Chitosan-alginate Scaffolds Research Articles

3D Printing of Alginate/Chitosan-Based Scaffold Empowered by Tyrosol-Loaded Niosome for Wound Healing Applications: In Vitro and In Vivo Performances.

This study introduces a tyrosol-loaded niosome integrated into a chitosan-alginate scaffold (Nio-Tyro@CS-AL), employing advanced electrospinning and 3D printing techniques for wound healing applications. The niosomes, measuring 185.40 ± 6.40 nm with a polydispersity index of 0.168 ± 0.012, encapsulated tyrosol with an efficiency of 77.54 ± 1.25%. The scaffold's microsized porous structure (600-900 μm) enhances water absorption, promoting cell adhesion, migration, and proliferation. Mechanical property assessments revealed the scaffold's enhanced resilience, with niosomes increasing the compressive strength, modulus, and strain to failure, indicative of its suitability for wound healing. Controlled tyrosol release was demonstrated in vitro, essential for therapeutic efficacy. The scaffold exhibited significant antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus, with substantial biofilm inhibition and downregulation of bacterial genes (ndvb and icab). A wound healing assay highlighted a notable increase in MMP-2 and MMP-9 mRNA expression and the wound closure area (69.35 ± 2.21%) in HFF cells treated with Nio-Tyro@CS-AL. In vivo studies in mice confirmed the scaffold's biocompatibility, showing no significant inflammatory response, hypertrophic scarring, or foreign body reaction. Histological evaluations revealed increased fibroblast and macrophage activity, enhanced re-epithelialization, and angiogenesis in wounds treated with Nio-Tyro@CS-AL, indicating effective tissue integration and repair. Overall, the Nio-Tyro@CS-AL scaffold presents a significant advancement in wound-healing materials, combining antibacterial properties with enhanced tissue regeneration, and holds promising potential for clinical applications in wound management.

Fabrication, Characterization, Antibacterial and Biocompatibility Studies of Graphene Oxide Loaded Alginate Chitosan Scaffolds for Potential Biomedical Applications

Graphene oxide nanomaterial possesses greater biocompatibility. Chitosan alginate is produced from chitin by deacetylation, it is a biodegradable and biocompatible biomaterial. Graphene has a large specific surface area that enhances the antibacterial effect by enabling biocompatible interactions with bacterial membranes. To fabricate a biocompatible graphene oxide loaded alginate chitosan scaffold for potential biomedical uses and perform the characterization, antibacterial, and biocompatibility properties of ALG-CHI-GO scaffolds. The scaffolds were prepared by mixing the solution of ChitosanHCl (5 %) and graphene oxide-oxidized alginate (10 %). This gelled mixture is freeze-dried (lyophilization) to form the scaffold and is later characterized using FTIR and SEM, The scaffolds were then tested for in biocompatibility towards peripheral blood mononuclear cells and antibacterial properties against Enterococcus faecalis and Streptococcus mutans. The biocompatibility towards peripheral blood mononuclear was checked using the annexin V PI assay. To conclude that the fabricated Graphene oxide loaded chitosan alginate scaffold was found to be biocompatible and showed antibacterial properties. Keywords: Antibacterial activity, Biocompatibility, Chitosan alginate scaffolds, Graphene oxide.

Physicochemical Characteristics of Chitosan–Alginate Scaffold Containing Atorvastatin

In addition to being a lipid-lowering medication, atorvastatin (ATV) is an anti-inflammatory agent. When there is a bone defect or inflammation of adjacent tissues, it aids in bone repair. This study aimed to develop a chitosan–alginate (CS/ALG)–tripolyphosphate (TPP)–ATV hybrid hydrogel as a drug delivery system, using a tissue engineering scaffold for the first time. For this purpose, a CS/ALG hydrogel crosslinked with TPP was developed. The delivery profile of ATV and its physicochemical properties such as particle size and hydrogel swelling percentage were determined. The structure and morphology of the hydrogels were analyzed using Fourier transform infrared spectroscopy and scanning electron microscopy. As a result, an alginate–chitosan hydrogel with a TPP crosslinker was prepared. The results revealed that drug loading was nearly complete, and the first hour revealed a 25% explosive release. The drug was gradually released over 10 hr at approximately 35%. The amount of crosslinker used significantly influenced the encapsulation gain and release profiles. Owing to its high porosity and swelling, the CS/ALG hydrogel crosslinked with PPT is an ideal scaffold for loading drugs, macromolecules, and cells.

