Chitosan-gelatin Scaffolds Research Articles

Novel Chitosan-Gelatin Scaffold with Valproic Acid Augments In Vitro Osteoblast Differentiation of Mesenchymal Stem Cells.

The study aimed to develop a biodegradable scaffold incorporating valproic acid (VPA) for improved human bone marrow-derived mesenchymal stem cell (hBMSC) proliferation, differentiation, and bone mineral synthesis. A chitosan-gelatin (CH-G) scaffold was fabricated and loaded with varying concentrations of VPA (1, 3, 5 mM/L). In vitro studies assessed drug release, cell proliferation, morphology, mineralization, and gene expression. VPA was rapidly released from the scaffold, with over 90% cumulative release within seven days. Cells cultured on VPA-loaded scaffolds exhibited significantly enhanced proliferation and mineralization compared to the control. VPA treatment upregulated osteocalcin and runt-related transcription factor 2 (Runx-2) expression, key markers of osteogenic differentiation. The CH-G scaffold, particularly with 1 mM/L VPA, demonstrates excellent biocompatibility and promotes hBMSC-mediated bone regeneration. This novel approach holds promise for future applications in bone tissue engineering.

Chitosan-Gelatin Scaffolds Loaded with Different Antibiotic Formulations for Regenerative Endodontic Procedures Promote Biocompatibility and Antibacterial Activity.

This study investigated the biocompatibility and antibacterial efficacy of chitosan-gelatin (CH-G) scaffolds loaded with slow-releasing antibiotic formulations used in regeneration endodontic procedures (REPs). Scaffolds were fabricated using freeze drying and loaded with varying concentrations of augmentin or modified triple antibiotic paste (mTAP). High-resolution scanning electron microscopy (SEM) was used to characterize the scaffold, while drug release was monitored via UV-Vis spectrophotometry. Immortalized human mesenchymal stem cells (hMSCs) were cultured on CH-G scaffolds alone (control), either 0.1 mg/mL or 1 mg/mL of augmentin or mTAP, and 10 mg/mL calcium hydroxide (Ca(OH)2). Cell viability and proliferation were assessed using the Alamar Blue assay and SEM, respectively, and live/dead staining further corroborated cell viability. Antibacterial activity against Enterococcus faecalis was evaluated using the MTT assay and confocal laser scanning microscopy (CLSM). Augmentin at 0.1 mg/mL appeared to promote better cell growth and attachment within the scaffolds than all other formulations, exhibiting acceptable viability. SEM revealed improved cell attachment in augmentin and mTAP groups compared to the Ca(OH)2 group. Augmentin at 1 mg/mL and mTAP groups significantly reduced viable bacteria compared to controls. Augmentin groups and mTAP at 1 mg/mL were highly effective in eliminating E. faecalis biofilms, with mTAP potentially causing more cell death within the remaining biofilm structures. This study suggests that CH-G scaffolds loaded with augmentin and mTAP, particularly at a concentration of 1 mg/mL, offer promising advantages for REPs due to their biocompatibility, antibacterial efficacy, and ability to promote cell attachment. Further research may explore the long-term effects in clinical settings.

Degradation of chitosan–gelatin and chitosan–gelatin–β-tricalcium phosphate scaffolds

Background: Fabrication of the composite scaffold was carried out by combining chitosan, gelatin, and β-tricalcium phosphate (βTCP) derived from limestone. The extraction of βTCP was based on the abundance of limestone containing calcium carbonate, which can be a source of βTCP synthesis. Purpose: This study evaluates the degradation of the combination of chitosan–gelatin (ChG) and chitosan–gelatin–βTCP (ChG-βTCP) composite scaffolds. Methods: The freeze-drying method was used to obtain the composite scaffold, which was a mixture of chitosan, gelatin, and βTCP. Degradation was measured by immersing the samples in a simulated body fluid solution at 37°C for 3, 7, 14, and 21 days. For statistical analysis, one-way analysis of variance (ANOVA) and post hoc Fisher’s least significant difference were performed. Results: The ChG scaffold shows better degradability than the ChG-βTCP scaffold. The ChG scaffold shows higher weight degradation than the ChG-βTCP scaffold up to 21 days. Conclusion: In conclusion, the scaffold containing βTCP has lower degradation than the ChG scaffold.

