Radiation synthesis of gelatin/CM-chitosan/β-tricalcium phosphate composite scaffold for bone tissue engineering

A series of biodegradable composite scaffolds was fabricated from an aqueous solution of gelatin, carboxymethyl chitosan (CM-chitosan) and β-tricalcium phosphate (β-TCP) by radiation-induced crosslinking at ambient temperature. Ultrasonic treatment on the polymer solutions significantly influenced the distribution of β-TCP particles. An ultrasonic time of 20 min, followed by 30 kGy irradiation induced a crosslinked scaffold with homogeneous distribution of β-TCP particles, interconnected porous structure, sound swelling capacity and mechanical strength. Fourier Transform Infrared Spectroscopy and X-ray Diffraction analysis indicated that β-TCP successfully incorporated with the network of gelatin and CM-chitosan. In vivo implantation of the scaffold into the mandible of beagle dog revealed that the scaffolds had excellent biocompatibility and the presence of β-TCP can accelerate bone regeneration. The comprehensive results of this study paved way for the application of gelatin/CM-chitosan/β-TCP composite scaffolds as candidate of bone tissue engineering material.     

Injectable melatonin-loaded carboxymethyl chitosan (CMCS)-based hydrogel accelerates wound healing by reducing inflammation and promoting angiogenesis and collagen deposition

Carboxymethyl chitosan(CMCS)-based hydrogels have antibacterial activity,and have shown the abilities of preventing wound infection,promoting cell proliferation,accelerating collagen deposition,and stimulating hyaluronic acid formation during wound healing.As a hormone produced by the pineal gland in humans and animals,melatonin promotes skin wound healing by regulating the release of inflammatory mediators and accelerating the proliferation and migration of cells,angiogenesis,and collagen deposition.However,the combined effects of CMCS and melatonin on wound healing remain unclear.Injectable CMCS-based hydrogels containing melatonin were prepared,and their healing effects were evaluated using a full-thickness cutaneous wound model in rats.Compared with the control and the hydrogel with no melatonin groups,the melatonin-loaded hydrogel significantly increased the percentage of wound closure,promoted the proliferation of granulation tissue and re-epithelialization,and accelerated collagen deposition.Additionally,the melatonin-loaded hydrogel promoted angiogenesis and vascular endothelial growth factor receptor protein expression and increased the expression of cyclooxygenase-2 and inducible nitric oxide synthase.The melatonin-loaded hydrogel also markedly increased the expression of collagen III,α-smooth muscle actin,and transforming growth factor-β1 proteins and reduced collagen I expression.These results suggest that the melatonin-loaded hydrogel promoted granulation tissue formation and accelerated wound healing by reducing inflammation and promoting angiogenesis and collagen deposition.     

Assessment of reinforced poly(ethylene glycol) chitosan hydrogels as dressings in a mouse skin wound defect model

Wound dressings of chitosan are biocompatible, biodegradable, antibacterial and hemostatic biomaterials. However, applications for chitosan are limited due to its poor mechanical properties. Here, we conducted an in vivo mouse angiogenesis study on reinforced poly(ethylene glycol) (PEG)-chitosan (RPC) hydrogels. RPC hydrogels were formed by cross-linking chitosan with PEGs of different molecular weights at various PEG to chitosan ratios in our previous paper. These dressings can keep the wound moist, had good gas exchange capacity, and was capable of absorbing or removing the wound exudate. We examined the ability of these RPC hydrogels and neat chitosan to heal small cuts and full-thickness skin defects on the backs of male Balb/c mice. Histological examination revealed that chitosan suppressed the infiltration of inflammatory cells and accelerated fibroblast proliferation, while PEG enhanced epithelial migration. The RPC hydrogels promoted wound healing in the small cuts and full layer wounds. The optimal RPC hydrogel had a swelling ratio of 100% and a water vapor transmission rate (WVTR) of about 2000 g/m²/day. In addition, they possess good mechanical property and appropriate degradation rates. Thus, the optimal RPC hydrogel formulation functioned effectively as a wound dressing and promoted wound healing.     

Nanofibers prepared by needleless electrospinning technology as scaffolds for wound healing

Electrospun gelatin and poly-ε-caprolactone (PCL) nanofibers were prepared using needleless technology and their biocompatibility and therapeutic efficacy have been characterized in vitro in cell cultures and in an experimental model of a skin wound. Human dermal fibroblasts, keratinocytes and mesenchymal stem cells seeded on the nanofibers revealed that both nanofibers promoted cell adhesion and proliferation. The effect of nanofibers on wound healing was examined using a full thickness wound model in rats and compared with a standard control treatment with gauze. Significantly faster wound closure was found with gelatin after 5 and 10 days of treatment, but no enhancement with PCL nanofibers was observed. Histological analysis revealed enhanced epithelialisation, increased depth of granulation tissue and increased density of myofibroblasts in the wound area with gelatin nanofibers. The results show that gelatin nanofibers produced by needleless technology accelerate wound healing and may be suitable as a scaffold for cell transfer and skin regeneration.     

