Efficacy and safety of chitosan-based bio-compatible dressing versus nanosilver (ActicoatTM ) dressing in treatment of recalcitrant diabetic wounds: A randomized clinical trial.

Chitosan has a biocompatible, biodegradable, and nontoxic nature. The effectiveness of Nano-chitosan films in the field of wound healing has been confirmed previously. The aim of this study was to compare the clinical efficacy and safety of two dressings (chitosan and nanosilver dressings) in the treatment of refractory diabetic wounds. A total of 25 eligible patients with chronic diabetic wound were included and randomly assigned to receive chitosan (13 patients) or nanosilver (12 cases) dressing. The dressings were applied on the wounds based on their protocols and patients were visited and examined by an experienced dermatologist every week. The clinical assessments and healing rates were recorded using diabetic-foot-infection (DFI) score at the 2nd, 4th, and 6th weeks during treatment. The study endpoint, safety and tolerability profile were also documented. The patterns of change in total 10-item-DFI wound scores did not differ significantly over time between the two groups. In both groups, the total 10-item-DFI wound score reduced continuously through the course of study. The mean percentage reduction of this score from baseline was 78.1% and 74.1% in the chitosan and nanosilver dressing groups, respectively. Both dressings were well tolerated and there were no adverse events. The relatively small sample size in both groups was the main limitation of the study. Our findings confirmed that chitosan may be safely and effectively used for the treatment of diabetic wounds just like the nanosilver (ActicoatTM ) dressing. Further studies are recommended with more volunteers and a longer follow-up period.

In Situ Forming, Cytocompatible, and Self-Recoverable Tough Hydrogels Based on Dual Ionic and Click Cross-Linked Alginate.

A dual cross-linking strategy was developed to answer the urgent need for fatigue-resistant, cytocompatible, and in situ forming tough hydrogels. Clickable, yet calcium-binding derivatives of alginate were synthesized by partial substitution of its carboxyl functionalities with furan, which could come into Diels-Alder click reaction with maleimide end groups of a four arm poly(ethylene glycol) cross-linker. Tuning the cooperative viscoelastic action of transient ionic and permanent click cross-links within the single network of alginate provided a soft tough hydrogel with a set of interesting features: (i) immediate self-recovery under cyclic loading, (ii) highly efficient and autonomous self-healing upon fracture, (iii) in situ forming ability for molding and minimally invasive injection, (iv) capability for viable cell encapsulation, and (v) reactivity for on-demand biomolecule conjugation. The facile strategy is applicable to a wide range of natural and synthetic polymers by introducing the calcium binding and click reacting functional groups and can broaden the use of tough hydrogels in load-bearing, cell-laden applications such as soft tissue engineering and bioactuators.

Rheological Study and Molecular Dynamics Simulation of Biopolymer Blend Thermogels of Tunable Strength.

The temperature-induced gelation of chitosan/glycerophosphate (Chs/GP) systems through physical interactions has shown great potential for various biomedical applications. In the present work, hydroxyethyl cellulose (HEC) was added to the thermosensitive Chs/GP solution to improve the mechanical strength and gel properties of the incipient Chs/HEC/GP gel in comparison with the Chs/GP hydrogel at body temperature. The physical features of the macromolecular complexes formed by the synergistic interaction between chitosan and hydroxyethyl cellulose in the presence of ß-glycerophosphate disodium salt solution have been studied essentially from a rheological point of view. The temperature and time sweep rheological characterizations of the thermogelling systems revealed that the sol-gel transition temperature of the Chs/HEC/GP blends is equal to 37 °C at neutral pH; with increasing HEC content in the solutions, more compact networks with considerably improved gel strength are formed without influencing the gelation time. The formed hydrogel matrix has enough mechanical integrity and adequate strength for using it as injectable in situ forming matrices for biomedical applications. The classical Winter-Chambon (W-C) and Fredrickson-Larson (F-L) theories were applied to determine the gel point. In view of the obtained results, it is shown that the F-L theory can be employed as a robust and less tedious method than the W-C approach to precisely determine the gel point in these systems. At the end, molecular simulation studies were conducted by using ab initio quantum mechanics (QM) calculations carried out on Chs and HEC models, and molecular dynamics (MD) simulations of solvated Chs/HEC blend systems showed the binding behavior of Chs/HEC polymers. Analyses of interaction energy, radial distribution function, and hydrogen bonding from simulation studies strongly supported the experimental results; they all disclosed that hydrogen-bond formation between Chs moieties with regard to HEC chains plays an important role for the stabilization of the complexes.