Macroporous Nanostructured Calcium Phosphate/Chitosan-Gelatin Composite Bone Tissue Engineering Scaffold

Background: Tissue engineering is an emerging technology developed for the therapeutic reconstruction of damaged tissue. Objective: In this study, a ceramic/polymer nanocomposite bone tissue engineering scaffold was prepared by coating a tetracalcium phosphate/dicalcium phosphate mixture slurry on a porous 3D chitosan-gelatin construction. Methods: The phase composition, structural groups, and morphological aspects of the samples were characterized. Furthermore, the 3D composite scaffold was immersed in simulated body fluid (SBF) solution at 37ºC for various periods to track its compositional and structural changes. Results: Based on the results, the coated layer is composed of needle-like carbonated apatite nanosized crystals with some tetracalcium phosphate/dicalcium phosphate initial materials. The nanocomposite was porous with an average macropore size of about 410 μm. The in vitro tests revealed that the composition of the coated layer tends to be apatite crystals, which are similar to natural bone in terms of chemistry and morphology. Conclusion: The results suggest that a simple coating of chitosan-gelatin scaffolds using reactive calcium phosphate particles may introduce a novel nanocomposite scaffold with improved mechanical strength, bioactivity, and osteoconductivity.

Silver-doped hydroxyapatite laden chitosan–gelatin nanocomposite scaffolds for bone tissue engineering: an in-vitro and in-ovo evaluation

Despite the advancements in bone tissue engineering, the majority of implant failures are caused due to microbial contamination. So, efforts are being made to develop biomaterial with antimicrobial property enhancing the regeneration of damaged bone tissue. In the present study, chitosan–gelatin (CG) scaffolds containing silver-doped hydroxyapatite (AgHAP) nanoparticles at 0.5%, 1.0% and 1.5% (w/v) were fabricated by lyophilization technique. The results confirmed the synthesis of AgHAP nanoparticles and showed interconnected porous structure of the nanocomposite scaffolds with 89%–75% porosity. Similarly, the swelling percentage, degradation behavior and compressive modulus of CG-AgHAP nanocomposite scaffolds were 1666%, 40% and 0.7 MPa, respectively. The developed nanocomposite scaffolds revealed better antimicrobial properties and bioactivity. The cell culture studies showed favorable viability of Wharton’s jelly stem cells on CG-AgHAP nanocomposite scaffolds. CAM (chorioallantoic membrane) assay determined the angiogenic potential with better visualization of blood vessels in the CAM area. Hence, the obtained results confirmed that CG-AgHAP3 nanocomposite scaffold was the most suitable for bone tissue engineering applications among all scaffolds.

DEVELOPMENT, CHARACTERIZATION, AND EVALUATION OF THE ANTIMICROBIAL PROPERTIES OF BIODEGRADABLE POROUS SCAFFOLDS LOADED WITH NATURAL VANILLIN

Objective: The study's objective is to create biodegradable porous scaffolds that are filled with natural vanillin and assess their in vitro antibacterial activity. Methods: Scaffolds were fabricated by blending different ratios of chitosan and gelatin along with vanillin using the freeze-drying method. Then the following characterization and evaluation of scaffolds, such as FTIR, SEM, porosity, swelling behaviour, degradation studies, in vitro drug release, and antibacterial studies, were carried out. Results: All of the scaffolds that were created had heterogeneous, well-connected pores and were pale yellow in color. This was validated by SEM, where the porosity is greater than 80% and the mean pore size ranges from 105.25±6.35 µm to 188.58±7.51 µm. With an increase in gelatin concentration, all of the scaffolds showed the maximum water absorption and retention capabilities of 760.15%±4.38% and 664.73%±5.82%. In the 7-day degradation investigation, all samples lost close to 60% of their mass. In the formulation CG11, the vanillin was released gradually over about 96 h. According to the present study, the developed scaffolds CG13-A and CG13-B, as well as CG11-A and CG11-B, displayed a higher zone of inhibition. Conclusion: Due to its potent antibacterial capabilities, it may be inferred from the current research that vanillin clothed in chitosan-gelatin scaffolds would be a superior option for treating various wound infections, including diabetic wounds.

