Multilayer composite scaffolds with mechanical properties similar to small intestinal submucosa

Use of biodegradable scaffolds to engineer new tissues has become an attractive option in various transplantation protocols. In particular, small intestinal submucosa (SIS) has generated immense interest in various tissue engineering applications because of its diverse favorable properties. However, it is a natural matrix, which leads to problems in large-scale preparations and contains sample to sample heterogeneity. In this study, we explored the formation of synthetic matrix mimicking the characteristics of the SIS. Three-dimensional composite structures were developed by sandwiching 50:50 PLGA film between porous chitosan matrices. The outer chitosan layers provide biological activity while the inner PLGA layer provides mechanical strength. PLGA films were initially perforated at 1 cm distance, and the porous chitosan matrix was formed sequentially on each side by controlled rate freezing and lyophilization technique at -80 degrees C. Scanning electron microscopy analysis showed a layered microarchitecture with chitosan filling the perforations of PLGA membrane. Urea permeability studies confirmed that the perforations were filled (negligible urea transfer across composite over 8 h). Tensile strength analysis showed that the matrices formed using 160 kDa PLGA had sufficient break stress ( approximately 4.5 MPa). Degradation analysis over 8 weeks in the presence of 10 mg/L lysozyme showed a 50% decrease in total weight and an 80% decrease in PLGA molecular weight. When cellular adhesion and actin distribution of mouse embryonic fibroblasts were evaluated, for 7 days, cells showed their typical spindle shape and redistribution of actin fibers on composite matrices. Viability studies and MMP-2/MMP-9 activity showed that the cells were viable and functional, similar to tissue culture plastic. Further, canine bladder smooth muscle cells also showed similar cell adhesion and spreading on the composite matrix. In summary, composite structures mimicking SIS were constructed and show potential as a tissue engineering material.     

缓慢释放bFGF壳聚糖多孔支架的构建及对细胞生长的影响

采用冷冻干燥制备壳聚糖支架,以牛血清白蛋白（BSA）和碱性成纤维细胞生长因子（bFGF）为模型药物,制备乳酸-乙醇酸共聚物（PLGA）微球,并将其包埋于壳聚糖支架中,考察药物在支架上的体外释放。以MTT法考察了缓慢释放的bFGF对L929细胞的影响。用扫描电镜观察包埋微球支架的形态和生长了细胞的支架。结果表明单用壳聚糖支架,药物释放得比较快,制成PLGA微球后,再包埋于壳聚糖支架中,则药物释放明显缓慢。缓慢释放的bFGF促进了细胞的生长。     

Gelatin/chitosan/hyaluronan scaffold integrated with PLGA microspheres for cartilage tissue engineering

Poly(lactide-co-glycotide) (PLGA) microspheres integrated into gelatin/chitosan/hyaluronan scaffolds were fabricated by freeze-drying and crosslinking with 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. The effects of the microspheres on porosity, density, compressive modulus, phosphate-buffered saline uptake ratio and weight loss of the scaffolds were evaluated. Generally, a scaffold with a higher PLGA content had a lower porosity and weight loss, and a medium uptake ratio, but a larger apparent density and compressive modulus. When the PLGA content was lower than 50%, the PLGA-integrated scaffolds had a similar pore size (approximately 200microm) as that of the control, and as much as 90% of their porosity could be preserved. In vitro chondrocyte culture in the 50% PLGA-integrated scaffold demonstrated that the cells could proliferate and secrete extracellular matrix at the same level as in the control gelatin/chitosan/hyaluronan scaffold.     

