Collagen-chitosan Scaffold Research Articles

Biopolymer collagen-chitosan scaffold containing Aloe vera for chondrogenic efficacy on cartilage tissue engineering

The chondrogenic efficacy of aloe vera blended collagen-chitosan (COL-CS-AV) porous scaffold was investigated using articular chondrocytes in a standard condition. Cytocompatibility was analyzed using fluorescent dyes (calcein AM/ethidium bromide) and the viable cells were quantified by MTT assay. Glycosaminoglycan (GAG) content of ECM was estimated by using 1, 9-Dimethyl methylene Blue (DMMB). The total RNA content was quantified and the cartilage specific genes (col2a1, Acan) were amplified by reverse transcription-PCR from the cell lysate of the scaffolds. Histological examination was made using Haematoxylin and Eosin (H&E), safranin-O, masson's trichrome, alcian blue, and alizarin red to stain the specific component of ECM secreted on the construct. The cartilage specific collagen type II was estimated by immunohistochemistry using monoclonal type II collagen antibody. The results of these studies proved that COL-CS-AV scaffold has more chondrogenic efficacy than COL-CS, thus the aloe vera blend COL-CS-AV scaffold might be used as suitable candidate for cartilage tissue engineering.

Nanosilver alleviates foreign body reaction and facilitates wound repair by regulating macrophage polarization.

Foreign body reactions induced by macrophages often cause delay or failure of wound healing in the application of tissue engineering scaffolds. This study explores the application of nanosilver (NAg) to reduce foreign body reactions during scaffold transplantation. An NAg hybrid collagen-chitosan scaffold (NAg-CCS) was prepared using the freeze-drying method. The NAg-CCS was implanted on the back of rats to evaluate the effects on foreign body reactions. Skin tissue samples were collected for histological and immunological evaluation at variable intervals. Miniature pigs were used to assess the effects of NAg on skin wound healing. The wounds were photographed, and tissue samples were collected for molecular biological analysis at different time points post-transplantation. NAg-CCS has a porous structure and the results showed that it could release NAg constantly for two weeks. The NAg-CCS group rarely developed a foreign body reaction, while the blank-CCS group showed granulomas or necrosis in the subcutaneous grafting experiment. Both matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced significantly in the NAg-CCS group. The NAg-CCS group had higher interleukin (IL)-10 and lower IL-6 than the blank CCS group. In the wound healing study, M1 macrophage activation and inflammatory-related proteins (inducible nitric oxide synthase (iNOS), IL-6, and interferon-‍γ (IFN-‍γ)) were inhibited by NAg. In contrast, M2 macrophage activation and proinflammatory proteins (arginase-1, major histocompatibility complex-II (MHC-II), and found in inflammatory zone-1 (FIZZ-1)) were promoted, and this was responsible for suppressing the foreign body responses and accelerating wound healing. In conclusion, dermal scaffolds containing NAg suppressed the foreign body reaction by regulating macrophages and the expression of inflammatory cytokines, thereby promoting wound healing.

Metabolic dependency of non-small cell lung cancer cells affected by three-dimensional scaffold and its stiffness.

Three-dimensional (3D) extracellular matrix (ECM) microenvironment is an important regulator of the stiffness of the tumors. Cancer cells require heterogeneous metabolic phenotypes to cope with resistance in the malignant process. However, how the stiffness of the matrix affects the metabolic phenotypes of cancer cells, is lacking. In this study, the young's modulus of the synthesized collagen-chitosan scaffolds was adjusted according to the percentage ratio of collagen to chitosan. We cultured non-small cell lung cancer (NSCLC) cells in four different microenvironments (two-dimensional (2D) plates, stiffest 0.5-0.5 porous collagen-chitosan scaffolds, middle stiff 0.5-1 porous collagen-chitosan scaffolds, and softest 0.5-2 porous collagen-chitosan scaffolds) to investigate the influence of the difference of 2D and 3D cultures as well as the 3D scaffolds with different stiffnesses on the metabolic dependency of NSCLC cells. The results revealed that NSCLC cells cultured in 3D collagen-chitosan scaffolds displayed higher capacity of mitochondrial metabolism and fatty acid metabolism than that cultured in 2D culture. The metabolic response of NSCLC cells is differential for 3D scaffolds with different stiffnesses. The cells cultured in middle stiff 0.5-1 scaffolds displayed a higher potential of mitochondrial metabolism than that of stiffer 0.5-0.5 scaffolds and softer 0.5-2 scaffolds. Furthermore, NSCLC cells culture in 3D scaffolds displayed drug resistance compared with that in 2D culture which maybe via the hyperactivation of the mTOR pathway. Moreover, the cells cultured in 0.5-1 scaffolds showed higher ROS levels, which were counterbalanced by an equally high expression of antioxidant enzymes when compared to the cells grown in 2D culture, which may be regulated by the increased expression of PGC-1α. Together, these results demonstrate that differences in the microenvironments of cancer cells profoundly impact their metabolic dependencies.