Physicochemical and in vivo evaluation of crosslinked carboxymethyl chitosan-gelatin scaffolds for wound healing application

This study intends to fabricate new antimicrobial scaffolds based on crosslinked Carboxymethyl chitosan (CMCh)/gelatin (Gel) biopolymers. While the physical and morphological characteristics of the crosslinked scaffolds were investigated using the swelling index, hydrolytic degradation study, moisture permeation study, surface roughness profiling, and tensile strength study, their chemical crosslinking was proved using FTIR spectroscopy and DSC analysis. Moreover, antibacterial study, MTT assay, and in vitro scratch assay were also conducted to get more information about the inherent antibacterial, cytocompatible and in vitro wound healing potential of the developed scaffolds. Further, the effectiveness of developed scaffolds was also assessed using an in-vivo incision wound study. FTIR and DSC studies confirmed the crosslinking of polymers at low concentrations of glut by the formation of Schiff base. Further, swelling index, hydrolytic degradation, tensile strength, and roughness of the scaffolds varied with both polymer ratio and concentration of the crosslinking agent. Additionally, scaffolds showed inherent antibacterial properties against gram-positive and gram-negative bacteria strains and exhibited excellent cell viability at lower concentrations of crosslinker. In-vitro wound healing scratch assay revealed proliferation and migration of cells when treated with scaffolds compared to control and standard treated formulation. Further, the TS of the healed wound indicated the quality of healing of the incision wound which was found to be higher for the group treated with an optimized scaffold in comparison to other groups. Thus, our findings show that developed scaffolds have a high applicative potential that can be studied further for biomedical applications such as skin tissue regeneration and wound healing.

LDH-doped gelatin-chitosan scaffold with aligned microchannels improves anti-inflammation and neuronal regeneration with guided axonal growth for effectively recovering spinal cord injury

Spinal cord injury (SCI) causes immune cell infiltration, neuronal death and permanent motor dysfunction. Effective treatment for SCI requires simultaneous suppression of the inhibitory and inflammatory microenvironment and enhancement of the pro-regenerative potential at SCI sites. However, realizing neuronal regeneration with guided axon growth and anti-inflammation simultaneously is currently challenging. In this study, a layered double hydroxides (LDH)-doped gelatin-chitosan scaffold with aligned microchannels (GC-LDH/A scaffold) combined with chemical and structural repair effects was developed to regulate immune microenvironment and promote directional neurogenesis. The GC-LDH/A scaffold was prepared by anisotropic freezing of a mixture of Mg-Fe layered double hydroxides (Mg-Fe LDH), gelatin and chitosan. The LDH, gelatin and aligned microchannel structure work together to promote neuronal regeneration. Chitosan and the aligned microchannel structure have a function of anti-inflammation while the latter also guides axonal growth. The repair effect of this scaffold was assessed by restoring a completely transected spinal cord injury, and the results showed that the GC-LDH/A scaffold effectively activated the anti-inflammatory polarization of macrophages and microglia, promoted neurogenesis and guided axonal growth from the rostral to caudal of the lesion site. The key mediator of functional recovery in SCI is Myosin II, which negatively regulates axonal growth; however, the aligned microchannel structure inhibits its activity and reverses the repair effect. This study provides a promising strategy for addressing the repair challenges for effective SCI treatment.