In vitro characterization of chitosan-gelatin scaffolds for tissue engineering

Recently, chitosan–gelatin scaffolds have gained much attention in various tissue engineering applications. However, the underlying cell–matrix interactions remain unclear in addition to the scaffold degradation and mechanical characteristics. In this study, we evaluated (i) the degradation kinetics of chitosan and chitosan–gelatin scaffolds in the presence of 10 mg/L of lysozyme for dimensional stability, weight loss, and pH changes for a period of 2 months, (ii) tensile and compressive properties of films and scaffolds in wet state at 37 °C, (iii) viability of fibroblasts and human umbilical vein endothelial cells (HUVECs) on scaffolds, and (iv) the alteration in cell spreading characteristics, cytoskeletal actin distribution, focal adhesion kinase (FAK) distribution and PECAM-1 expression of HUVECs under static and 4.5, 8.5, 13 and 18 dyn/cm² shear stress conditions. Degradation results showed that gelatin-containing chitosan scaffolds had faster degradation rate and significant loss of material than chitosan. Mechanical properties of chitosan are affected by the addition of gelatin although there was no clear trend. Three-dimensional chitosan and chitosan–gelatin scaffolds supported fibroblast viability equally. However, chitosan membranes decreased cell-spreading area, disrupted F-actin and localized FAK in the nucleus of HUVECs. Importantly, the lowest shear stress tested (4.5 dyn/cm²) for 3 h washed away cells on chitosan suggesting weak cell adhesion. In the blends, effect of gelatin was dominant; actin and FAK distribution were comparable to gelatin in static culture. However, at higher shear stresses, presence of chitosan inhibited shear-induced increase in cell spreading and weakened cell adhesive strength. No significant differences were observed in PECAM-1 expression. In summary, these results showed significant influence of blending gelatin with chitosan on scaffold properties and cellular behavior.     

Hybrid nanofibrous membranes of PLGA/chitosan fabricated via an electrospinning array

Hybrid nanofibrous membranes of poly(lactic-co-glycolic acid) (PLGA) and chitosan with different chitosan amounts (32.3, 62.7, and 86.5%) were fabricated via a specially designed electrospinning setup consisting of two sets of separate syringe pumps and power supplies. After soaking in chloroform overnight to dissolve PLGA, the amount of chitosan in the hybrid membranes was determined. The structure, mechanical properties, water uptake, and cytocompatibilities of the nanofibrous membranes were investigated by scanning electron microscopy, tensile testing, incubation in phosphate buffer solution, and human embryo skin fibroblasts culturing. Results showed that the chitosan amount in PLGA/chitosan membranes could be well controlled by adjusting the number of syringe for electrospinning of PLGA or chitosan, respectively. Because of the introduction of chitosan, which is a naturally hydrophilic polymer, the hybrid PLGA/chitosan membranes after chitosan crosslinking exhibited good mechanical and water absorption properties. The cytocompatibility of hybrid PLGA/chitosan membranes was better than that of the electrospun PLGA membrane. The electrospun hybrid nanofibrous membranes of PLGA and chitosan appear to be promising for skin tissue engineering. The concept of using an electrospinning array to form multicomponent nanofibrous membranes will lead to the creation of novel scaffolds for tissue engineering applications.     

Gradual pore formation in natural origin scaffolds throughout subcutaneous implantation

This study used a rat subcutaneous implantation model to investigate gradual in situ pore formation in a self-regulating degradable chitosan-based material, which comprises lysozyme incorporated into biomimetic calcium phosphate (CaP) coatings at the surface to control the scaffold degradation and subsequent pore formation. Specifically, the in vivo degradation of the scaffolds, the in situ pore formation, and the tissue response were investigated. Chitosan or chitosan/starch scaffolds were studied with and without a CaP coating in the presence or absence of lysozyme for a total of six experimental groups. Twenty-four scaffolds per group were implanted, and eight scaffolds were retrieved at each of three time points (3, 6, and 12 weeks). Harvested samples were analyzed for weight loss, microcomputed tomography, and histological analysis. All scaffolds showed pronounced weight loss and pore formation as a function of time. The highest weight loss was 29.8% ± 1.5%, obtained at week 12 for CaP chitosan/starch scaffolds with lysozyme incorporated. Moreover, all experimental groups showed a significant increase in porosity after 12 weeks. At all time points no adverse tissue reaction was observed, and as degradation increased, histological analysis showed cellular ingrowth throughout the implants. Using this innovative methodology, the ability to gradually generate pores in situ was clearly demonstrated in vivo.     

Characterization of knitted polymeric scaffolds for potential use in ligament tissue engineering

Different scaffolds have been designed for ligament tissue engineering. Knitted scaffolds of poly-L-lactic acid (PLLA) yarns and co-polymeric yarns of PLLA and poly(glycolic acid) (PLGA) were characterized in the current study. The knitted scaffolds were immersed in medium for 20 weeks, before mass loss, molecular weight, pH value change in medium were tested; changes in mechanical properties were evaluated at different time points. Results showed that the knitted scaffolds had 44% porosity. There was no significant pH value change during degradation, while there was obvious mass loss at initial 4 week, as well as smooth molecular weight drop of PLLA. PLGA degraded more quickly, while PLLA kept its integrity for at least 20 weeks. Young's modulus increased while tensile strength and strain at break decreased with degradation time; however, all of them could maintain the basic requirements for ACL reconstruction. It showed that the knitted polymeric structures could serve as potential scaffolds for tissue-engineered ligaments.     