Studies on gelatin-based sponges. Part III: A comparative study of cross-linked gelatin/alginate, gelatin/hyaluronate and chitosan/hyaluronate sponges and their application as a wound dressing in full-thickness skin defect of rat

Novel cross-linked sponges composed of gelatin/alginate and gelatin/hyaluronate and chitosan/hyaluronate (GH, GA and CH, respectively) were prepared and compared. Six different sponges with or without silver sulfadiazine (AgSD) were applied on the full-thickness dorsal skin defect of Wistar rat. The histology and epidermal wound healing rates of the skin defects were investigated by light microscopy and computerized morphometry 5 and 12 days post-operatively. In our full-thickness wound model (diameter 1 cm), the AgSD-impregnated sponges showed good wound healing performances on the whole. However, there appeared meaningful differences of wound healing between the gelatin-based sponges (GH, GA) and the CH. GH with AgSD was found to show the best wound healing properties as a wound dressing resulting from histological findings and computerized morphometric analysis of epidermal healing.     

Moisture-Adaptive Contractile Biopolymer-Derived Fibers for Wound Healing Promotion

The advancement in thermosensitive active hydrogels has opened promising opportunities to dynamic full-thickness skin wound healing. However, conventional hydrogels lack breathability to avoid wound infection and cannot adapt to wounds with different shapes due to the isotropic contraction. Herein, a moisture-adaptive fiber that rapidly absorbs wound tissue fluid and produces a large lengthwise contractile force during the drying process is reported. The incorporation of hydroxyl-rich silica nanoparticles in the sodium alginate/gelatin composite fiber greatly improves the hydrophilicity, toughness, and axial contraction performance of the fiber. This fiber exhibits a dynamic contractile behavior as a function of humidity, generating ≈15% maximum contraction strain or ≈24 MPa maximum isometric contractile stress. The textile knitted by the fibers features excellent breathability and generates adaptive contraction in the target direction during the natural desorption of tissue fluid from the wounds. In vivo animal experiments further demonstrate the advantages of the textiles over traditional dressings in accelerating wound healing.     

Biological evaluation of human hair keratin scaffolds for skin wound repair and regeneration

The cytocompatibility, in vivo biodegradation and wound healing of keratin biomaterials were investigated. For the purposes, three groups of keratin scaffolds were fabricated by freeze-drying reduced solutions at 2 wt.%, 4 wt.% and 8 wt.% keratins extracted from human hairs. These scaffolds exhibited evenly distributed high porous structures with pore size of 120–220 μm and the porosity > 90%. NIH3T3 cells proliferated well on these scaffolds in culture lasting up to 22 days. Confocal micrographs stained with AO visually revealed cell attachment and infiltration as well as scaffold architectural stability. In vivo animal experiments were conducted with 4 wt.% keratin scaffolds. Early degradation of subcutaneously implanted scaffolds occurred at 3 weeks in the outermost surface, in concomitant with inflammatory response. At 5 weeks, the overall porous structure of scaffolds severely deteriorated while the early inflammatory response in the outermost surface obviously subsided. A faster keratin biodegradation was observed in repairing full-thickness skin defects. Compared with the blank control, keratin scaffolds gave rise to more blood vessels at 2 weeks and better complete wound repair at 3 weeks with a thicker epidermis, less contraction and newly formed hair follicles. These preliminary results suggest that human hair keratin scaffolds are promising dermal substitutes for skin regeneration.     

Novel three-dimensional cocoon-like hydrogels for soft tissue regeneration

Large three-dimensional hydrogels (> 150 cm³) of bacterial cellulose (BC) were synthesized by using Gluconacetobacter hansenii ATCC 23769 under controlled agitated culture conditions. The macroscopic cocoon-like structures are gelatinous and translucent and may find applications in several areas, particularly in tissue and organ engineering. Internal microstructure was investigated by scanning electron microscopy (SEM) and field emission scanning electron microscopy (FESEM), which revealed that the cocoons are composed of cellulosic nanofibres randomly and three-dimensionally dispersed. The macroscopic bodies are delimited by a dense semi-permeable membrane with thickness between 0.2 and 2 mm, also composed of cellulosic nanofibres. Endothelial cells were seeded on the hydrogels and incubated for 7 days. HUVECs grew and migrated into the inner part of the structure. The three-dimensional BC hydrogel structures can be directly implanted in tissue deficient regions as scaffolds containing the appropriate cultured cells.     