Preparation, characterization, and bioactivity of reinforced monetite with chitosan-gelatin electrospun composite scaffold for bone tissue engineering

In this study, chitosan-gelatin-monetite (CGM)-based electrospun scaffolds have been developed that closely mimicked the microstructure and chemical composition of the extracellular matrix of natural bone. CGM-based nanofibrous composite scaffolds were prepared with the help of the electrospinning technique, post-cross-linked using ethyl(dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide solution to improve their stability in an aqueous environment. The prepared chitosan/gelatin (CG) scaffold showed an average fiber diameter of 308 ± 17 nm, whereas 5 and 7 wt% monetite containing CGM5 and CGM7 scaffolds, exhibited an average fiber diameter of 287 ± 13 and 265 ± 9 nm, respectively, revealing the fine distribution of monetite particles on the fibrous surface. The distribution of monetite nanoparticles onto the CG nanofibrous surface was confirmed using x-ray diffraction, Fourier transform infrared, and EDAX. Moreover, the addition of 7 wt% monetite into the CG electrospun matrix increased their ultimate tensile strength from 7.62 ± 0.13 MPa in the CG scaffold to 14.34 ± 0.39 MPa in the CGM7 scaffold. Simulated body fluid study and staining with alizarin red S (ARS) confirmed the higher mineralization ability of monetite-containing scaffolds compared to that revealed by the CG scaffold. The monetite incorporation into the CG matrix improved its osteogenic properties, including pre-osteoblast MG-63 cell adhesion, proliferation, and differentiation, when seeded with the cells. A higher degree of cellular adhesion, spreading, and migration was observed on the monetite-incorporated CG scaffold than that on the CG scaffold. From 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) MTT assay, alkaline phosphatase activity, ARS staining, and immunocytochemistry study, the cultured cells discovered a more conducive microenvironment to proliferate and subsequently differentiate into osteoblast lineage in contact with CGM7 nanofibers rather than that in CGM0 and CGM5. In-vitro results indicated that electrospun CGM-based composite scaffolds could be used as a potential candidate to repair and regenerate new bone tissues.

Cerium oxide nanoparticles disseminated chitosan gelatin scaffold for bone tissue engineering applications

Cell-free and cell-loaded constructs are used to bridge the critical-sized bone defect. Oxidative stress at the site of the bone defects is a major interference that slows bone healing. Recently, there has been an increase in interest in enhancing the properties of three-dimensional scaffolds with free radical scavenging materials. Cerium oxide nanoparticles (CNPs) can scavenge free radicals due to their redox-modulating property. In this study, freeze-drying was used to fabricate CG-CNPs nanocomposite scaffolds using gelatin (G), chitosan (C), and cerium oxide nanoparticles. Physico-chemical, mechanical, and biological characterization of CG-CNPs scaffolds were studied. CG-CNPs scaffolds demonstrated better results in terms of physicochemical, mechanical, and biological properties as compared to CG-scaffold. CG-CNPs scaffolds were cyto-friendly to MC3T3-E1 cells studied by performing in-vitro and in-ovo studies. The scaffold's antimicrobial study revealed high inhibition zones against Gram-positive and Gram-negative bacteria. With 79 % porosity, 45.99 % weight loss, 178.25 kPa compressive modulus, and 1.83 Ca/P ratio, the CG-CNP2 scaffold displays the best characteristics. As a result, the CG-CNP2 scaffolds are highly biocompatible and could be applied to repair bone defects.

Therapeutic Efficiency of Nasal Mucosa-Derived Ectodermal Mesenchymal Stem Cells in Rats with Acute Hepatic Failure.