Solvent effects on the microstructure and properties of 75/25 poly(D,L-lactide-co-glycolide) tissue scaffolds

Poly(lactide-co-glycolide) (PLGA) is used in many biomedical applications because it is biodegradable, biocompatible, and FDA approved. PLGA can also be processed into porous tissue scaffolds, often through the use of organic solvents. A static light scattering experiment showed that 75/25 PLGA is well solvated in acetone and methylene chloride, but forms aggregates in chloroform. This led to an investigation of whether the mechanical properties of the scaffolds were affected by solvent choice. Porous 75/25 PLGA scaffolds were created with the use of the solvent casting/particulate leaching technique with three different solvents: acetone, chloroform, and methylene chloride. Compression testing resulted in stiffness values of 21.7 +/- 4.8 N/mm for acetone, 18.9 +/- 4.2 N/mm for chloroform, and 30.2 +/- 9.6 N/mm for methylene chloride. Permeability testing found values of 3.9 +/- 1.9 x 10(-12) m2 for acetone, 3.6 +/- 1.3 x 10(-12) m2 for chloroform, and 2.4 +/- 1.0 x 10(-12) m2 for methylene chloride. Additional work was conducted to uncouple polymer/solvent interactions from evaporation dynamics, both of which may affect the scaffold properties. The results suggest that solvent choice creates small but significant differences in scaffold properties, and that the rate of evaporation is more important in affecting scaffold microstructure than polymer/solvent interactions.     

Differentiation of bone marrow stromal cells in poly(lactide-co-glycolide)/chitosan scaffolds

This study investigates the physicochemical properties of poly(lactide-co-glycolide) (PLGA)/chitosan scaffolds and the neuron growth factor (NGF)-guided differentiation of bone marrow stromal cells (BMSCs) in the scaffolds. The scaffolds were prepared by the crosslinking of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and genipin, and the differentiating BMSCs were characterized against CD44, CD90 and NeuN. The scaffold with 20% PLGA yielded 95% porosity, Young's modulus of 13 MPa, 70% adhesion of BMSCs and 1.6-fold increase in the cell viability over 7-day cultivation. BMSCs without guidance in the PLGA/chitosan scaffolds were prone to differentiate toward osteoblasts with apparent deposition of calcium. When NGF was introduced, an increased weight percentage of PLGA yielded more identified neurons. In addition, mature neurons emerged from the PLGA-rich biomaterials after induction with NGF over 2 days. A proper control over the physical and biomedical property of the scaffolds and the NGF-guided differentiation of BMSCs can be promising for nerve regeneration.     

Bone tissue engineering evaluation based on rat calvaria stromal cells cultured on modified PLGA scaffolds

Using natural materials to coat the scaffolds used for tissue-engineered bone-repair techniques is expected to increase osteoblast adhesion to the scaffold and to express normal physiological function. To test this hypothesis, we therefore modified poly(DL-lactic-co-glycolic acid) (PLGA) substrate by coating it with natural biomaterial solutions of collagen, chitosan, or N-succinyl-chitosan, and then used these three combinations as scaffolds to evaluate their effects on osteoblast attachment, proliferation, and differentiation. The results demonstrated that the pore size of scaffolds ranging from 125-500 microm did not affect the osteoblast phenotype; however, the surface modification of the scaffolds coated with these natural biomaterials did. Collagen increased cell attachment and proliferation, but chitosan and N-succinyl-chitosan decreased them. Chitosan and N-succinyl-chitosan increased differentiation, but collagen decreased it. These results provide us a new strategy for modifying microenvironments to increase osteoblast adhesion, proliferation, and differentiation on PLGA scaffolds, a strategy that might be useful for tissue regeneration.     