In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1_WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1_WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1_WSC constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1_WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1_WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.     

Cross-linked nanocomposite hydrogels based on cellulose nanocrystals and PVA: Mechanical properties and creep recovery

Cellulose nanocrystal (CNC) reinforced poly(vinyl alcohol) (PVA) hydrogels with a water content of ∼92% were successfully prepared with glutaraldehyde (GA) as a cross-linker. The effects of the CNC content on the thermal stability, swelling ratio and mechanical and viscoelastic properties of the cross-linked hydrogels were investigated. The compressive strength at 60% strain for the hydrogels with 1 wt% CNCs increased by 303%, from 17.5 kPa to 53 kPa. The creep results showed that the addition of CNCs decreased the creep elasticity due to molecular chain restriction. The almost complete strain recovery (∼97%) after fixed load removal for 15 min was observed from the hydrogels with CNCs, compared with 92% strain recovery of the neat cross-linked PVA hydrogels. The incorporation of CNCs did not affect the swelling ratio and thermal stability of the hydrogels. These results suggest the cross-linked CNC-PVA hydrogels have potential for use in biomedical and tissue engineering applications.     

Effect of crosslinking temperature on compression strength of gelatin scaffold for articular cartilage tissue engineering

A crosslinking method with directly crosslinking the gelatin gel by genipin was performed at 10–25 °C. The microstructure of the gelatin scaffold can be modulated by the crosslinking temperature. During repeated compression–swelling test, a plateau stress occurred. This plateau stress was relatively constant during the cyclic test. The cartilage tissue developed in the scaffold can largely enhance the compression strength of the scaffold and the scaffold with largest pore (350–500 μm, 25 °C-crosslinked) developed largest amount of the cartilage tissue after cell culture. With or without cell culture, the scaffold with smallest pore (50–150 μm, 10 °C-crosslinked) showed the highest compression strength. The pure gelatin scaffold can undergo reversible deformation for the motion of the articular cartilage.     

Preparation of fluoride-containing gelatin nanofiber scaffold

A novel material, fluoride-containing gelatin nanofiber scaffold, was prepared by electrospinning process successfully. Scanning electron microscopy (SEM) image showed that the morphology of nanofibers was uniform and smooth, and the average diameter was about 200 nm. An even distribution in the fiber matrix of some nanoparticles which is about 20 nm was also observed in transmission electron microscopy (TEM) images. X-ray diffraction (XRD) demonstrated that the nanoparticles dispersed in the gelatin fiber matrix were CaF₂ crystals. This scaffold was crosslinked with glutaraldehyde (GTA) vapor at room temperature for 4 days in order to improve the water-resistant ability. After soaking in Dulbecco's Modified Eagle's Medium (DMEM) solution (37 °C) for 4 weeks the crosslinked scaffold still maintained a good appearance and morphology. All the properties of this novel material show a good potential to be a bone tissue engineering scaffold.     

X-ray computed tomography proof of bacterial-based self-healing in concrete

Self-healing strategies are regarded as a promising solution to reduce the high maintenance and repair cost of concrete infrastructures. In the present work, a bacterial-based self-healing by use of hydrogel encapsulated bacterial spores (bio-hydrogels) was investigated. The crack closure behavior of the specimens with/without bio-hydrogels was studied quantitatively by light microscopy. To have a view of the self-healing inside the specimens, a high resolution X-ray computed microtomography (X-ray μCT) was used. The total amount and the distribution of the healing products in the whole matrix were investigated. This study indicates that the specimens incorporated with bio-hydrogels had distinct improved healing efficiency compared to the reference ones with pure hydrogel only. The healing ratios in the specimens with bio-hydrogels were in the range from 70% to 100% for the cracks smaller than 0.3 mm, which is more than 50% higher than for the ones with pure hydrogel; and the maximum crack bridging was about 0.5 mm (in 7 d), while pure hydrogels only allowed healing of cracks of about 0.18 mm. The total volume ratio of the healing product in the specimens with bio-hydrogels amounted to 2.2%, which was about 60% higher than for the ones with pure hydrogel (1.37%).     