Optimization and evaluation of ciprofloxacin-loaded collagen/chitosan scaffolds for skin tissue engineering.

A novel ciprofloxacin-loaded collagen-chitosan scaffold was developed for the treatment of wound using freeze drying method. The average pore size and porosity of developed scaffold was found to be around 125µm and 91 ± 0.56%. Moreover, swelling, degradation and mechanical tests profile supported the suitability of scaffold for wound healing process. The scaffold has high degree of hemocompatibility towards the blood and promotes the growth, migration and proliferation of fibroblast. The developed scaffold exhibits antibacterial properties and was found to be efficient against the Gram-negative (E.coli) and Gram-positive (Staphylococcus aureus) bacteria hence can be used for wound healing applications. In vivo study demonstrated that the scaffold not only escalated the tissue regeneration time but also accelerated the wound healing process compared to control. The histological studies revealed better granulation, vascularization, and remodeling of extracellular matrix along with regeneration of epidermal and dermal layer of skin. Overall, the obtained results suggested that the developed skin tissue constructs possess the enormous potential for tissue regeneration and might be a suitable biomaterial for skin tissue engineering applications.

Design and evaluation of ciprofloxacin loaded collagen chitosan oxygenating scaffold for skin tissue engineering

Hypoxia and sepsis are key concerns towards modern regenerative medicine. Oxygen generating biomaterials having antibacterial property aims to answer these concerns. Hypoxia promotes reactive oxygen species at the implant site that delays wound healing. Sepsis in wound also contributes to delay in wound healing. Therefore, scaffold with antibacterial property and oxygen-producing capacities have shown ability to promote wound healing. In the present study oxygen releasing, ciprofloxacin loaded collagen chitosan scaffold was fabricated for sustained oxygen delivery. Calcium peroxide (CPO) acted as a chemical oxygen source. Oxygen release pattern exhibited a sustained release of oxygen with uniform deposition of CPO on the scaffold. The drug release study shows a prolonged, continuous, and sustained release of ciprofloxacin. Cell culture studies depict that scaffold has suitable cell attachment and migration properties for fibroblasts. In vivo studies performed in the skin flip model visually shows better wound healing and less necrosis. Histological studies show the maintenance of tissue architecture and the deposition of collagen. The results demonstrate that the proposed CPO coated ciprofloxacin loaded collagen-chitosan scaffold can be a promising candidate for skin tissue engineering.

COLLAGEN CHITOSAN SCAFFOLDS ON INDUCED SKIN DEFECT IN A RAT MODEL (AN EXPERIMENTAL STUDY)

BACKGROUND: Over the past ten years, an alternative for maxillofacial reconstruction was offered through regenerative medicine. It provided a new scope for improving the reconstruction of the oral and maxillofacial structures whether they are hard tissues which include the teeth and bone or the soft tissues like the oral mucosa, skin, nerves and blood vessels.OBJECTIVES: The aim of this study is to evaluate the effect of the collagen chitosan scaffolds on the duration and quality of skin surface healing.MATERIALS AND METHODS: An experimental in vivo comparative study was conducted, on twelve adult male Sprague-Dawley rats, where an induced skin wound was made on the backs of rats and then classified into two groups; 1-control group and 2-scaffold group. The effect of the collagen-chitosan scaffolds on the duration and quality of skin surface healing were assessed using Masson’s trichrome staining for collagen fibers and the imageJ software to calculate % area after wound healing also the number of CD 68 macrophages was counted using histological sections then the two groups were compared using student t-test (p < 0.05).RESULTS: The scaffold group showed accelerated wound healing in comparison to the control group and with a significant result. Quality of healed skin was significantly better in the scaffold treated group, which is attributed to the better collagen alignment and deposition in the stained histological sections indicated by the % area after wound healing. The number of CD 68 macrophages was higher in the scaffold group compared to the control group.CONCLUSION: The collagen chitosan scaffolds can be considered as a treatment regimen for treating skin wounds.