Liver transplantation is limited by the insufficiency of liver organ donors when treating end-stage liver disease or acute liver failure (ALF). Ectodermal mesenchymal stem cells (EMSCs) derived from nasal mucosa have emerged as an alternative cell-based therapy. However, the role of EMSCs in acute liver failure remains unclear. EMSCs were obtained from the nasal mucosa tissue of rats. First, EMSCs were seeded on the gelatin-chitosan scaffolds, and the biocompatibility was evaluated. Next, the protective effects of EMSCs were investigated in carbon tetrachloride- (CCl4-) induced ALF rats. Finally, we applied an indirect coculture system to analyze the paracrine effects of EMSCs on damaged hepatocytes. A three-step nontransgenic technique was performed to transform EMSCs into hepatocyte-like cells (HLCs) in vitro. EMSCs exhibited a similar phenotype to other mesenchymal stem cells along with self-renewal and multilineage differentiation capabilities. EMSC-seeded gelatin-chitosan scaffolds can increase survival rates and ameliorate liver function and pathology of ALF rat models. Moreover, transplanted EMSCs can secrete paracrine factors to promote hepatocyte regeneration, targeted migration, and transdifferentiate into HLCs in response to the liver's microenvironment, which will then repair or replace the damaged hepatocytes. Similar to mature hepatocytes, HLCs generated from EMSCs possess functions of expressing specific hepatic markers, storing glycogen, and producing urea. These results confirmed the feasibility of EMSCs in acute hepatic failure treatment. To our knowledge, this is the first time that EMSCs are used in the therapy of liver diseases. EMSCs are expected to be a novel and promising cell source in liver tissue engineering.

Evaluation of bioactivity and biodegradability of a biomimetic soft tissue scaffold for clinical use: An in vitro study.

Autogenous soft-tissue graft is the gold-standard approach to augment oral soft tissues. However, tissue engineering is increasingly surveyed to overcome its substantial drawbacks, including the secondary site of operation, patient's pain and discomfort, limited tissue of donor site, and so on. Chitosan and gelatin have been utilized in this field over the years due to their great biological virtues. Zeolite, another remarkable candidate for tissue engineering, possesses outstanding biological and mechanical properties, thanks to its nanostructure. Therefore, this study aimed to investigate the biodegradability and DNA content of seeded human gingival fibroblasts on a New Chitosan-Gelatin-Zeolite Scaffold for the perspective of oral and mucosal soft tissue augmentation. DNA contents of the human gingival fibroblast cell line (HGF.1) seeded on the chitosan-gelatin (CG) and CGZ scaffolds were evaluated by propidium iodide staining on days 1, 5, and 8. Scaffolds' biodegradations were investigated on days 1, 7, 14, 28, 42, and 60. Although both scaffolds provided appropriate substrates for HGF.1 growth, significantly higher DNA contents were recorded for the CGZ scaffold. Among experimental groups, the highest mean value was recorded in the CGZ on day 8. CGZ showed a significantly lower biodegradation percentage at all time points. The incorporation of zeolite into the CG scaffold at a ratio of 1:10 improved the cell proliferation and stability of the composite scaffold. CGZ scaffold may offer a promising alternative to soft-tissue grafts due to its suitable biological features.

Effect of Glutaraldehyde Concentration Variation on Diclofenac Sodium Scaffolds as Cross-Linking Agent

Chitosan and gelatin were used as polymer scaffolds for cartilage tissue engineering. The scaffold was used as a biodegradable drug delivery system for diclofenac sodium to treat cartilage defects on osteoarthritis (OA). The materials were composed of diclofenac sodium, chitosan, gelatin, and cross-linking agent-glutaraldehyde (GTA) were form as scaffold. The purpose of this study to investigate the effect of GTA concentration variations (0.00%; 0.25%; 0.50%; 1.00%; 2.50%) on characteristics and the release of diclofenac sodium from chitosan-gelatin scaffold. The scaffolds were made by using the pre-freezing method with a temperature of -56 ± 5°C for 24 hours and characterized by porosity, pore size, swelling, degradation, toxicity test, and diclofenac sodium released from chitosan-gelatin scaffolds at pH and temperature body. The results showed, the addition of GTA increased the swelling ratio from 195.79 ± 7.04% to 793.49 ± 6.92% and minimized weight loss up to 50.98 ± 0.82%, percentage of living cells >60%, optimal porosity at 106.94 ± 9.38 % with pore size 135.48 ± 89.70 µm, diclofenac sodium as sustained release drug completed in 542 hours and the release was following zero-order kinetic. Chitosan-gelatin scaffold is a potential candidate for cartilage tissue engineering and drug delivery system for diclofenac sodium.