Effect of chondroitin sulfate on osteogenetic differentiation of human mesenchymal stem cells

Chondroitin sulfate (CS) has anti-inflammatory properties and increases the regeneration ability of injured bone. In different in vivo investigations on bone defects the addition of CS to calcium phosphate bone cement has lead to an enhanced bone remodeling and increased new bone formation. The goal of this study was to evaluate the cellular effects of CS on human mesenchymal stem cells (hMSCs). In cell culture experiments hMSCs were incubated on calcium phosphate bone cements with and without CS and cultivated in a proliferation and an osteogenetic differentiation media. Alkaline phosphatase and the proliferation rate were determined on days 1, 7 and 14. Concerning the proliferation rates, no significant differences were detected. On days 1, 7 and 14 a significantly higher activity of alkaline phosphatase, an early marker of osteogenesis, was detected around CS modified cements in both types of media. The addition of CS leads to a significant increase of osteogenetic differentiation of hMSCs. To evaluate the influence of the osteoconductive potency of CS in twelve adult male Wistar rats, the interface reaction of cancellous bone to a nanocrystalline hydroxyapatite cement containing type I collagen (CDHA/Coll) without and with CS (CDHA/Coll/CS) was evaluated. Cylindrical implants were inserted press-fit into a defect of the tibial head. 28 days after the operation the direct bone contact and the percentage of newly formed bone were significantly higher on CDHA/Coll/CS-implants (p < 0.05). The addition of CS appears to enhance new bone formation on CDHA/Coll-composites in the early stages of bone healing. Possible mechanisms are discussed.     

Adhesive Hemostatic Conducting Injectable Composite Hydrogels with Sustained Drug Release and Photothermal Antibacterial Activity to Promote Full‐Thickness Skin Regeneration During Wound Healing

Developing injectable nanocomposite conductive hydrogel dressings with multifunctions including adhesiveness, antibacterial, and radical scavenging ability and good mechanical property to enhance full‐thickness skin wound regeneration is highly desirable in clinical application. Herein, a series of adhesive hemostatic antioxidant conductive photothermal antibacterial hydrogels based on hyaluronic acid‐graft‐dopamine and reduced graphene oxide (rGO) using a H₂O₂/HPR (horseradish peroxidase) system are prepared for wound dressing. These hydrogels exhibit high swelling, degradability, tunable rheological property, and similar or superior mechanical properties to human skin. The polydopamine endowed antioxidant activity, tissue adhesiveness and hemostatic ability, self‐healing ability, conductivity, and NIR irradiation enhanced in vivo antibacterial behavior of the hydrogels are investigated. Moreover, drug release and zone of inhibition tests confirm sustained drug release capacity of the hydrogels. Furthermore, the hydrogel dressings significantly enhance vascularization by upregulating growth factor expression of CD31 and improve the granulation tissue thickness and collagen deposition, all of which promote wound closure and contribute to a better therapeutic effect than the commercial Tegaderm films group in a mouse full‐thickness wounds model. In summary, these adhesive hemostatic antioxidative conductive hydrogels with sustained drug release property to promote complete skin regeneration are an excellent wound dressing for full‐thickness skin repair.     

Recognition of four structurally resembled benzoic acid derivatives by albumin-crosslinked poly(acrylamide) hydrogel

Poly(acrylamide) hydrogels crosslinked by bovine serum albumin (BSA) were prepared by the introduction of vinyl groups into BSA and subsequent co-polymerization with acrylamide (AAm). The hydrogels were loaded with four structurally resembled benzoic acid derivatives such as salicylic acid, o-anisic acid, salicylamide and sodium benzoate, and their release from the hydrogel was investigated. The affinity of these four compounds for BSA gradually decreased in a following order; salicylic acid > o-anisic acid > salicylamide > sodium benzoate. The amounts of compounds loaded on a hydrogel of high BSA content and the duration of their release were found to be dependent on the affinity for BSA clearly reflecting the subtle difference in BSA affinity of each compound. Therefore, this hydrogel would be used not only as a sustained drug release carrier, but also a facile tool to evaluate the binding of various compounds to serum albumin.     

Mg ion implantation on SLA-treated titanium surface and its effects on the behavior of mesenchymal stem cell