Porous Scaffold Implantation for Bone Fractures Healing

The objective of this research was to evaluate the efficacy of Low Level Therapy of Continuous Diode Laser on porous Hydroxyapatite–Chitosan–Collagen scaffold implantation by daily clinical observation, weekly radiographic findings and histopathological examination at the end of 3rd, 6th, 9th and 12th weeks after surgery. Fifty Healthy adult rabbits of both sexes were used to induced diaphyseal Femur and to remove about (1±0.05 cm) of bone, as empty space filler.

Bone marrow-derived mesenchymal stem cells and extracellular vesicles enriched collagen chitosan scaffold in skin wound healing (a rat model).

Over the past ten years, regenerative medicine has focused on the regeneration and the reconstruction of damaged, diseased, or lost tissues and organs. Skin, being the largest organ in the human body, had attained a good attraction in this field. Delayed wound healing is one of the most challenging clinical medicine complications. This study aimed to evaluate the collagen chitosan scaffold's effect alone, or enriched with either bone marrow-derived mesenchymal stem cells (BM-MSCs) or their secreted extracellular vesicles (EVs) on the duration and quality of skin wound healing. A full-thickness skin wound was induced on the back of 32 adult male Sprague-Dawley rats. The wounds were either covered with collagen chitosan scaffolds alone, scaffolds enriched with stem cells, or extracellular vesicles. Unprotected wounds were used as control. Healing duration, collagen deposition and alignment, CD 68+ macrophage count, and functional tensile strength of healed skin were assessed (α = 0.05, n = 8). The rate of skin healing was significantly accelerated in all treated groups compared to the control. Immuno-histochemical assessment of CD68+ macrophages showed enhanced macrophages count, in addition to higher collagen deposition and better collagen alignment in EVs and BM-MSCs treated groups compared to the control group. Higher tensile strength values reflected the better collagen deposition and alignment for these groups. EVs showed higher amounts of collagen deposition and better alignment compared to MSCs treated group. The collagen chitosan scaffolds enriched with MSCs or their EVs improved wound healing and improved the quantity and remodeling of collagen with a better assignment to EVs.

Preparation and characterization of biomedical collagen–chitosan scaffolds with entrapped ibuprofen and silver nanoparticles

In this work, the preparation and characterization of a novel multifunctional collagen–chitosan material containing silver nanoparticles and nonsteroid anti‐inflammatory drug ibuprofen is described. As a starting material, the commercially available collagen–chitosan scaffold was used. Drug‐loading procedure was performed via supercritical CO2‐assisted impregnation technique. Silver nanoparticles were prepared via metal vapor synthesis and introduced into the composite biopolymer matrix. An analysis of the small‐angle X‐ray scattering profiles revealed that silver nanoparticles are characterized by having a logarithmic normal size distribution with the maximum at 25 nm. The average size of particles determined from the broadening of diffraction peaks is in a good agreement with this result. The surface of the materials was characterized by X‐ray photoelectron spectroscopy. The in vitro release kinetics of ibuprofen in phosphate buffer solution with pH = 7.4 from prepared materials was studied. The drug release to solution is governed by Fickian diffusion and can be described within the Ritger–Peppas model. Introduction of silver nanoparticles has no effect on the diffusion mechanism. The as‐prepared materials are promising for the medical applications such as dressings for treatments of infected wounds and infection preventing. These materials can act as a matrix for transdermal drug administration. POLYM. ENG. SCI., 59:2479–2487, 2019. © 2019 Society of Plastics Engineers

Effect of two crosslinking methods on the physicochemical and biological properties of the collagen-chitosan scaffolds