Chitosan-alginate porous scaffold incorporated with hydroalcoholic Zingiber officinale Roscoe extract for neural tissue engineering

Context: Preparing a suitable substrate for the culture of neural stem cells and their proliferation in neural tissue engineering is of paramount importance. Aims: To evaluate the effect of the hydroalcoholic Zingiber officinale extract incorporated in the chitosan-alginate scaffold (Chi-Alg-Zo) on nerve tissue. Methods: The porous scaffolds developed in the present study were investigated in terms of their surface properties, chemical interaction, crystallinity, thermal stability, porosity percentage, pore sizes, degradability, and water absorption properties. To this end, the following tests were performed: Field emission scanning electron microscope (FE-SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), thermal gravimetric analysis (TGA), porosity based on liquid replacement, Image-J analysis, and water degradation and absorption test. Mouse neural stem/progenitor cells (NS/PCs) were harvested from the embryonic mouse brain. NSCs were seeded on scaffolds incorporated with hydroalcoholic Z. officinale extract. The MTT assay was done for the survival and the proliferation of neural stem/progenitor cells (NS/PCs) on scaffolds. Results: Results indicated the good capacity of Chi-Alg-Zo for proliferation and differentiation into glial (astrocytes and oligodendrocytes) lineages. A suitable surface, which was provided for cellular interaction, led to the advancement of cell survival, connectivity, proliferation, and separation of NSCs. Conclusions: The present study evaluated the separation of stem cells on the scaffold, finding that the expression of the glial fibrillary acidic protein (GFAP) and Oligo4 markers was higher in Chi-Alg scaffolds containing hydroalcoholic Z. officinale extract. Chi-Alg-Zo scaffolds could be suitable candidates for neural tissue engineering.

Evaluation of the effect of 3D porous Chitosan-alginate scaffold stiffness on breast cancer proliferation and migration.

Breast cancer (BCa) is one of the most common cancers for women and metastatic BCa causes the majority of deaths. The extracellular matrix (ECM) stiffens during cancer progression and provides biophysical signals to modulate proliferation, morphology, and metastasis. Cells utilize mechanotransduction and integrins to sense and respond to ECM stiffness. Chitosan-alginate (CA) scaffolds have been used for 3D culture, but lack integrin binding ligands, resulting in round cell morphology and limited cell-material interaction. In this study, 2, 4, and 6wt% CA scaffolds were produced to mimic the stages of BCa progression and evaluate the BCa response to CA scaffold stiffness. All three CA scaffold compositions highly porous with interconnected pores and scaffold stiffness increased with increasing polymer concentration. MDA-MB-231 (231) cells were cultured in CA scaffolds and 2D cultures for 7 d. All CA scaffold cultures had similar cell numbers at 7 d and the 231 cells formed clusters that increased in size during the culture. The 2wt% CA had the largest clusters throughout the 7 d culture compared with the 4 and 6wt% CA. The 231 cell migration was evaluated on 2D surfaces after 7 d culture. The 6wt% CA cultured cells had the greatest migration speed, followed by 4wt% CA, 2D cultures, and 2wt% CA. These results suggest that 231 cells sensed the stiffness of CA scaffolds without the presence of focal adhesions. This indicates that a non-integrin-based mechanism may explain the observed mechanotransduction response.

Flake-like glass nanoparticles reinforced natural polymeric scaffold as a promising substrate for bone regeneration

Natural polymeric scaffolds with unique characteristics can be considered as the best candidates for tissue engineering applications. But improve the mechanical properties of these scaffolds is a critical factor. In the current study glass nanoparticles with a flake-like morphology were used as filler in the chitosan-alginate scaffold. To understand the effect of surface modification, silane groups were created on these nanoparticles. SEM, FTIR, TGA, and XRD were used for scaffolds characterization. Swelling, degradation and mechanical trials are the other analyses to scaffold examination. Grafting of silane on nanoparticles was successful and the grafting ratio was 2.2 wt%. Pore shape and size were changed by using pure and modified nanoparticles. The swelling and degradation trend of the scaffold had a decreasing manner by applying nanoparticles. The polymeric scaffold compression modulus has improved 2.09 times. Our results show that, although glass nanoparticles with plate structure can make scaffolds with better mechanical features the presence of other functionalities on their surface is the appropriate means to gain better mechanical behavior.

