diameter blood vessel tissue engineering applications.     

Immobilization of a keratinolytic protease from Purpureocillium lilacinum on genipin activated-chitosan beads

Keratinase from Purpureocillium lilacinum LPSC # 876 was immobilized on chitosan beads using two different cross-linking agents: glutaraldehyde and genipin. For its immobilization certain parameters were optimized such as cross-linker concentration, activation time and activation temperature. Under optimum conditions, enzyme immobilization resulted to be 96 and 92.8% for glutaraldehyde and genipin, respectively, with an activity recovery reaching up to 81% when genipin was used. The immobilized keratinase showed better thermal and pH stabilities compared to the soluble form, retaining more than 85% of its activity at pH 11 and 74% at 50 °C after 1 h of incubation. The residual activity of immobilized keratinase remained more than 60% of its initial value after five hydrolytic cycles. The results in this study support that glutaraldehyde could be replaced by genipin as an alternative cross-linking eco-friendly agent for enzyme immobilization.     

Chemical cross-linking of thioredoxin to hybrid membrane fraction in Escherichia coli

Thioredoxin was cross-linked to a membrane fraction in vivo using the heterobifunctional photoreactive cross-linking reagent p-azidophenacyl bromide, chosen to couple thioredoxin via its highly reactive thiol. Under mild reaction conditions, a significant amount of thioredoxin (30%) was rapidly cross-linked to the crude membrane fraction. The cross-linking reaction was selective, with thioredoxin purified 15-fold in the cross-linked membrane fraction. Membrane fractionation studies showed that thioredoxin associated with the inner membrane and with a hybrid membrane fraction. This hybrid membrane fraction banded at a density between the inner and outer membranes. This result is consistent with the localization of thioredoxin in association with the bacterial membrane adhesion sites first described by Bayer (Bayer, M. (1968) J. Gen. Microbiol. 53, 395-404). Association of thioredoxin with the membrane adhesion sites defines a structure corresponding to the osmotically sensitive cytoplasmic compartment (Lunn, C. A., and Pigiet, V. (1982) J. Biol. Chem. 257, 11424-11430).     

Chitosan-modified poly(acrylonitrile-co-acrylic acid) nanofibrous membranes for the immobilization of concanavalin A

Lectin affinity membranes have been receiving much attention for the separation and detection of various glycoconjugates. In this work, we present a simple and efficient method for the preparation of lectin affinity nanofibrous membranes. Chitosan-modified poly(acrylonitrile-co-acrylic acid) (PANCAA) nanofibrous membranes were first prepared by a coupling reaction between the primary amino groups of chitosan and the carboxyl groups of PANCAA electrospun membranes. Surface characterizations by attenuated total reflectance Fourier transform infrared spectroscopy (FT-IR/ATR), X-ray photoelectron spectroscopy (XPS) and field-emission scanning electron microscopy (FESEM) confirm the chemical and morphological changes of the studied nanofibrous membranes. Fluorescence-labeled concanavalin A (FL-Con A) was then immobilized on these membranes via noncovalent binding. Analyses by fluorescence spectrophotometer (FS) and confocal laser scanning microscopy (CLSM) reveal that the immobilization of Con A onto the modified nanofibrous membranes has been successfully achieved on the basis of the electrostatic interaction and the specific recognition between Con A and chitosan. The results show that the amount of adsorbed FL-Con A increases dramatically with the increasing coupling degree of chitosan (CDC) on the nanofibrous membrane. Moreover, Con A immobilized on the chitosan-modified nanofibrous membranes (CMNMs) can remain relatively stable at pH 5.3. Therefore, it is believed that this work may provide a new kind of material for affinity application.     

Studies on effect of pH on cross-linking of chitosan with sodium tripolyphosphate: A technical note

The ionotropic gelation method for formation of crosslinked chitosan particles can be easily modified from ionic cross-linking to deprotonation by adjusting the pH of TPP. Chitosan was cross-linked ionically with TPP at lower pH and by deprotonation mechanism at higher pH. The swelling behavior of cross-linked chitosan appeared to depend on the pH of TPP. The ionically cross-linked chitosan showed higher swelling ability. Thus the nature of crosslinked chitosan can be tailor made to obtain the desired properties in terms of cross-linking density, crystallinity, and hydrophilicity.     

Phosphorylated chitosan membranes for the separation of ethanol-water mixtures by pervaporation

Dense membranes of chitosan were prepared and ionically crosslinked with phosphoric acid for varying intervals of time. The membranes were characterized by FTIR and XRD to confirm cross-linking. TGA and IEC studies were conducted to assess the thermal stability and estimate the number of interactive groups left in the membrane after crosslinking. Sorption studies were carried out to evaluate the extent of interaction and degree of swelling of the membranes in pure liquids as well as binary mixtures. The phosphorylated chitosan membrane crosslinked for 2 h showed good mechanical strength and strong potential for breaking the azeotrope of 95.58 wt% ethanol by exhibiting a high pervaporation selectivity of 213 with substantial water flux of 0.58 kg/(m² h). Pervaporation experimental parameters such as feed composition, membrane thickness and permeate pressure were varied to identify optimum operating conditions.     