Chemical and physical crosslinking methods using glutaraldehyde (GA) and ammonium hydroxide (AH), respectively, were utilized to prepare chitosan (CS) and chitosan/collagen (CS-Co) hydrogels; these materials were then subjected to freeze-drying process to obtain 3D porous scaffolds. The effect of crosslinking methods on the physicochemical and biological properties of the scaffolds was studied. Results indicated that crosslinking method plays a key role on morphology, average pore size, pore size distribution, degradation and cytotoxicity of the scaffolds. Physically crosslinked scaffolds exhibited a homogeneous morphology with higher pore size and interconnectivity in comparison to other prepared scaffolds; also, these samples, showed a good biocompatibility. Scaffolds derived from hydrogels treated with acetone (AC) showed shrinkage, smaller pore size and higher degradation rates; finally, materials chemically crosslinked with glutaraldehyde presented cytotoxicity and exhibited a heterogeneous morphology. Overall, these results suggest that the scaffolds physically cross-linked by means of AH, containing or not collagen (CS-AH and CS-Co-AH), are suitable for tissue engineering.

Effect of Collagen-Chitosan-Glycerol Composition in Scaffold for Gingival Recession Therapy

The case of gingival recession has a high prevalence, about 88% of the population of the United States in one or more locations suffering from gingival recession. One of the handling cases of gingival recession is to use scaffold that includes the development of tissue and cell engineering. This study aims to determine the best composition variation scaffold of collagen-chitosan with the addition of glycerol. The process of synthesis of collagen-chitosan-glycerol scaffold using freeze dry method that can form pores on the scaffold. Characterization was also carried out on the results of the synthesis of collagen-chitosan scaffold with the addition of glycerol include the morphological characterization, tensile, cytotoxicity, swelling, degradation, and thickness. The results of morphological characterization showed pore size ranged from 26.68 - 191.7 μm with a thickness of 0.51 - 0.65 mm which was suitable for handling of gingival recession cases. The result of tensile test showed that the variation of 9: 1 has the lowest value that is 2.87 MPa where the value is close to tensile strength value for periodontal which has a value ranging from 2.75 to 5.13. The characterization of cytotoxicity shows a value that is less in line with the literature, where live cells &lt;50%. This is because collagen and chitosan have an acidic pH so that the cells cannot reproduce. Characterization of degradation shows all the variations experienced a severe reduction process from day to day. The characterization of the swelling of all samples was equilibrated at 7 minute. Chitosan-collagen scaffold with the addition of glycerol has good potential as a scaffold candidate for gingival recession therapy based on morphological characterization (thickness and surface structure), the mechanical strength (tensile strength), degradation, and the degree of swelling.

Curcumin nanoparticles incorporated collagen-chitosan scaffold promotes cutaneous wound healing through regulation of TGF-β1/Smad7 gene expression

Wound healing is a tissue regeneration process which is regulated by a complex interaction of multiple growth factors, primarily transforming growth factor-β1 (TGF-β1). The natural antagonist of transforming growth factor-β (TGF-β) signaling is Smad7. It has been shown that curcumin (an antioxidant) and some biocompatible polymers like collagen and chitosan enhance cutaneous wound healing. In this study, three scaffolds made with curcumin-nanoparticles (CNs) and using collagen and chitosan with various ratios of collagen and chitosan were used for evaluation of wound healing activity on full thickness punch wound model using male Wistar rats. The wound healing in terms of histology and morphology was assessed at different time points post-wounding and the expression pattern of TGF-β1 and Smad7 was studied. CNs incorporated collagen-chitosan scaffolds significantly accelerated the healing of the wounds, as revealed by a significant change in the wound area, the epidermal thickness, the density of granulation tissue, the number of new vessels and a higher collagen content compared to the control group. However, blank collagen-chitosan scaffolds did not cause any significant change in the above parameters, except for epidermal thickness compared to the control group. Incorporation of CNs into collagen-chitosan scaffold changed expression of TGF-β1 and Smad7 mRNAs in the healing wounds compared to the control group. Indeed, blank collagen-chitosan scaffold did not cause any significant up-regulation either in TGF-β1 mRNA expression or in Smad7 mRNA expression (except for day 3 post-wounding), compared to the control group. This study indicates that topical application of CNs-incorporated collagen-chitosan scaffold promotes wound healing via a regulatory effect on the expression of TGF-β1 and Smad7 mRNA in the cutaneous wound-healing model.