Development of osteogenic chitosan/alginate scaffolds reinforced with silicocarnotite containing apatitic fibers

Porous composite scaffolds of chitosan-alginate (CH-AL) reinforced by biphasic calcium phosphate fibers containing silicon (Si) were prepared using the freeze-drying method. The fibers were synthesized using a homogenous precipitation method with differing reaction times and were characterized by XRD, FTIR, SEM, and ICP-OES. Fibers produced with no Si incorporation using two different reaction times of 4 d and 8 d comprised two phases of hydroxyapatite (∼93-96 wt%) and β-tricalcium phosphate (β-TCP). No new phases were observed by adding 0.8 wt% of Si during 4 d of precipitation. However, the addition of Si to fibers synthesized within 8 d under reflux conditions produced biphasic fibers with 1.9 wt% Si which consisted of a new phase of silicocarnotite (∼94 wt%) associated with the β-TCP phase. The whisker-like fibers were 10–200 µm in length and 0.2–5 µm in width. The physicochemical, mechanical, and biological properties of composite scaffolds fabricated by adding different fiber contents and types were investigated. The scaffolds exhibited favorable microstructures with a high porosity (66–88%) and the interconnected pores varied in size between 40 and 250 µm. Scaffolds containing silicocarnotite showed a significant improvement in their mechanical properties and in vitro bioactivity (using SBF testing and characterization of the apatite layer by ATR-FTIR and SEM/EDS) as well as proliferation, mineralization and adhesion of MG63 cells, when evaluated by MTT assay, alkaline phosphatase, and SEM. Scaffolds reinforced with silicocarnotite fibers also exhibited better mechanical properties and water uptake, compared to ones containing incorporated fibers made of Si. Composite scaffolds reinforced by 50 wt% fibers precipitated after 8 d were superior in terms of their mechanical properties and achieved a compressive strength and modulus of 272 kPa and 4.9 MPa, respectively, which is 400% greater than CH-AL scaffolds. The results indicate that the addition of Si into biphasic fibers, leading to the formation of silicocarnotite, makes silicocarnotite a potential candidate for the bioactive reinforcement of composite scaffolds for bone tissue engineering.

Effect of Mold Geometry on Pore Size in Freeze-Cast Chitosan-Alginate Scaffolds for Tissue Engineering.

Freeze-casting is a popular method to produce biomaterial scaffolds with highly porous structures. The pore structure of freeze-cast biomaterial scaffolds is influenced by processing parameters but has mostly been controlled experimentally. A mathematical model integrating Computational Fluid Dynamics with Population Balance Model was developed to predict average pore size (APS) of 3D porous chitosan-alginate scaffolds and to assess the influence of the geometrical parameters of mold on scaffold pore structure. The model predicted the crystallization pattern and APS for scaffolds cast in different diameter molds and filled to different heights. The predictions demonstrated that the temperature gradient and solidification pattern affect ice crystal nucleation and growth, subsequently influencing APS homogeneity. The predicted APS compared favorably with APS measurements from a corresponding experimental dataset, validating the model. Sensitivity analysis was performed to assess the response of the APS to the three geometrical parameters of the mold: well radius; solution fill height; and spacing between wells. The pore size was most sensitive to the distance between the wells and least sensitive to solution height. This validated model demonstrates a method for optimizing the APS of freeze-cast biomaterial scaffolds that could be applied to other compositions or applications.

3D porous chitosan-alginate scaffold stiffness promotes differential responses in prostate cancer cell lines

Prostate cancer (PCa) is a leading cause of death for men worldwide. Most PCa patients die from metastasis and bone is the most common metastatic site. Three dimensional (3D) porous chitosan-alginate (CA) scaffolds were developed for bone tissue engineering and demonstrated for culture of cancer cells and enrichment of cancer stem cells. However, only a single scaffold composition was studied. Three compositions of 3D porous CA scaffolds (2, 4, and 6 wt%) were used to investigate the effect of scaffold stiffness on PCa cell response with PC-3, C4-2B, and 22Rv1 cell lines. The PC-3 cells formed cell clusters while the C4-2B and 22Rv1 cells formed multicellular spheroids. The three cell lines demonstrated stiffness independent cell growth and expressed phenotypic PCa biomarkers. The osteoblastic PCa lines C4-2B and 22Rv1 mineralized in basal media, while the osteolytic PC-3 line did not, demonstrating that CA scaffold cultures revealed differences in PCa phenotypes. The CA scaffolds are a 3D culture platform that supports PCa growth and phenotypic expression with adjustable scaffold stiffness to mimic stages of metastatic progression. Further investigation of the scaffolds for co-culture of PCa cells with fibroblasts and primary PCa cell culture should be conducted to develop a platform for screening chemotherapies.