Characterization of chitosan-polycaprolactone blends for tissue engineering applications

The objective of this work was to study the effect of blending chitosan with poly(epsilon-caprolactone) (PCL) on their biomechanical properties. After testing the effect of molecular weight (MW), temperature, and humidity on the tensile properties in dry, wet at 25 degrees C and wet at 37 degrees C conditions, chitosan with a MW>310 kD was selected for use in the blend. Homogeneous blends of 25%, 50% and 75% PCL compositions were formed by dissolving chitosan and 80 kD PCL in a common solvent of approximately 77% aqueous acetic acid. Taking advantage of the low melting point of PCL, blend membranes were processed at 25, 37, 55 degrees C water bath or 55 degrees C oven into films. Also, membranes were solvent annealed using chloroform vapors. Tensile properties were analyzed in wet conditions at 25 degrees C. Support for cell viability and distribution of cytoskeletal actin were analyzed by in vitro cell culture of mouse embryonic fibroblasts (MEFs). Differential scanning calorimetry studies indicated the miscibility of the two components when approximated using Nishi-Wang equation. Drying the films at 55 degrees C in an oven formed membranes without separation of two phases. However, the analyzed tensile properties showed no significant alterations relative to chitosan. On the contrary, significant improvements were observed after solvent annealing. Interestingly, increased viability and redistribution of actin fibers was observed on blends formed with 50% PCL and 75% PCL relative to individual polymers. In summary, 50:50 blends when processed at 55 degrees C in an oven showed significant improvement in mechanical properties as well as support for cellular activity relative to chitosan.     

Emulsion Self‐Assembly Synthesis of Chitosan/Poly(lactic‐co‐glycolic acid) Stimuli‐Responsive Amphiphiles

An amphiphilic graft copolymer using chitosan (CS) as a hydrophilic main chain and poly(lactic‐co‐glycolic acid) (PLGA) as a hydrophobic side chain is prepared through an emulsion self‐assembly synthesis. CS aqueous solution is used as a water phase and PLGA in chloroform is served as an oil phase. A water‐in‐oil (W/O) emulsion is fabricated in the presence of the surfactant span‐80. The self‐assembly reaction is performed between PLGA and CS under the condensation of EDC. Fourier transform IR (FTIR) spectroscopy reveals that PLGA is grafted onto the backbone of CS through the interactions between end carboxyl and amino groups of the two components. ¹H NMR spectroscopy directly indicates the grafting content of PLGA in the CS‐graft‐PLGA (CS‐g‐PLGA) copolymer is close to 25%. X‐ray diffraction (XRD) confirms that the copolymer exhibits an amorphous structure. The CS‐g‐PLGA amphiphile can self‐assemble to form micelles with size in the range of ≈100–300 nm, which makes it easy to apply in various targeted‐drug‐release and biomaterial fields.     

Degradation behaviors of biodegradable macroporous scaffolds prepared by gas foaming of effervescent salts

Biodegradable polymeric scaffolds for tissue engineering were fabricated by a gas-foaming/salt-leaching method using a combination of two effervescent salts, ammonium bicarbonate and citric acid. Poly(D,L-lactic-co-glycolic acid) (PLGA) in a state of gel-like paste was first produced by precipitation of PLGA dissolved in chloroform into ethanol. The polymer slurry was mixed with sieved particles of ammonium bicarbonate, molded, and then immersed in an aqueous solution of citric acid to generate macroporous scaffolds. The scaffolds had relatively homogeneous pore structures throughout the matrix and showed an average pore size of 200 microm and over 90% porosity. By adjusting the concentration of citric acid in the aqueous medium, it was possible to control porosity as well as mechanical strength of the scaffolds. In vitro degradation studies of three different scaffolds having lactic/glycolic acid molar ratios of 75/25, 65/35, and 50/50 exhibited marked swelling behaviors at different critical time points. The swollen matrices had a hydrogel-like internal structure. It was found that massive water uptake into the degrading scaffolds induced matrix swelling, which facilitated the hydrolytic scission of PLGA chains with concomitant disintegration of the matrices.     

Enzymatic degradation behavior and mechanism of poly(lactide-co-glycolide) foams by trypsin

The purpose of this study is to investigate the enzymatic degradation behaviors of porous poly(lactide-co-glycolide) (PLGA) foams in the presence of trypsin, in comparison with their hydrolytic degradation. To inspect the effect of trypsin on the degradation of PLGA, both the hydrolytic and enzymatic degradation of non-porous PLGA samples were also performed. The changes of molecular weight and molecular weight distribution (polydispersity) during the degradation were determined by gel permeation chromatograph. And the changes of weight, thickness and morphology with the degradation were also measured. The degradation of PLGA displayed as two stages. In the first stage, the molecular weight of PLGA decreased continuously with degradation time, whereas little weight loss occurred. But in the second stage, the molecular weight of PLGA had decreased to a low value and was almost unchanged with time, while the sample experienced significant weight loss. And it was found that the presence of trypsin could significantly accelerate the weight loss rates of all the PLGA samples, but it caused little difference in the decrease of molecular weight and the change of PLGA composition between the enzymatic and hydrolytic degradation. Therefore, the enzymatic degradation of PLGA was still primarily a hydrolysis process. A mechanism of enzymatic degradation was proposed that the trypsin could enhance the weight loss of PLGA by acting as surfactant to push the dispersion of degradation products into water even though they could not dissolve in water.     