Collagen-chitosan scaffold impregnated with bone marrow mesenchymal stem cells for treatment of traumatic brain injury.

Combinations of biomaterials and cells can effectively target delivery of cells or other therapeutic factors to the brain to rebuild damaged nerve pathways after brain injury. Porous collagen-chitosan scaffolds were prepared by a freeze-drying method based on brain tissue engineering. The scaffolds were impregnated with rat bone marrow mesenchymal stem cells. A traumatic brain injury rat model was established using the 300 g weight free fall impact method. Bone marrow mesenchymal stem cells/collagen-chitosan scaffolds were implanted into the injured brain. Modified neurological severity scores were used to assess the recovery of neurological function. The Morris water maze was employed to determine spatial learning and memory abilities. Hematoxylin-eosin staining was performed to measure pathological changes in brain tissue. Immunohistochemistry was performed for vascular endothelial growth factor and for 5-bromo-2-deoxyuridine (BrdU)/neuron specific enolase and BrdU/glial fibrillary acidic protein. Our results demonstrated that the transplantation of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds to traumatic brain injury rats remarkably reduced modified neurological severity scores, shortened the average latency of the Morris water maze, increased the number of platform crossings, diminished the degeneration of damaged brain tissue, and increased the positive reaction of vascular endothelial growth factor in the transplantation and surrounding areas. At 14 days after transplantation, increased BrdU/glial fibrillary acidic protein expression and decreased BrdU/neuron specific enolase expression were observed in bone marrow mesenchymal stem cells in the injured area. The therapeutic effect of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds was superior to stereotactic injection of bone marrow mesenchymal stem cells alone. To test the biocompatibility and immunogenicity of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds, immunosuppressive cyclosporine was intravenously injected 12 hours before transplantation and 1-5 days after transplantation. The above indicators were similar to those of rats treated with bone marrow mesenchymal stem cells and collagen-chitosan scaffolds only. These findings indicate that transplantation of bone marrow mesenchymal stem cells in a collagen-chitosan scaffold can promote the recovery of neuropathological injury in rats with traumatic brain injury. This approach has the potential to be developed as a treatment for traumatic brain injury in humans. All experimental procedures were approved by the Institutional Animal Investigation Committee of Capital Medical University, China (approval No. AEEI-2015-035) in December 2015.

A Three-Dimensional Human Tissue-Engineered Lung Model to Study Influenza A Infection.

Influenza A virus (IAV) claims ∼250,000-500,000 lives annually worldwide. Currently, there are a few in vitro models available to study IAV immunopathology. Monolayer cultures of cell lines and primary lung cells (two-dimensional [2D] cell culture) is the most commonly used tool, however, this system does not have the in vivo-like structure of the lung and immune responses to IAV as it lacks the three-dimensional (3D) tissue structure. To recapitulate the lung physiology in vitro, a system that contains multiple cell types within a 3D environment that allows cell movement and interaction would provide a critical tool. In this study, as a first step in designing a 3D-Human Tissue-Engineered Lung Model (3D-HTLM), we describe the 3D culture of primary human small airway epithelial cells (HSAEpCs) and determined the immunophenotype of this system in response to IAV infections. We constructed a 3D chitosan-collagen scaffold and cultured HSAEpCs on these scaffolds at air-liquid interface (ALI). These 3D cultures were compared with 2D-cultured HSAEpCs for viability, morphology, marker protein expression, and cell differentiation. Results showed that the 3D-cultured HSAEpCs at ALI yielded maximum viable cells and morphologically resembled the in vivo lower airway epithelium. There were also significant increases in aquaporin-5 and cytokeratin-14 expression for HSAEpCs cultured in 3D compared to 2D. The 3D culture system was used to study the infection of HSAEpCs with two major IAV strains, H1N1 and H3N2. The HSAEpCs showed distinct changes in marker protein expression, both at mRNA and protein levels, and the release of proinflammatory cytokines. This study is the first step in the development of the 3D-HTLM, which will have wide applicability in studying pulmonary pathophysiology and therapeutics development.