Using 3-Dimensional Printing to Construct Chitosan Alginate Scaffolds for Nasal Alar Cartilage Tissue Engineering

Alba, Brandon; Swami, Pooja MS; Tanna, Neil MD, MBA, FACS; Grande, Daniel PhD Author Information

Immobilization of β-galactosidase in Chitosan-Alginate composite scaffolds and optimization of lactose hydrolysis

The immobilization of lactase (s-galactosidase) enzyme was achieved by entrapping it in chitosan-alginate composite scaffolds, which were formed by ionotropic pre-gelation followed by a pH increase to synthesize polyelectrolyte complex scaffolds, and subsequent cross-linking and activation by glutaraldehyde. The operational stability upon storage of the enzyme entrapped was checked by performing batch hydrolysis of lactose concentrations ranging from 1mg/ml to 5mg/ml and the resulting glucose produced in every reaction was determined using the glucose oxidase-peroxidase assay. This study also investigates the stability of the free and immobilized enzymes at altered sets of pH and temperature at a fixed concentration of enzyme for the reaction medium. Morphology and structure characterization of lyophilized composite scaffolds were examined using scanning electron microscope and Fourier transform infra-red spectra, respectively.

Synthesis and characterization of chitosan-alginate scaffolds for seeding human umbilical cord derived mesenchymal stem cells.

Chitosan and alginate are two natural and accessible polymers that are known to be biocompatible, biodegradable and possesses good antimicrobial activity. When combined, they exhibit desirable characteristics and can be created into a scaffold for cell culture. In this study interaction of chitosan-alginate scaffolds with mesenchymal stem cells are studied. Mesenchymal stem cells were derived from human umbilical cord tissues, characterized by flow cytometry and other growth parameters studied as well. Proliferation and viability of cultured cells were studied by MTT Assay and Trypan Blue dye exclusion assay. Besides chitosan-alginate scaffold was prepared by freeze-drying method and characterized by FTIR, SEM and Rheological properties. The obtained 3D porous structure allowed very efficient seeding of hUMSCs that are able to inhabit the whole volume of the scaffold, showing good adhesion and proliferation. These materials showed desirable rheological properties for facile injection as tissue scaffolds. The results of this study demonstrated that chitosan-alginate scaffold may be promising biomaterial in the field of tissue engineering, which is currently under a great deal of examination for the development and/or restoration of tissue and organs. It combines the stem cell therapy and biomaterials.

Evaluation of Extracted Chitosan from Portunus Pelagicus for the Preparation of Chitosan Alginate Blend Scaffolds

In recent years there is a growing need in generating a biocompatible and cost effective porous scaffold for tissue engineering purposes. Therefore, this study focused on conversion of the shell waste of locally available crab variety P.pelagicus (Blue swimming crab) into the chitosan scaffold. As the poor mechanical strength of chitosan limits its usage in tissue engineering, it was blended with alginate. The scaffolds were prepared by the freeze gelation method which requires less time and minimum energy, with fewer residual solvent and easier to scale up. To the best of our knowledge there are no reports on scaffold preparation from the extracted chitosan, blended with alginate by freeze gelation method. The biological properties of chitosan-alginate scaffolds (Cts–Alg) were evaluated and compared with those of chitosan scaffolds. The prepared scaffolds were characterized by SEM, swelling property, in vitro enzymatic degradation, and hemo, biocompatibility properties. Chitosan-alginate scaffolds had an average pore size of 40 μm and tensile strength of 0.564 ± 0.0.018 % MPa. Its swelling ratio was 27.5 ± 0.28 %, with mass loss percentage of 10 ± 0.33 % after 4 weeks of degradation. It has exhibited good hemocompatible properties too. Mouse fibroblast 3T3 cells were able to adhere and proliferate well in the blended scaffold. All these results indicated that chitosan-alginate scaffold is a suitable alternative substitute for tissue engineering.