Novel 3D scaffold with enhanced physical and cell response properties for bone tissue regeneration, fabricated by patterned electrospinning/electrospraying.

Developing three dimensional scaffolds mimicking the nanoscale structure of native extracellular matrix is a key parameter in tissue regeneration. In this study, we aimed to introduce a novel 3D structures composed of nanofibers (NF) and micro particles (MP) and compare their efficiency with 2D nanofibrous scaffold. The conventional nanofibrous PCL scaffolds are 2D mats fabricated by the electrospinning technique, whereas the NF/MP and patterned NF/MP PCL scaffolds are three dimensional structures fabricated by a modified electrospinning/electrospraying technique. The mentioned method was carried out by varying the electrospinning solution parameters and use of a metal mesh as the collector. Detailed fabrication process and morphological properties of the fabricated structures is discussed and porosity, pore size and PBS solution absorption value of the prepared structures are reported. Compared with the 2D structure, 3D scaffolds possessed enhanced porosity and pore size which led to the significant increase in their water uptake capacity. In vitro cell experiments were carried out on the prepared structures by the use of MG-63 osteosarcoma cell line. The fabricated 3D structures offered significantly increased cell attachment, spread and diffusion which were confirmed by SEM analysis. In vitro cytocompatibility assessed by MTT colorimetric assay indicated a continuous cell proliferation over 21 days on the innovative 3D structure, while on 2D mat cell proliferation stopped at early time points. Enhanced osteogenic differentiation of the seeded MG-63 cells on 3D scaffold was confirmed by the remarkable ALP activity together with increased and accelerated calcium deposition on this structure compared to 2D mat. Massive and well distributed bone minerals formed on patterned 3D structure were shown by EDX analysis. In comparison between NF/MP quasi-3D and Patterned NF/MP 3D scaffolds, patterned structures proceeded in all of the above properties. As such, the innovative Patterned NF/MP 3D scaffold could be considered as a proper bone graft substitute for bone tissue regeneration.

Cellulose nanocrystals-reinforced dual crosslinked double network GelMA/hyaluronic acid injectable nanocomposite cryogels with improved mechanical properties for cartilage tissue regeneration.

Improvement of mechanical properties of injectable tissue engineering scaffolds is a current challenge. The objective of the current study is to produce a highly porous injectable scaffold with improved mechanical properties. For this aim, cellulose nanocrystals-reinforced dual crosslinked porous nanocomposite cryogels were prepared using chemically crosslinked methacrylated gelatin (GelMA) and ionically crosslinked hyaluronic acid (HA) through the cryogelation process. The resulting nanocomposites showed highly porous structures with interconnected porosity (>90%) and mean pore size in the range of 130-296 µm. The prepared nanocomposite containing 3%w/v of GelMA, 20 w/w% of HA, and 1%w/v of CNC showed the highest Young's modulus (10 kPa) and excellent reversibility after 90% compression and could regain its initial shape after injection by a 16-gauge needle in the aqueous media. The in vitro results demonstrated acceptable viability (>90%) and migration of the human chondrocyte cell line (C28/I2), and chondrogenic differentiation of human adipose stem cells. A two-month in vivo assay on a rabbit's ear model confirmed that the regeneration potential of the prepared cryogel is comparable to the natural autologous cartilage graft, suggesting it is a promising alternative for autografts in the treatment of cartilage defects.

PCL-based 3D nanofibrous structure with well-designed morphology and enhanced specific surface area for tissue engineering application.

Tissue engineering opens a new horizon for biological tissue replacement applications. Scaffolds, appropriate cells, and signaling induction are the main three determinant parameters in any tissue engineering applications. Designing a suitable scaffold which can mimic the cellular inherent and natural habitation is of great importance for cellular growth and proliferation. Just like a natural extracellular matrix (ECM), scaffolds provide the cells with an environment for performing biological functions. Accordingly, vast surface area and three-dimensional nanofibrous structures are among the pivotal characteristics of functional scaffolds in tissue engineering, and enhancement of their properties is the main purpose of the present research. In our previous study, a patterned structure composed of continuous nanofibers and microparticles was introduced. In this work, a new modification is applied for adjustment of the surface area of an electrospun/electrosprayed scaffold. For this purpose, at predetermined stages during electrospinning/electrospraying, the nitrogen gas is flushed through the mesh holes of the collector in the opposite direction of the jet movement. This method has led to the formation of very thin nanofibrous layers at nitrogen flush intervals by providing a cooling effect of the sweeping nitrogen. As a consequence, a straticulated structure has been fabricated which possesses extremely high surface/volume ratio. The porosity, water absorption, and morphological analysis were conducted on the obtained scaffold. In vitro cytocompatibility assessments as well as histological analysis demonstrated that the fabricated scaffold provides a proper substrate for cellular attachment, proliferation and infiltration. These findings can be advantageous in three-dimensional tissue engineering such as bone tissue engineering applications. Furthermore, according to the advanced microstructure and vast surface area of the fabricated samples, they can be applied in many other applications, such as membrane, filtration, etc.