Beagle sciatic nerve regeneration across a 30 mm defect bridged by chitosan/PGA artificial nerve grafts

Longitudinally oriented microstructures are essential for a nerve scaffold to promote the significant regeneration of injured peripheral axons across nerve gaps. In the current study, we present a novel nerve-guiding collagen-chitosan (CCH) scaffold that facilitated the repair of 30 mm-long sciatic nerve defects in beagles. The CCH scaffolds were observed with a scanning electron microscope. Eighteen beagles were equally divided into CCH group, autograft group and non-graft group. The posture and gait of each dog was recorded at 12 and 24 weeks after surgery. Electrophysiological tests, Fluoro-Gold retrograde tracing test, Histological assessment of gastrocnemius and immunofluorescent staining of nerve regeneration were performed. Our investigation of regenerated sciatic nerves indicated that a CCH scaffold strongly supported directed axon regeneration in a manner similar to that achieved by autologous nerve transplantation. In vivo animal experiments showed that the CCH scaffold achieved nerve regeneration and functional recovery equivalent to that achieved by an autograft but without requiring the exogenous delivery of regenerative agents or cell transplantation. We conclude that CCH nerve guides show great promise as a method for repairing peripheral nerve defects.

Ellagic acid containing collagen-chitosan scaffolds as potential antioxidative bio-materials for tissue engineering applications

ABSTRACTIn this study, chitosan and collagen based scaffolds loaded with ellagic acid were synthesized with an aim to incorporate antioxidant properties within the supporting matrix. Structural characterization of the scaffolds by X-ray diffraction and Fourier transform infrared spectroscopy confirmed the presence of ellagic acid within the chitosan-collagen matrix. The ‘fabricated’ scaffolds exhibited sustained release of ellagic acid in phosphate buffered saline for 120 h. The scaffolds were biocompatible and supported the recovery of COS 7 cells from the oxidative stress arising due to UV irradiation. Such antioxidant incorporated scaffolds present a potential approach in managing chronic wounds by eliminating ROS.

Mechanical enhancement and in vitro biocompatibility of nanofibrous collagen-chitosan scaffolds for tissue engineering

The collagen–chitosan complex with a three-dimensional nanofiber structure was fabricated to mimic native ECM for tissue repair and biomedical applications. Though the three-dimensional hierarchical fibrous structures of collagen–chitosan composites could provide more adequate stimulus to facilitate cell adhesion, migrate and proliferation, and thus have the potential as tissue engineering scaffolding, there are still limitations in their applications due to the insufficient mechanical properties of natural materials. Because poly (vinyl alcohol) (PVA) and thermoplastic polyurethane (TPU) as biocompatible synthetic polymers can offer excellent mechanical properties, they were introduced into the collagen–chitosan composites to fabricate the mixed collagen/chitosan/PVA fibers and a sandwich structure (collagen/chitosan-TPU-collagen/chitosan) of nanofiber in order to enhance the mechanical properties of the nanofibrous collagen–chitosan scaffold. The results showed that the tensile behavior of materials was enhanced to different degrees with the difference of collagen content in the fibers. Besides the Young’s modulus had no obvious changes, both the break strength and the break elongation of materials were heightened after reinforced by PVA. For the collagen–chitosan nanofiber reinforced by TPU, both the break strength and the Young’s modulus of materials were heightened in different degrees with the variety of collagen content in the fibers despite the decrease of the break elongation of materials to some extent. In vitro cell test demonstrated that the materials could provide adequate environment for cell adhesion and proliferation. All these indicated that the reinforced collagen–chitosan nanofiber could be as potential scaffold for tissue engineering according to the different mechanical requirements in clinic.

Scaffold combination of chitosan and collagen synthesized from chicken feet induces osteoblast and osteoprotegerin expression in bone healing process of mice