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein.

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine epsilon-NH2 groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.     

In situ forming chitosan hydrogels prepared via ionic/covalent co-cross-linking

In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.     

Ketal cross-linked poly(ethylene glycol)-poly(amino acid)s copolymer micelles for efficient intracellular delivery of doxorubicin

A biocompatible, robust polymer micelle bearing pH-hydrolyzable shell cross-links was developed for efficient intracellular delivery of doxorubicin (DOX). The rationally designed triblock copolymer of poly(ethylene glycol)-poly(L-aspartic acid)-poly(L-phenylalanine) (PEG-PAsp-PPhe) self-assembled to form polymer micelles with three distinct domains of the PEG outer corona, the PAsp middle shell, and the PPhe inner core. Shell cross-linking was performed by the reaction of ketal-containing cross-linkers with Asp moieties in the middle shells. The shell cross-linking did not change the micelle size and the spherical morphology. Fluorescence quenching experiments confirmed the formation of shell cross-linked diffusion barrier, as judged by the reduced Stern-Volmer quenching constant (K(SV)). Dynamic light scattering and fluorescence spectroscopy experiments showed that shell cross-linking improved the micellar physical stability even in the presence of micelle disrupting surfactants, sodium dodecyl sulfate (SDS). The hydrolysis kinetics study showed that the hydrolysis half-life (t(1/2)) of ketal cross-links was estimated to be 52 h at pH 7.4, whereas 0.7 h at pH 5.0, indicating the 74-fold faster hydrolysis at endosomal pH. Ketal cross-linked micelles showed the rapid DOX release at endosomal pH, compared to physiological pH. Confocal laser scanning microscopy (CLSM) showed that ketal cross-linked micelles were taken up by the MCF-7 breast cancer cells via endocytosis and transferred into endosomes to hydrolyze the cross-links by lowered pH and finally facilitate the DOX release to inhibit proliferation of cancer cells. This ketal cross-linked polymer micelle is promising for enhanced intracellular delivery efficiency of many hydrophobic anticancer drugs.     

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine ?-NH₂ groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.     

Cross-linked collagen sponges loaded with plant polyphenols with inhibitory activity towards chronic wound enzymes

Collagen sponges loaded with polyphenols from Hamamelis virginiana were investigated as active materials for chronic wound dressings, evaluating in vitro the inhibition of two major enzymes that impair the wound healing process - myeloperoxidase (MPO) and collagenase. Prior to polyphenols loading, collagen was cross-linked with genipin to improve its biostability. The effect of genipin cross-linking and polyphenol concentration in the development of mechanically and enzymatically stable sponges was studied. The tensile strength of the cross-linked collagen increased with the increase of the cross-linking degree, coupled to decrease in the elongation and the swelling capacity of the sponges. The stability of the sponges to collagenase digestion reached maximum when 1 mM genipin was used. However, the biostability decreased more than 10-fold after loading the sponges with polyphenols (0.5 mg/mL), nevertheless, this effect was partially overcome using higher concentration of polyphenols (1 and 2 mg/mL) to inhibit collagenase. Moreover, the polyphenols released from the sponges were sufficient for complete inhibition of MPO activity. No considerable cytotoxicity of the genipin cross-linked collagen loaded with polyphenols was observed evaluating the NIH 3T3 fibroblasts viability.     

Superior Cell Delivery Features of Genipin Crosslinked Polymeric Microcapsules: Preparation,In Vitro Characterization and Pro-Angiogenic Applications Using Human Adipose Stem Cells

The ability of mesenchymal stem cells to self-renew and differentiate into specialized cell lineages makes them promising tools for regenerative medicine. Local injection and use of scaffolds had been employed earlier to deliver these cells; yet, an optimal delivery system remains to be identified. Here, using genipin, which is a non-toxic natural cross linker for proteins, we prepared alginate–chitosan polymeric microcapsules (GCAC) to develop an efficient stem cell delivery system. We investigated the properties of this membrane along with the encapsulated adipose tissue-derived stem cells (ASCs) and compared that with the widely used alginate poly-lysine (APA) membranes. The GCAC membrane was able to support cell viability, augment cell growth, and showed better results under external rotational and osmotic pressures with about 30% of the ruptured capsules in comparison to 60% ruptured APA capsules. The membrane also provided immune-protection to the entrapped cells as demonstrated by the lymphocyte proliferation assay. The capsule also has potential for long-term storage. The encapsulated four million ASCs also showed steady secretion of approximately 4600 pg vascular endothelial growth factor (VEGF) over 15-day time period comparable to that of free cells. Furthermore, the encapsulated ASCs showed around 3.8-fold increase in VEGF secretion after 72 h hypoxic conditions in comparison to normoxic conditions. This increased VEGF expression resulted in improved angiogenic potential of the bioactive capsules as noted by enhanced endothelial cell growth. GCAC encapsulation also did not show any effect on their differentiation ability. Thus, because of these biocompatible and bioactive attributes, genipin cross-linked polymeric microcapsules can emerge as a potentially important tool for improved stem cell-based therapy and cell delivery applications.