Mechanical improvement of chitosan–gelatin scaffolds reinforced by β-tricalcium phosphate bioceramic

This study aimed to fabricate and evaluate the properties of a chitosan–gelatin scaffold reinforced by beta-tricalcium phosphate (βTCP) powder derived from limestone as a composite scaffold for application in bone tissue engineering. Preparation of βTCP powder involved obtaining calcium hydroxide from limestone and mixing it with phosphate solution through wet precipitation and calcination. To fabricate the scaffold, chitosan was dissolved in acetic acid solution and mixed with gelatin-in-water solution. βTCP powder was then added to the mixture and crosslinked with glutaraldehyde. The slurry was dispensed into a mold and then freeze-dried. Dried chitosan–gelatin–βTCP scaffolds were then characterized. X-ray diffractometer (XRD) results showed that chitosan–gelatin scaffolds incorporated with βTCP displayed the phase composition corresponding to the βTCP reference. Fourier-transform infrared spectroscopy results showed increasing intensity of amide I and amide II at a lower βTCP amount because of the increasing amount of chitosan and gelatin where amide I and amide II groups were a characteristic of both materials. A porous structure was present in all scaffolds, as shown by scanning electron microscopy observation. The pore size as well as porosity (85.76% ± 0.77%) decreased with increased βTCP amount up to 70%, affecting the compressive strength of the scaffolds, with the highest value up to 3.27 ± 0.29 MPa. In conclusion, porous scaffolds suitable for bone tissue engineering applications were fabricated using a combination of limestone-derived βTCP powder, chitosan, and gelatin.

EFFECT OF Β-TRICALCIUM PHOSPHATE ELEMENTAL IMPURITIES ON THE ANTIBACTERIAL ACTIVITY OF CHITOSAN–GELATIN–ΒTCP COMPOSITE SCAFFOLD

This study aims to fabricate composite a scaffold using a mixture of chitosan-gelatin and limestone-extracted βTCP for better antibacterial properties. Limestone is abundantly available in nature and can provide calcium in the production of calcium phosphate (CaP) materials. However, the elemental impurities present in limestone-extracted βTCP may affect the properties of the CaP-fabricated scaffolds. βTCP powder could be used to fabricate bone scaffold using calcination and wet precipitation method. For example, the preparation of βTCP powder involved the transformation of calcium carbonate in limestone to calcium hydroxide, followed by a reaction with phosphoric acid and sintering treatment to form βTCP. In order to fabricate the scaffold, chitosan was first dissolved in acetic acid and then mixed with a gelatin-in-water solution, followed by adding βTCP powder (limestone-extracted βTCP and commercial βTCP). Then, the mixture was placed in a mold and freeze-dried. Our method improved the antibacterial activity of chitosan–gelatin–βTCP compared to the chitosan–gelatin scaffold. Limestone-extracted βTCP also exhibited higher inhibitory action against bacteria than commercial βTCP. This method's effectiveness evaluation was confirmed by the measurement of X-ray fluorescence analysis, which showed that βTCP scaffolds had more antibacterial elements than the chitosan–gelatin scaffold. These new research results contribute to enhanced antibacterial properties of the material due to elemental impurities in βTCP, especially the limestone-extracted βTCP. However, further evaluation on controlling βTCP impurities is essential for better outcomes in clinical applications.

Copper crosslinked carboxymethyl chitosan–gelatin scaffolds: A potential antibacterial and cytocompatible material for biomedical applications

The study delineates the development and characterization of carboxymethyl chitosan–gelatin-copper oxide (CuO) crosslinked scaffolds for biomedical applications. Four scaffolds were developed using lyophilization technique using two stage crosslinking and evaluated using FT-IR, TGA to ascertain the crosslinking. Further, swelling index (SI), hydrolytic degradation, water vapour transmission rate (WVTR) as well as morphological characterization of scaffolds were carried out using SEM and optical profilometer. Additionally, modified disc diffusion method and MTT assay was carried out to check antibacterial action and cyto-compatibility. The results revealed that SI, hydrolytic degradation, and WVTR changes with the change in percentage of CuO. Further, effect of CuO concentration was evident from roughness variations of the developed scaffolds. Additionally SEM images shows presence of micro pores and the scaffolds exhibited good antibacterial action both against Gram positive and Gram negative bacteria. Lastly cytotoxicity assay exhibited good cell viability even at higher concentration of CuO nanoparticles. Thus, this work represents a facile approach to develop CuO crosslinked scaffolds of CMCh-gelatin that may potentially be explored further for biomedical applications.