Background: Over 500.000 of the 2,3 million surgical treatments requiring bone grafting procedures that are performed annually are likely to be necessitated by or will result in bone defects that will not regenerate. Treatment to regenerate new tissues is needed, especially for hard tissue repair, which not only relies on a natural osseointegration process, but also requires a physical support to guide the differentiation and proliferation of cells into the targeted functional tissue. Chitosan and collagen extracted from chicken feet combinations are expected to enhance the bioactive surface and provide mechanical strength as a bone graft scaffold. Purpose: The aim of this study was to investigate the role of chitosan and collagen scaffold synthesized from chicken feet applications to increase the expression of Osteoprotegerin (OPG) and osteoblast cells on the fourteenth day of bone healing. Methods: Eighteen three-month old, adult, male, Rattus novergicu strain rodents with a body weight ranging from 200-350 g were kept under controlled environmental conditions. The mice were randomly divided into three groups consisting of three subjects, each treated with collagen, chitosan, chitosancollagen combination (50:50) scaffolds. On the 14th post-treatment day, three members of each group were sacrificed. Examination of Osteoprotegerin (OPG) expression was conducted by means of immunohistochemistry staining with anti-OPG polyclonal antibodies. Meanwhile, osteoblast cell examination was performed by means of hematoxilin-eosin (HE) staining. Results: The mice treated with collagen and a chitosan-collagen combination scaffold presented an increase in the expression of Osteoprotegerin (OPG) and the number of osteoblast cells respectively. Conclusion: A combination of chitosan-collagen (50:50) scaffold extracted from chicken feet increased the expression of OPG and the number of osteoblasts in the bone healing process. The combination scaffolds demonstrated the highest OPG expression and number of osteoblasts compared to the other groups.

Induced migration of endothelial cells into 3D scaffolds by chemoattractants secreted by pro-inflammatory macrophages in situ.

Cell migration in scaffolds plays a crucial role in tissue regeneration, which can better mimic cell behaviors in vivo. In this study, a novel model has been proposed on controlling 3D cell migration in porous collagen-chitosan scaffolds with various pore structures under the stimulation of inflammatory cells to mimic the angiogenesis process. Endothelial cells (ECs) cultured atop the scaffolds in the Transwell molds which were placed into a well of a 24-well culture plate were promoted to migrate into the scaffolds by chemoattractants such as vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-α) secreted by the pro-inflammatory macrophages incubated in the well culture plate. The phenotype of macrophages was mediated by 50 ng/ml interferon-gamma (IFN-γ) and different concentrations of lipopolysaccharide (LPS, 150–300 ng/ml). The cell migration depth had a positive correlation with LPS concentration, and thereby the TNF-α concentration. The ECs migrated easier to a deeper zone of the scaffolds prepared at − 10ºC (187 μm in pore diameter) than that at − 20ºC (108 μm in pore diameter) as well. The method provides a useful strategy to study the 3D cell migration, and is helpful to reveal the vascularization process during wound healing in the long run.

Odontogenic differentiation potential of human dental pulp cells cultured on a calcium-aluminate enriched chitosan-collagen scaffold.

The study aims to evaluate the odontogenic potential of human dental pulp cells (HDPCs) in contact with an experimental porous chitosan-collagen scaffold (CHC) enriched or not with a mineral phase of calcium-aluminate (CHC-CA). To assess the chemotactic effect of the materials, we placed HDPCs seeded on transwell membranes in intimate contact with the CHC or CHC-CA surface, and the cell migration was monitored for 48h. Additionally, cells were seeded onto the material surface, and the viability and proliferation were evaluated at several time points. To assess the odontoblastic differentiation, we evaluated ALP activity, DSPP/DMP-1 gene expression, and mineralized matrix deposition. HDPCs cultured onto a polystyrene surface (monolayer) were used as negative control group. The experimental CHC-CA scaffold induced intense migration of HDPCs through transwell membranes, with cells attaching to and spreading on the material surface after 24-h incubation. Also, the HDPCs seeded onto the CHC-CA scaffold were capable of migrating inside it, remaining viable and featuring a proliferative rate more rapid than that of CHC and control groups at 7 and 14days of cell culture. At long-term culture, cells in the CHC-CA scaffold featured the highest deposition of mineralized matrix and expression of odontoblastic markers (ALP activity and DSPP/DMP-1 gene expression). According to the results, the CHC-CA scaffold is a bioactive and cytocompatible material capable of increasing the odontogenic potential of human pulp cells. Based on analysis of the positive data obtained in this study, one can suggest that the CHC-CA scaffold is an interesting future candidate for the treatment of exposed pulps. The experimental scaffold composed by a chitosan-collagen matrix mineralized with calcium aluminate seems to be an interesting candidate for in vivo application as a cell-free approach to dentin tissue engineering, which may open a new perspective for the treatment of exposed pulp tissue.