Polysaccharide-based polyampholyte complex formation: Investigating the role of intra-chain interactions.

The difference in inter-chain and intra-chain electrostatic attraction was investigated in polyelectrolyte and polyampholyte electrostatic complex formation. Three polymers with similar backbone molecular structures including chitosan (Ch) polycation, carboxymethyl cellulose (CMCe) polyanion, and carboxymethyl chitosan (CMCh) polyampholyte were used for this purpose. The turbidimetric, water content, and rheological measurements for polyampholyte self-complex showed more dependence on the ionic strength rather than the polyelectrolyte one. The degree of dissociation (α), dissociation constant (pKa), and intrinsic persistence length were calculated by applying the Katchalsky-Lifson model to potentiometric data. We studied the gyration radii as a function of Debye length and observed the polyampholyte chain contractions due to the intra-chain electrostatic attractions, which minimize the entropic gain of the inter-chain complex formation. This is in accordance with the decrease in pKa by αc for CMCh which is the opposite of that for the Ch and CMCe samples. We also found that the polyampholyte has less intrinsic and electrostatic persistence length compared with both polyanion and polycation with similar chain structures indicating the impact of the inter-chain electrostatic interaction on the complex properties. This study deepens our insight about the behavior of CMCh and the nature of difference between CMCh and Ch/CMCe electrostatic complexes.

Bioinspired scaffolds based on aligned polyurethane nanofibers mimic tendon and ligament fascicles.

Topographical factors of scaffolds play an important role in regulating cell functions. Although the effects of alignment topography and three-dimensional (3D) configuration of nanofibers as well as surface stiffness on cell behavior have been investigated, there are relatively few reports that attempt to understand the relationship between synergistic effects of these parameters and cell responses. Herein, the influence of biophysical and biomechanical cues of electrospun polyurethane (PU) scaffolds on mesenchymal stem cells (MSCs) activities was evaluated. To this aim, multiscale bundles were developed by rolling up the aligned electrospun mats mimicking the fascicles of tendons/ligaments and other similar tissues. Compared to mats, the 3D bundles not only maintained the desirable topographical features (i.e., fiber diameter, fiber orientation, and pore size), but also boosted tensile strength (∼40 MPa), tensile strain (∼260%), and surface stiffness (∼1.75 MPa). Alignment topography of nanofibers noticeably dictated cell elongation and a uniaxial orientation, resulting in tenogenic commitment of MSCs. MSCs seeded on the bundles expressed higher levels of tenogenic markers compared to mats. Moreover, the biomimetic bundle scaffolds improved synthesis of extracellular matrix components compared to mats. These results suggest that biophysical and biomechanical cues modulate cell-scaffold interactions, providing new insights into hierarchical scaffold design for further studies.

Antibacterial aligned nanofibrous chitosan/PVA patch for repairing chronic tympanic membrane perforations.

Chronic tympanic membrane (TM) perforation is a consequence of trauma or chronic otitis media, and these chronic TM perforations often lead to conduction hearing loss. This study focuses on the development of a patch using a combination of chitosan (CS) and polyvinyl alcohol (PVA) as graft material for repairing chronic tympanic membrane (TM) perforations. Aligned nanofibers were created using a specially designed collector (SDC) through the electrospinning method. The scanning electron microscopy (SEM) analysis revealed that the CS/PVA ratio of (15:85) resulted in uniform and bead-free nanofibers. The aligned nanofibers had a diameter of 131.11 ± 28 nm, indicating that the influence of the electrostatic field introduced by the SDC affected not only the nanofiber alignment but also the nanofiber diameter. The nanofiber angles demonstrated effective alignment. This patch is infused with thyme essential oil (TEO) for antibacterial properties. The results showed that its antibacterial property for Pseudomonas aeruginosa bacteria was enhanced in such a way that the diameter of the antibacterial halo increased from zero to 25 mm. Cell viability assays showed >80 % viability. A preclinical case study on six patients demonstrated the biocompatibility and promising potential of the fabricated patch for eardrum repair.