Sequential release of bioactive IGF-I and TGF-beta 1 from PLGA microsphere-based scaffolds

Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta 1) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-beta 1 from modular designed poly(l,d-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-beta 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-beta 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-beta 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-beta 1 released continuously, TGF-beta 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-beta 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-beta 1 sequentially makes them very useful for cartilage tissue engineering applications.     

Electrospun PLGA nanofiber scaffolds for articular cartilage reconstruction: mechanical stability,degradation and cellular responses under mechanical stimulation in vitro

We investigated the potential of a nanofiber-based poly(DL-lactide-co-glycolide) (PLGA) scaffold to be used for cartilage reconstruction. The mechanical properties of the nanofiber scaffold, degradation of the scaffold and cellular responses to the scaffold under mechanical stimulation were studied. Three different types of scaffold (lactic acid/glycolic acid content ratio = 75 : 25, 50 : 50, or a blend of 75 : 25 and 50 : 50) were tested. The tensile modulus, ultimate tensile stress and corresponding strain of the scaffolds were similar to those of skin and were slightly lower than those of human cartilage. This suggested that the nanofiber scaffold was sufficiently mechanically stable to withstand implantation and to support regenerated cartilage. The 50 : 50 PLGA scaffold was degraded faster than 75 : 25 PLGA, probably due to the higher hydrophilic glycolic acid content in the former. The nanofiber scaffold was degraded faster than a block-type scaffold that had a similar molecular weight. Therefore, degradation of the scaffold depended on the lactic acid/glycolic acid content ratio and might be controlled by mixing ratio of blend PLGA. Cellular responses were evaluated by examining toxicity, cell proliferation and extracellular matrix (ECM) formation using freshly isolated chondrocytes from porcine articular cartilage. The scaffolds were non-toxic, and cell proliferation and ECM formation in nanofiber scaffolds were superior to those in membrane-type scaffolds. Intermittent hydrostatic pressure applied to cell-seeded nanofiber scaffolds increased chondrocyte proliferation and ECM formation. In conclusion, our nanofiber-based PLGA scaffold has the potential to be used for cartilage reconstruction.     

Electrospun PLGA nanofiber scaffolds for articular cartilage reconstruction: mechanical stability, degradation and cellular responses under mechanical stimulation in vitro

We investigated the potential of a nanofiber-based poly(DL-lactide-co-glycolide) (PLGA) scaffold to be used for cartilage reconstruction. The mechanical properties of the nanofiber scaffold, degradation of the scaffold and cellular responses to the scaffold under mechanical stimulation were studied. Three different types of scaffold (lactic acid/glycolic acid content ratio = 75 : 25, 50 : 50, or a blend of 75 : 25 and 50 : 50) were tested. The tensile modulus, ultimate tensile stress and corresponding strain of the scaffolds were similar to those of skin and were slightly lower than those of human cartilage. This suggested that the nanofiber scaffold was sufficiently mechanically stable to withstand implantation and to support regenerated cartilage. The 50 : 50 PLGA scaffold was degraded faster than 75 : 25 PLGA, probably due to the higher hydrophilic glycolic acid content in the former. The nanofiber scaffold was degraded faster than a block-type scaffold that had a similar molecular weight. Therefore, degradation of the scaffold depended on the lactic acid/glycolic acid content ratio and might be controlled by mixing ratio of blend PLGA. Cellular responses were evaluated by examining toxicity, cell proliferation and extracellular matrix (ECM) formation using freshly isolated chondrocytes from porcine articular cartilage. The scaffolds were non-toxic, and cell proliferation and ECM formation in nanofiber scaffolds were superior to those in membrane-type scaffolds. Intermittent hydrostatic pressure applied to cell-seeded nanofiber scaffolds increased chondrocyte proliferation and ECM formation. In conclusion, our nanofiber-based PLGA scaffold has the potential to be used for cartilage reconstruction.     