Probiotic bacteria cell surface-associated protein mineralized hydroxyapatite incorporated in porous scaffold: In vitro evaluation for bone cell growth and differentiation

There is a high demand for synthesis of biocompatible hydroxyapatite nanoparticle (HANP), which is a key component in bone tissue engineering scaffolds. The present study describes a facile route of HANP synthesis through mineralization of the cell surface-associated protein (CSP) from the human probiotic lactic acid bacteria (LAB) Lactobacillus rhamnosus GG. CSP extract from the LAB (consisting of ~66 kDa, ~47 kDa, ~40 kDa and ~25 kDa protein) was mineralized to yield spindle-shaped HANPs having an average particle length of 371 nm as evidenced in FETEM analysis. CSP-mineralized HANPs (CSP-HANPs) were characterized by FTIR and BET analysis, while XRD and SAED analysis indicated their crystalline nature. Mechanistic studies suggested the key role of ~25 kDa CSP (F4SP) in mineralization. In contrast to CSP-HANPs, F4SP-mineralized crystalline HA was plate-shaped having an average length of 1.68 μm and breadth of 0.95 μm. HANP mineralization at the whole-cell (WC) level resulted in clusters of aggregated HANPs (WC-HANPs) adhering onto L. rhamnosus GG cells as evident in FETEM, FESEM and AFM analysis. FETEM analysis revealed that the desorbed WC-HANPs recovered by cell lysis were needle-shaped, with a particle size distribution of 70–110 nm. Given that CSP-HANPs were non-toxic to cultured HEK 293 cells and osteoblast-like MG-63 cells, chitosan-gelatin (CG) scaffold incorporated with 15% w/v CSP-HANP (H-CG) was generated and tested for bone cell growth. H-CG exhibited a favorable pore size distribution (160–230 μm), overall porosity (~84%) and biodegradation profile. H-CG scaffold was conducive to osteogenesis and rendered enhanced proliferation, alkaline phosphatase (ALP) activity, calcium mineralization and heightened marker gene expression (ALP, Col I, Runx2 and OCN) in seeded MG-63 cells. CSP sourced from a safe probiotic LAB is thus a viable and effective mineralization template for synthesis of biocompatible HANPs that can be leveraged for bone tissue engineering applications.

In vivo cartilage regeneration in a multi-layered articular cartilage architecture mimicking scaffold.

AimsExtracellular matrix (ECM) and its architecture have a vital role in articular cartilage (AC) structure and function. We hypothesized that a multi-layered chitosan-gelatin (CG) scaffold that resembles ECM, as well as native collagen architecture of AC, will achieve superior chondrogenesis and AC regeneration. We also compared its in vitro and in vivo outcomes with randomly aligned CG scaffold.MethodsRabbit bone marrow mesenchymal stem cells (MSCs) were differentiated into the chondrogenic lineage on scaffolds. Quality of in vitro regenerated cartilage was assessed by cell viability, growth, matrix synthesis, and differentiation. Bilateral osteochondral defects were created in 15 four-month-old male New Zealand white rabbits and segregated into three treatment groups with five in each. The groups were: 1) untreated and allogeneic chondrocytes; 2) multi-layered scaffold with and without cells; and 3) randomly aligned scaffold with and without cells. After four months of follow-up, the outcome was assessed using histology and immunostaining.ResultsIn vitro testing showed that the secreted ECM oriented itself along the fibre in multi-layered scaffolds. Both types of CG scaffolds supported cell viability, growth, and matrix synthesis. In vitro chondrogenesis on scaffold showed an around 400-fold increase in collagen type 2 (COL2A1) expression in both CG scaffolds, but the total glycosaminoglycan (GAG)/DNA deposition was 1.39-fold higher in the multi-layered scaffold than the randomly aligned scaffold. In vivo cartilage formation occurred in both multi-layered and randomly aligned scaffolds treated with and without cells, and was shown to be of hyaline phenotype on immunostaining. The defects treated with multi-layered + cells, however, showed significantly thicker cartilage formation than the randomly aligned scaffold.ConclusionWe demonstrated that MSCs loaded CG scaffold with multi-layered zonal architecture promoted superior hyaline AC regeneration.Cite this article: Bone Joint Res 2020;9(9):601–612.