Injectable hydrogels for bone and cartilage tissue engineering: a review.

Tissue engineering, using a combination of living cells, bioactive molecules, and three-dimensional porous scaffolds, is a promising alternative to traditional treatments such as the use of autografts and allografts for bone and cartilage tissue regeneration. Scaffolds, in this combination, can be applied either through surgery by implantation of cell-seeded pre-fabricated scaffolds, or through injection of a solidifying precursor and cell mixture, or as an injectable cell-seeded pre-fabricated scaffold. In situ forming and pre-fabricated injectable scaffolds can be injected directly into the defect site with complex shape and critical size in a minimally invasive manner. Proper and homogeneous distribution of cells, biological factors, and molecular signals in these injectable scaffolds is another advantage over pre-fabricated scaffolds. Due to the importance of injectable scaffolds in tissue engineering, here different types of injectable scaffolds, their design challenges, and applications in bone and cartilage tissue regeneration are reviewed.

Shape memory injectable cryogel based on carboxymethyl chitosan/gelatin for minimally invasive tissue engineering: In vitro and in vivo assays.

Shape-memory cryogels have drawn attention as an injectable system to minimize the risks associated with surgical implantation in tissue engineering. To achieve shape memory behavior with hydration as an external stimulus, it is necessary to have a porous elastic network. To achieve this, it is crucial to control the crosslinking process at the time of pore formation, especially for natural-based polymers. In this study, a versatile method using a cryogelation method in the presence of chemical and physical crosslinkers is investigated to obtain an injectable super macroporous elastic structure based on a poly(ampholyte) (carboxymethyl chitosan) and a protein (gelatin). Mechanical, swelling, shape memorizing behavior, injectability, and in vitro and in vivo behavior of cryogels were studied. Cryogelation in a subzero temperature led to the formation of scaffolds with interconnected pores of the size of 350 µm which swelled completely after 3 min. Cryogels had crosslink density up to 22% and elastic modulus in the hydrated state up to 0.054 and 1.733 MPa at low and high strains, respectively, and low hysteresis (<30 kPa). Injectability studies confirmed the ability of the cryogels to be injected through a 16G needle. In vitro studies demonstrated good cellular penetration, cell adhesion, and high cell viability (>100%). In vivo studies using mice showed that the body's response was befitting without inflammation and any side effect for the liver and kidneys.

Application of plasma surface modification techniques to improve hemocompatibility of vascular grafts: A review.

Surface modification using plasma processing can significantly change the chemical and physical characteristics of biomaterial surfaces. When used in combination with additional modification techniques such as direct chemical or biochemical methods, it can produce novel biomaterial surfaces, which are anticoagulant, bioactive, and biomimetic in nature. This article reviews recent advances in improving hemocompatibility of biomaterials by plasma surface modification (PSM). The focus of this review is on PSM of the most commonly used polymers for vascular prostheses such as expanded polytetrafluoroethylene (PTFE), polyethylene terephthalate (Dacron(®) ), and next generation of biomaterials, including polyhedral oligomeric silsesquioxane nanocomposite.

Surface modification of POSS-nanocomposite biomaterials using reactive oxygen plasma treatment for cardiovascular surgical implant applications.

In this study, central composite design (CCD) was used to develop predictive models to optimize operating conditions of plasma surface modification. It was concluded that out of the two process variables, power and duration of plasma exposure, the latter was significantly affecting the surface energy (γ(s) ), chemistry, and topography of polyhedral oligomeric silsesquioxane-poly(carbonate-urea)urethane (POSS-PCU) films. On the basis of experimental data, CCD was used to model the γ(s) using a quadratic modeling of the process variables to achieve optimum surface energy to improve the interaction between endothelial cells (ECs). It was found that optimal water θ for EC adhesion and retention, which was reported 55° from supporting literature (equivalent to γ(s) = 51 mN/m), was easily achievable using the following experimental conditions: (1) power output at 30 W for 75 Sec, (2) 90 W for 40 Sec, and (3) 90 W for 55 Sec in oxygen. In vitro cell culture and metabolic activity studies on optimized films [as in (1)] demonstrate increased adhesion, coverage, and growth of human umbilical vein endothelial cells that were confluent over a shorter time period (<24 H) than controls. Such materials enhanced the EC response and promoted endothelialization on optimized films, thus demonstrating their use as bypass graft materials.