Magnesium (Mg) is one of the most important ions associated with bone osseointegration. The aim of this study was to evaluate the cellular effects of Mg implantation in titanium (Ti) surfaces treated with sand blast using large grit and acid etching (SLA). Mg ions were implanted into the surface via vacuum arc source ion implantation. The surface morphology, chemical properties, and the amount of Mg ion release were evaluated by scanning electron microscopy (SEM), Auger electron spectroscopy (AES), Rutherford backscattering spectroscopy (RBS), and inductively coupled plasma-optical emission spectrometer (ICP-OES). Human mesenchymal stem cells (hMSCs) were used to evaluate cellular parameters such as proliferation, cytotoxicity, and adhesion morphology by MTS assay, live/dead assay, and SEM. Furthermore, osteoblast differentiation was determined on the basis of alkaline phosphatase (ALP) activity and the degree of calcium accumulation. In the Mg ion-implanted disk, 2.3 × 10¹⁶ ions/cm² was retained. However, after Mg ion implantation, the surface morphology did not change. Implanted Mg ions were rapidly released during the first 7 days in vitro. The MTS assay, live/dead assay, and SEM demonstrated increased cell attachment and growth on the Mg ion-implanted surface. In particular, Mg ion implantation increased the initial cell adhesion, and in an osteoblast differentiation assay, ALP activity and calcium accumulation. These findings suggest that Mg ion implantation using the plasma source ion implantation (PSII) technique may be useful for SLA-treated Ti dental implants to improve their osseointegration capacity.     

Investigation of UV-photon induced hydrophilicity of titanium ion-implanted soda-lime silicate glasses

The UV-induced wetting effect on titanium oxide surface is well-known; however, the UV-induced hydrophilicity of titanium implanted soda-lime silicate glass has not been investigated. Hence the contact angle of water droplet under the indoor fluorescent lights on titanium-ion implanted soda-lime silicate glasses was investigated. The silicate glasses were implanted by MEVVA ion implanter by 40 keV titanium ions with a fluence of 10¹⁵ ions cm^?2. The contact angle, the chemical bonding environment, and surface morphologies were examined. Results show the formation of TiO₂, the increase of surface roughness, and the reduction of the contact angle after the ion implantation. Further enhancement of hydrophilicity after the 254 nm pre-UV irradiation for 1 h on the implanted sample surface was observed. The enhancement of the wetting effect after ion implantation could be attributed to rougher TiO₂ content surface. However, according to the mechanisms of UV photo-induced hydrophilicity on TiO₂ proposed previously, the enhancement of hydrophilicity of titanium implanted surface with and without 254 nm pre-photon radiation can be attributed to not only the reduction of hydrocarbon on surface during the UV radiation but also to the oxygen vacancies produced by 254 nm UV photon irradiation.     

Detection of growth factor binding to gelatin and heparin using a photonic crystal optical biosensor

Drug–carrier interactions are important to protein controlled release systems to protect the protein from denaturation and ensure properly timed release. A novel photonic crystal biosensor was used to investigate a gelatin–protein controlled release system to determine the amount of protein bound to the carrier at physiological conditions. The Biomolecular Interaction Detection (BIND) system reflects a narrow band of wavelengths when white light is shone incident to the grating. As mass is deposited onto the surface, the peak wavelength value is shifted due to changes in the optical density of the biosensor. The BIND system was used to detect the binding of growth factors onto acidic gelatin, basic gelatin, and heparin on the sensor surface. Through a series of experiments, including functionalizing the sensor, adjusting the ionic strength of the solution, adjusting the substrate concentration, and minimizing non-specific signal, the adsorption of the gelatins and heparin on the sensor was enhanced. The binding interaction of recombinant human transforming growth factor (rhTGF)-β1 and bone morphogenetic protein (rhBMP)-2 with the two types of gelatin and heparin were investigated. The strength of the interaction between rhTGF-β1 and the substrates is in the following order: heparin > acidic gelatin > basic gelatin. RhBMP-2 bound to the substrates but with less intensity than TGF-β1: heparin > basic gelatin > acidic gelatin. This work provides support for the controlled release mechanism through degradation of the gelatin carrier.     

Thermosensitive in situ-forming dextran–pluronic hydrogels through Michael addition

A thiolated dextran (Dex-SH) with a degree of substitution of 10 was synthesized and used for in situ hydrogel formation via Michael-type addition using vinyl sulfone functionalized Pluronic 127 (PL-VS) or acrylated Pluronic 127 (PL-Ar). Dextran/Pluronic hydrogels were rapidly formed in situ under physiological conditions upon mixing the solutions of Dex-SH and PL-VS or PL-Ar at a PL concentration of 10 or 20 w/v%. Rheological studies showed that these hydrogels with a broad range of storage moduli of 0.3 to 80 kPa could be obtained by varying PL concentration from 5% to 20 w/v%. Moreover, the hydrogels at a PL concentration of 10% or 20 w/v% revealed thermosensitive property with a temperature increase from 10 to 37 °C. Dex-SH/PL-Ar hydrogels were degradable under physiological conditions and had lower cytotoxicity than Dex-SH/PL-VS hydrogels. These thermosensitive injectable hydrogels show promise for biomedical applications.