Macro- and micro-designed chitosan-alginate scaffold architecture by three-dimensional printing and directional freezing

While many tissue-engineered constructs aim to treat cartilage defects, most involve chondrocyte or stem cell seeding on scaffolds. The clinical application of cell-based techniques is limited due to the cost of maintaining cellular constructs on the shelf, potential immune response to allogeneic cell lines, and autologous chondrocyte sources requiring biopsy from already diseased or injured, scarce tissue. An acellular scaffold that can induce endogenous influx and homogeneous distribution of native stem cells from bone marrow holds great promise for cartilage regeneration. This study aims to develop such an acellular scaffold using designed, channeled architecture that simultaneously models the native zones of articular cartilage and subchondral bone. Highly porous, hydrophilic chitosan-alginate (Ch-Al) scaffolds were fabricated in three-dimensionally printed (3DP) molds designed to create millimeter scale macro-channels. Different polymer preform casting techniques were employed to produce scaffolds from both negative and positive 3DP molds. Macro-channeled scaffolds improved cell suspension distribution and uptake overly randomly porous scaffolds, with a wicking volumetric flow rate of 445.6 ± 30.3 mm3 s−1 for aqueous solutions and 177 ± 16 mm3 s−1 for blood. Additionally, directional freezing was applied to Ch-Al scaffolds, resulting in lamellar pores measuring 300 μm and 50 μm on the long and short axes, thus creating micrometer scale micro-channels. After directionally freezing Ch-Al solution cast in 3DP molds, the combined macro- and micro-channeled scaffold architecture enhanced cell suspension uptake beyond either macro- or micro-channels alone, reaching a volumetric flow rate of 1782.1 ± 48 mm3 s−1 for aqueous solutions and 440.9 ± 0.5 mm3 s−1 for blood. By combining 3DP and directional freezing, we can control the micro- and macro-architecture of Ch-Al to drastically improve cell influx into and distribution within the scaffold, while achieving porous zones that mimic articular cartilage zonal architecture. In future applications, precisely controlled micro- and macro-channels have the potential to assist immediate endogenous bone marrow uptake, stimulate chondrogenesis, and encourage vascularization of bone in an osteochondral scaffold.

3D Porous Chitosan-Alginate Scaffolds Promote Proliferation and Enrichment of Cancer Stem-Like Cells.

Cancer stem cells are increasingly becoming a primary target for new cancer treatment development. The ability to study their transient behavior in vitro will provide the opportunity for high-throughput testing of more effective therapies. We have previously demonstrated the use of 3D porous chitosan-alginate (CA) scaffolds to promote cancer stem-like cell (CSC) proliferation and enrichment in glioblastoma. Here we use 3D porous CA scaffolds to promote cancer stem-like cell enrichment in cell lines from prostate, liver, and breast cancers, and investigate the proliferation, morphology, and gene expressions of cells cultured in CA scaffolds as compared to 2D controls. The 3D CA scaffold cultures for all three cancer types showed reduced proliferation, formation of tumor spheroids, and increased expression of CSC associated mark genes (CD133 and NANOG), as opposed to monolayers. Additionally, we present a putative mechanism for the cancer stem-like cell enrichment on CA scaffolds. This study demonstrates that the cancer stem-like cell enrichment in CA scaffolds is a robust process that is not restricted to particular cancer types.

Modeling the tumor microenvironment using chitosan-alginate scaffolds to control the stem-like state of glioblastoma cells.

Better prediction of in vivo drug efficacy using in vitro models should greatly improve in vivo success. Here we utilize 3D highly porous chitosan-alginate complex scaffolds to probe how various components of the glioblastoma microenvironment including extracellular matrix and stromal cells affect tumor cell stem-like state.

3D Porous Chitosan-Alginate Scaffolds as an In Vitro Model for Evaluating Nanoparticle-Mediated Tumor Targeting and Gene Delivery to Prostate Cancer.

Cationic nanoparticles (NPs) for targeted gene delivery are conventionally evaluated using 2D in vitro cultures. However, this does not translate well to corresponding in vivo studies because of the marked difference in NP behavior in the presence of the tumor microenvironment. In this study, we investigated whether prostate cancer (PCa) cells cultured in three-dimensional (3D) chitosan-alginate (CA) porous scaffolds could model cationic NP-mediated gene targeted delivery to tumors in vitro. We assessed in vitro tumor cell proliferation, formation of tumor spheroids, and expression of marker genes that promote tumor malignancy in CA scaffolds. The efficacy of NP-targeted gene delivery was evaluated in PCa cells in 2D cultures, PCa tumor spheroids grown in CA scaffolds, and PCa tumors in a mouse TRAMP-C2 flank tumor model. PCa cells cultured in CA scaffolds grew into tumor spheroids and displayed characteristics of higher malignancy as compared to those in 2D cultures. Significantly, targeted gene delivery was only observed in cells cultured in CA scaffolds, whereas cells cultured on 2D plates showed no difference in gene delivery between targeted and nontarget control NPs. In vivo NP evaluation confirmed targeted gene delivery, indicating that only CA scaffolds correctly modeled NP-mediated targeted delivery in vivo. These findings suggest that CA scaffolds serve as a better in vitro platform than 2D cultures for evaluation of NP-mediated targeted gene delivery to PCa.