Development of biodegradable electrospun scaffolds for dermal replacement

Our objective is to develop a synthetic biodegradable replacement dermal substitute for tissue engineering of skin and oral mucosa. Our in vivo criteria were that candidate scaffolds should allow surrounding cells to migrate fully into the scaffolds, enabling vasculogenesis and remodelling without invoking a chronic inflammatory response. We examined a total of six experimental electrospun polymer scaffolds: (1) poly-l-lactide (PLLA); (2) PLLA+10% oligolactide; (3) PLLA+rhodamine and (4-6) three poly(d,l)-lactide-co-glycolide (PLGA) random multiblock copolymers, with decreasing lactide/glycolide mole fractions (85:15, 75:25 and 50:50). These were evaluated for degradation in vitro up to 108 days and in vivo in adult male Wistar rats from 4 weeks to 12 months. In vivo, all scaffolds permitted good cellular penetration, with no adverse inflammatory response outside the scaffold margin and with no capsule formation around the periphery. The breakdown rate for each scaffold in vitro versus in vivo was similar, and an increase in the ratio of polyglycolide to polylactide correlated with an increase in breakdown rate, as expected. Scaffolds of PLLA were stable in vivo even after 12 months whereas scaffolds fabricated from PLGA 85:15 and 75:25 revealed a 50% loss of mass after 4 and 3 months, respectively. In vitro PLGA 85:15 and 75:25 scaffolds were able to support keratinocyte, fibroblast and endothelial cell growth and extracellular matrix production, with evidence of new collagen production after 7 days. In conclusion, the data supports the development of PLGA 85:15 and 75:25 electrospun polymer scaffolds as potential degradable biomaterials for dermal replacement.     

The influence of molecular weight of chitosan on the physical and biological properties of collagen/chitosan scaffolds

Biopolymer blends between collagen and chitosan have the potential to produce cell scaffolds with biocompatible properties. However, the relationship between the molecular weight of chitosan and its effect on physical and biological properties of collagen/chitosan scaffolds has not been elucidated yet. Porous scaffolds were fabricated by freeze-drying the solution of collagen and chitosan, followed by cross-linking by dehydrothermal treatment. Various types of scaffolds were prepared using chitosan with various molecular weights and blending ratios. Fourier transform infrared spectroscopy proved that collagen and chitosan scaffolds at all blending ratios contained mainly electrostatic interactions at the molecular level. The compressive modulus decreased with increasing the concentration of chitosan. Equilibrium swelling ratios of approximately 6-8, determined in phosphate-buffered saline at physiological pH (7.4), were found in case of collagen-dominated scaffolds. The lysozyme biodegradation test demonstrated that the presence of chitosan, especially the high-molecular-weight species, could significantly prolong the biodegradation of collagen/chitosan scaffolds. In vitro culture of L929 mouse connective tissue fibroblast evidenced that low-molecular-weight chitosan was more effective to promote and accelerate cell proliferation, particularly for scaffolds containing 30 wt% chitosan. The results elucidated that the blends of collagen with low-molecular-weight chitosan have a high potential to be applied as new materials for skin-tissue engineering.     

A multilayered scaffold of a chitosan and gelatin hydrogel supported by a PCL core for cardiac tissue engineering

A three-dimensional scaffold composed of self-assembled polycaprolactone (PCL) sandwiched in a gelatin–chitosan hydrogel was developed for use as a biodegradable patch with a potential for surgical reconstruction of congenital heart defects. The PCL core provides surgical handling, suturability and high initial tensile strength, while the gelatin–chitosan scaffold allows for cell attachment, with pore size and mechanical properties conducive to cardiomyocyte migration and function. The ultimate tensile stress of the PCL core, made from blends of 10, 46 and 80 kDa (Mn) PCL, was controllable in the range of 2–4 MPa, with lower average molecular weight PCL blends correlating with lower tensile stress. Blends with lower molecular weight PCL also had faster degradation (controllable from 0% to 7% weight loss in saline over 30 days) and larger pores. PCL scaffolds supporting a gelatin–chitosan emulsion gel showed no significant alteration in tensile stress, strain or tensile modulus. However, the compressive modulus of the composite tissue was similar to that of native tissue (～15 kPa for 50% gelatin and 50% chitosan). Electron microscopy revealed that the gelatin–chitosan gel had a three-dimensional porous structure, with a mean pore diameter of ～80 μm, showed migration of neonatal rat ventricular myocytes (NRVM), maintained NRVM viability for over 7 days, and resulted in spontaneously beating scaffolds. This multi-layered scaffold has sufficient tensile strength and surgical handling for use as a cardiac patch, while allowing migration or pre-loading of cardiac cells in a biomimetic environment to allow for eventual degradation of the patch and incorporation into native tissue.