Injectable and reversible preformed cryogels based on chemically crosslinked gelatin methacrylate (GelMA) and physically crosslinked hyaluronic acid (HA) for soft tissue engineering.

Hydrogels are a promising choice for soft tissue (cartilage, skin and adipose) engineering and repair. However, lack of interconnected porosity and poor mechanical performance have hindered their application, especially in natural polymer-based hydrogels. Cryogels with the potential to overcome the shortcomings of hydrogels have drawn attention in the last few years. Thus, in this study, highly porous and mechanically robust cryogels based on interpenetrating polymer network (IPN) of gelatin methacrylate (GelMA) and hyaluronic acid (HA) were fabricated for soft tissue engineering application. Cryogels have a constant amount of GelMA (3% wt) with different concentrations of HA (from 5% to 20 % w/w). In fact, crosslinking through cryogelation in subzero temperature facilitates the formation of interconnected pores with 90 % porosity percentage without external progen. On the other hand, high mechanical stability (no failure up to 90 % compression) was achieved due to the cryogelation and chemical crosslinking of GelMA as well as physical crosslinking of HA. Furthermore, the porous and hydrophile nature of the cryogels resulted in shape memory properties under compression, which can reverse to initial shape after retaining the water. Although increasing the HA concentration followed by the density of physical crosslinking boosted the mechanical performance of cryogels under compression, it limited the reversibility properties. Nevertheless, all cryogels with different HA concentrations showed acceptable gel strength and Young's modulus (G-H-20, E = 6kPa) and had appropriate pore size for cell infiltration and nutrient transportation with good cell adhesion and high cell viability (more than 90 %). The unique property of fabricated cryogels that facilitate less invasive delivery makes them a promising alternative for the soft tissue application.

Fabrication of nanocomposite/nanofibrous functionally graded biomimetic scaffolds for osteochondral tissue regeneration.

One of the main challenges in treating osteochondral lesions via tissue engineering approach is providing scaffolds with unique characteristics to mimic the complexity. It has led to application of heterogeneous scaffolds as a potential candidate for engineering of osteochondral tissues, in which graded multilayered-structure should promote bone and cartilage growth. By designing three-dimensional (3D)-nanofibrous scaffolds mimicking the native extracellular matrix's nanoscale structure, cells can grow in controlled conditions and regenerate the damaged tissue. In this study, novel 3D-functionality graded nanofibrous scaffolds composed of five layers based on different compositions containing polycaprolactone(PCL)/gelatin(Gel)/nanohydroxyapatite (nHA) for osteoregeneration and chitosan(Cs)/polyvinylalcohol(PVA) for chondral regeneration are introduced. This scaffold is fabricated by electrospinning technique using spring as collector to create 3D-nanofibrous scaffolds. Fourier-transform infrared spectroscopy, X-ray diffraction, energy dispersive X-ray spectroscopy, scanning electron microscopy, mechanical compression test, porosimetry, and water uptake studies were applied to study each layer's physicochemical properties and whole functionally graded scaffold. Besides, biodegradation and biological studies were done to investigate biological performance of scaffold. Results showed that each layer has a fibrous structure with continuous nanofibers with improved pore size and porosity of novel 3D scaffold (6-13 µm and 90%) compared with two-dimensional (2D) mat (2.2 µm and 19.3%) with higher water uptake capacity (about 100 times of 2D mat). Compression modulus of electrospun scaffold was increased to 78 MPa by adding nHA. The biological studies revealed that the layer designed for osteoregeneration could improve cell proliferation rate in comparison to the layer designed for chondral regeneration. These results showed such structure possesses a promising potential for the treatment of osteochondral defects.

Crosslinking strategies for silk fibroin hydrogels: promising biomedical materials.

Due to their strong biomimetic potential, silk fibroin (SF) hydrogels are impressive candidates for tissue engineering, due to their tunable mechanical properties, biocompatibility, low immunotoxicity, controllable biodegradability, and a remarkable capacity for biomaterial modification and the realization of a specific molecular structure. The fundamental chemical and physical structure of SF allows its structure to be altered using various crosslinking strategies. The established crosslinking methods enable the formation of three-dimensional (3D) networks under physiological conditions. There are different chemical and physical crosslinking mechanisms available for the generation of SF hydrogels (SFHs). These methods, either chemical or physical, change the structure of SF and improve its mechanical stability, although each method has its advantages and disadvantages. While chemical crosslinking agents guarantee the mechanical strength of SFH through the generation of covalent bonds, they could cause some toxicity, and their usage is not compatible with a cell-friendly technology. On the other hand, physical crosslinking approaches have been implemented in the absence of chemical solvents by the induction of ß-sheet conformation in the SF structure. Unfortunately, it is not easy to control the shape and properties of SFHs when using this method. The current review discusses the different crosslinking mechanisms of SFH in detail, in order to support the development of engineered SFHs for biomedical applications.