Antibiotic-loaded chitosan-gelatin scaffolds for infected seawater immersion wound healing

Skin damaged during sea battles is vulnerable to seawater immersion and bacterial infection. Scaffolds with effective biological function are highly desired for treatment of naval combat wound injuries. Herein, we prepared composite scaffolds of CS/GEL/GMs-CIP. The chitosan (CS) and gelatin (GEL) were cross-linked by genipin as matrix, and then gelatin microspheres loading ciprofloxacin hydrochloride (GMs-CIP) were add. From in vitro characterization results, CS/GEL/GMs-CIP had high water absorption ability, proper porosity, satisfactory fracture resistance, and flexibility. Furthermore, CS/GEL/GMs-CIP composite scaffold had excellent biocompatibility. Antibacterial experiments confirmed that CS/GEL/GMs-CIP had a significant inhibitory effect on E. coli, S. aureus and P. aeruginosa. The in vivo wound healing was evaluated using animal wound infection model of seawater immersion, and it was observed that the prepared composite scaffolds accelerated wound healing, reepithelialization, collagen deposition. Further analysis of wound tissue indicated that the expression of anti-inflammatory factor (TGF-β1) was up-regulated, but the serum endotoxin levels and expression of pro-inflammatory factor (TNF-a, IL-6, and IL-1β) were down-regulated. In summary, we believe that CS/GEL/GMs-CIP composite scaffold may serve as a promising multifunctional dressing for healing with open trauma wound infections and wound with seawater immersion.

The opposite functions of miR-24 in the osteogenesis and adipogenesis of adipose-derived mesenchymal stem cells are mediated by the HOXB7/β-catenin complex.

Adipose-derived mesenchymal stem cells (ADMSCs) used in combination with nanoparticles or scaffolds represent promising candidates for bone engineering. Compared to bone marrow-derived MSCs (BMMSCs), ADMSCs show a relatively low capacity for osteogenesis. In the current study, miR-24 was identified as an osteogenesis- and adipogenesis-related miRNA that performs opposing roles (inhibition in osteogenesis and promotion in adipogenesis) during these two differentiation processes. Through bioinformatics analysis and luciferase reporter assays, homeobox protein Hox-B7 (HOXB7) was identified as a potential novel downstream target of miR-24 that contains a miR-24 binding site in the 3'-UTR of its mRNA. Overexpression of HOXB7 could partly halt the inhibitory effect of miR-24 on osteogenesis, and downregulation of HOXB7 could also partly suppress the positive effect of miR-24 on adipogenesis. Furthermore, immunoprecipitation experiments found that HOXB7 and β-catenin formed a functional complex that acted as an essential modulator during osteogenesis and adipogenesis of ADMSCs. After transfecting ADMSCs with an MSNs-PEI-miR-24 agomir or antagomir and loading the cells onto gelatin-chitosan scaffolds, the compounds were assessed for their abilities to repair the critical-sized calvarial defects in rats. Comprehensive evaluation, including micro-CT, sequential fluorescent labeling, and immunohistochemistry analysis, revealed that silencing miR-24 distinctly promoted in vivo bone remolding, whereas overexpression of miR-24 significantly repressed bone formation. Taken together, our findings demonstrated opposite roles for the miR-24/HOXB7/β-catenin signaling pathway in the osteogenesis and adipogenesis of ADMSCs, which may provide a novel mechanism for determining the balance between these two biological processes.