Proliferation and enrichment of CD133+ glioblastoma cancer stem cells on 3D chitosan-alginate scaffolds

Emerging evidence implicates cancer stem cells (CSCs) as primary determinants of the clinical behavior of human cancers, representing an ideal target for next-generation anti-cancer therapies. However CSCs are difficult to propagate in vitro, severely limiting the study of CSC biology and drug development. Here we report that growing cells from glioblastoma (GBM) cell lines on three dimensional (3D) porous chitosan-alginate (CA) scaffolds dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. CA scaffold-grown cells were found more tumorigenic in nude mouse xenografts than cells grown from monolayers. Growing in CA scaffolds rapidly promoted expression of genes involved in the epithelial-to-mesenchymal transition that has been implicated in the genesis of CSCs. Our results indicate that CA scaffolds have utility as a simple and inexpensive means to cultivate CSCs in vitro in support of studies to understand CSC biology and develop more effective anti-cancer therapies.

CCL21 and IFNγ Recruit and Activate Tumor Specific T cells in 3D Scaffold Model of Breast Cancer

Effective elicitation of endogenous immunity is associated with improved prognosis for cancer patients. Clinical evidence in hematological and solid cancers shows that intratumoral injection of immunostimulatory genes primes and augments endogenous T cell responses. The ability of pro-inflammatory chemokines/cytokines to facilitate migration/activation of antigen-presenting cells (APC) and lymphocytes prompted our modeling of intratumoral delivery of a chemokine/cytokine combination for breast cancer treatment. Here, we demonstrate that expression of chemokine ligand 21 (CCL21) and interferon gamma (IFNγ) in tumors improves tumor specific T cell recruitment to tumor and activation in the tumor milieu. IFNγ and CCL21 were delivered into tumor cells via plasmids, and transfected cells were seeded to form spheroids on three-dimensional (3D) chitosan-alginate (CA) scaffolds. Co-expression of CCL21 and IFNγ, as evidenced by qRT-PCR and ELISA, induced increased recruitment, binding, and infiltration of anti-neu (p98) peptide specific T cells into the breast tumors as determined by SEM and immunofluorescence assays. The co-expression promoted recruitment of only p98 T cells, but not naïve T cells, demonstrating an antigen-restricted activation. Furthermore, the co-expression impacted T helper (Th) cell immunity, promoting an increase in secretion of pro-inflammatory Th-associated cytokine, tumor necrosis factor alpha (TNFα), and cytotoxic T lymphocyte (CTL)-associated protease, Granzyme B (GzB). Therefore, 3D CA scaffolds may be a useful breast cancer tumor microenvironment model to evaluate T cell function. Further characterization of CCL21-IFNγ mediated anti-tumor immunity will potentially benefit the development of chemokine/cytokine combination platforms as anti-cancer agents.

Evaluation of three‐dimensional porous chitosan–alginate scaffolds in rat calvarial defects for bone regeneration applications

This study investigated the use of three-dimensional porous chitosan-alginate (CA) scaffolds for critical size calvarial defect (diameter, 5.0 mm) repair in Sprague-Dawley rats. CA scaffolds have been used for in vitro culture of many cell types and demonstrated osteogenesis in ectopic locations in vivo, but have yet to be evaluated for functional bone tissue engineering applications. CA scaffolds demonstrated the ability to support undifferentiated mesenchymal stem cells (MSCs) in culture for 14 days in vitro and promoted spherical morphology. In vivo tests were performed using CA scaffolds and CA scaffolds with treatments including undifferentiated MSCs, bone marrow aspirate, and bone morphogenetic protein-2 (BMP-2) growth factor in comparison to unfilled bone defect used as a control. The samples were analyzed with MicroCT, histology, and immunohistochemical staining at 4 and 16 weeks. Partial defect closure was observed in all experimental groups at 16 weeks, with the greatest defect closure (71.56 ± 19.74%) in the animal group treated with CA scaffolds with BMP-2 (CA + BMP-2). The experimental samples demonstrated osteogenesis in histology and immunohistochemical staining, with the CA + BMP-2 group, showing the greatest level of osteogenesis. Tissue engineered CA scaffolds show promise in reconstruction of critical size bone defects.