PEGylated curcumin-loaded nanofibrous mats with controlled burst release through bead knot-on-spring design.

APEGylatedcurcumin (PCU) loaded electrospuns based on poly(ε-caprolactone) (PCL) andpolyvinyl alcohol (PVA) were fabricated for wound dressing applications. The main reason for this wound dressing design is antibacterialactivity enhancement, and wound exudates management. PEGylation increases curcuminsantibacterial properties and PVA can help exudates management. For optimal wound dressing, first, response surface methodology (RSM) was applied to optimize the electrospinning parameters to achieve appropriate nanofibrous mats. Then a three-layer electrospun was designed by considering the water absorbability, PCU release profile as well as antibacterial and biocompatibility of the final wound dressing. The burst release in controlled release systems could be evaluated for prevention of the higher initial drug release and control the effective life time. The PCU release results illustrated that the bead knot plays a positive role in controlling the release profile andby increase in the number of beads per unit area from 3000 to 9000 mm-2,the PCU burst release will be reduced; Also in vitro studies show that optimized three-layer dressing based on PCL/PVA/PCU can support water vapour transmission rate in optimal range and also absorb more than three times exudates in comparison with mono-layerdressing. Antibacterial tests show that the electrospun wound dressing containing 5% PCU exhibits100% antibacterial activityas well as cell viability level within an acceptable range.

Mechanical Characteristics of SPG-178 Hydrogels: Optimizing Viscoelastic Properties through Microrheology and Response Surface Methodology

Background: Self-assembling peptides (SApeptides) have growing applications in tissue engineering and regenerative medicine. The application of SApeptide-based hydrogels depends strongly on their viscoelastic properties. Optimizing the properties is of importance in tuning the characteristics of the hydrogels for a variety of applications. Methods: In this study, we employed statistical modeling, conducted with the response surface methodology (RSM) and particle tracking microrheology, to investigate the effects of self-assembling SPG-178 peptide and added NaCl salt concentrations and milieu type (deionized water or blood serum) on the viscoelastic properties of SPG-178 hydrogels. A central composite RSM model was employed for finding the optimum value of the parameters to achieve the highest storage modulus and the lowest tan δ. Results: Viscoelastic properties of each sample, including storage modulus, loss modulus, and tan δ, were determined. Storage modulus and tan δ were modeled, accounting for the impact of the SPG-178 peptide and NaCl concentrations and milieu type on the viscoelastic properties. It was found that the SPG-178 hydrogel storage modulus was positively influenced by the SPG-178 peptide concentration and the serum. Conclusion: A combination of microrheology and RSM is a useful test method for statistical modeling and analysis of rheological behavior of solid-like gels, which could be applied in various biomedical applications such as hemostasis.

Influence of poly (lactide-co-glycolide) type and gamma irradiation on the betamethasone acetate release from the in situ forming systems.

In situ forming biodegradable polymeric systems were prepared from Poly (DL-lactide-co-glycolide), RG504H (50:50, lactide:glycolide), RG756 (75:25) and mixture of them. They were dissolved in N-methyl-2-pyrrolidone (33% w/w) and mixed with betamethasone acetate (BTMA, 5 and 10% w/w) and ethyl heptanoate (5% w/w, as an additive). The effects of gamma irradiation, drug loading, type of polymers and solvent removal were evaluated on release profiles. Scanning electron microscopy (SEM) of RG756 samples loaded by BTMA did not show any degradation until two weeks. Differential scanning calorimeter (DSC) experiments confirmed insignificant decrease in T(g), and consequently release rate. Declining T(g) of RG504H and RG756 after gamma irradiation was about 0.4 and 1.46 degrees C, respectively. High performance liquid chromatography (HPLC) revealed that BTMA release is more rapid from the formulations prepared using the RG504H with lower molecular weight. The formulations prepared by RG756 had lower burst release (2.5-41%) than the samples based on RG504H (60-67%) and mixture of them (30-33%). Regarding this research three different kinds of steriled in situ forming systems were developed which can release BTMA for 24, 90 and 60 days.