Enhanced Healing of Rat Calvarial Defects with MSCs Loaded on BMP-2 Releasing Chitosan/Alginate/Hydroxyapatite Scaffolds

In this study, we designed a chitosan/alginate/hydroxyapatite scaffold as a carrier for recombinant BMP-2 (CAH/B2), and evaluated the release kinetics of BMP-2. We evaluated the effect of the CAH/B2 scaffold on the viability and differentiation of bone marrow mesenchymal stem cells (MSCs) by scanning electron microscopy, MTS, ALP assay, alizarin-red staining and qRT-PCR. Moreover, MSCs were seeded on scaffolds and used in a 8 mm rat calvarial defect model. New bone formation was assessed by radiology, hematoxylin and eosin staining 12 weeks postoperatively. We found the release kinetics of BMP-2 from the CAH/B2 scaffold were delayed compared with those from collagen gel, which is widely used for BMP-2 delivery. The BMP-2 released from the scaffold increased MSC differentiation and did not show any cytotoxicity. MSCs exhibited greater ALP activity as well as stronger calcium mineral deposition, and the bone-related markers Col1α, osteopontin, and osteocalcin were upregulated. Analysis of in vivo bone formation showed that the CAH/B2 scaffold induced more bone formation than other groups. This study demonstrates that CAH/B2 scaffolds might be useful for delivering osteogenic BMP-2 protein and present a promising bone regeneration strategy.     

Three-dimensional scaffold containing EGF incorporated biodegradable polymeric nanoparticles for stem cell based tissue engineering applications

Stem cell-based tissue engineering holds much hope for the development of multifunctional tissues to replace diseased organs. The attachment and survival of stem cells on a three-dimensional (3D) scaffold must be enhanced for faster progression of stem cell based tissue engineering. This study evaluate the stability of mesenchymal stem cells (MSCs) in 3D porous scaffolds composed of a collagen and chitosan blend impregnated with epidermal growth factor incorporated chitosan nanoparticles (EGF-CNP). The EGF-CNP scaffolds were characterized by transmission electron microscopy, which revealed that the nanoparticles were round in shape and 20 ∼ 50 nm in size. The scaffolds were prepared by freeze drying. A Fourier-transform infrared spectrum study revealed that the linkage between collagen and chitosan was through an ionic interaction. Thermal analysis and degradation studies showed that the scaffold could be used in tissue engineering application. MSCs proliferated well in the EGF-CNP impregnated scaffold. A scanning electron microscope study showed anchored and elongated MSCs on the EGF-CNP impregnated scaffold. A 3D biodegradable collagen chitosan scaffold impregnated with EGF-CNP is a promising transportable candidate for MSC-based tissue engineering, and this scaffold could be used as an in vitro model for subsequent clinical applications.     

Biological behavior of the curcumin incorporated chitosan/poly(vinyl alcohol) nanofibers for biomedical applications

Electrospun composite scaffolds show high ability to be used in regenerative medicine and drug delivery, due to the nanofibrous structure and high surface area to volume ratio. In this study, we used nanofibrous scaffolds fabricated by chitosan (CS), poly(vinyl alcohol) (PVA), carbopol, and polycaprolactone using a dual electrospinning technique while curcumin (Cur) incorporated inside of the CS/PVA fibers. Scaffolds were fully characterized via scanning electron microscopy, water contact angle, tensile measurement, hydration, protein adsorption, and wrinkled tests. Furthermore, viability of the buccal fat pad-derived mesenchymal stem cells (BFP-MSCs) was also investigated using MTT assay for up to 14 days while cultured on these scaffolds. Cell cycle assay was also performed to more detailed evaluation of the stem cells growth when grown on scaffolds (with and without Cur) compared with the culture plate. Results demonstrated that Cur loaded nanofibrous scaffold had more suitable capability for water absorption and mechanical properties compared with the scaffold without Cur and it could also support the stem cells viability and proliferation. Cur release profile showed a decreasing effect on BFP-MSCs viability in the initial stage, but it showed a positive effect on stem cell viability in a long-term manner. In general, the results indicated that this nanofibrous scaffold has great potential as a delivery of the Cur and BFP-MSCs simultaneously, and so holds the promising potential for use in various regenerative medicine applications.     

Porous bioactive glass scaffolds for local drug delivery in osteomyelitis: development and in vitro characterization

A new bioactive glass-based scaffold was developed for local delivery of drugs in case of osteomyelitis. Bioactive glass having a new composition was prepared and converted into porous scaffold. The bioactivity of the resulting scaffold was examined by in vitro acellular method. The scaffolds were loaded with two different drugs, an antibacterial or antifungal drug. The effects of the size of the scaffold, drug concentration, and dissolution medium on drug release were studied. The scaffolds were further coated with a degradable natural polymer, chitosan, to further control the drug release. Both the glass and scaffold were bioactive. The scaffolds released both the drugs for 6 weeks, in vitro. The results indicated that the bigger the size and the higher the drug concentration, the better was the release profile. The scaffolds appeared to be suitable for local delivery of the drugs in cases of osteomyelitis.     

Different Porosities of Chitosan Can Influence the Osteogenic Differentiation Potential of Stem Cells

Scaffolds porosity has an important role in in vitro and in vivo differentiation process of stem cells with given the amount of space available to the cells to proliferate and differentiate. In the present study, chitosan with three porosities including 10%, 15%, and 20% that created by gelatin were used for investigation of the proliferation and osteogenic differentiation potential of adipose‐derived stem cells (ADSCs). In order to be more like the scaffold to natural bone tissue, freeze‐drying method was used in the scaffold preparation. Scaffold morphology, cell attachment, and toxicity were evaluated using scanning electron microscopy and MTT assay. Then, osteogenic differentiation potential of ADSCs cultured on chitosan with different porosities was evaluated by common osteogenic markers such as Alizarin red staining, ALP activity, calcium content, and osteogenic‐related genes expression via real‐time RT‐PCR. Although all scaffolds supported the proliferation and differentiation of ADSCs, but 10% scaffold demonstrated higher amount of osteogenic markers in comparison with the other porosities and control groups. Taking together, it can be concluded that osteogenic differentiation well done in the scaffolds with lower porosity because density of the cells will increase by forcing resulted from the scaffold, so osteogenic differentiation of the stem cells have an inverse association with scaffold porosity. J. Cell. Biochem. 119: 625–633, 2018. © 2017 Wiley Periodicals, Inc.     

Sodium hyaluronate/chitosan polyelectrolyte complex scaffolds for dental pulp regeneration: synthesis and characterization

In the present study, small-sized porous scaffolds were obtained from the freeze-drying of sodium hyaluronate/chitosan polyelectrolyte complexes. The obtained materials were characterized by a set of techniques including attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, swelling determination and weight loss studies. The morphology of the scaffolds was observed using scanning electron microscopy. Thermal characterization of the scaffolds was also performed by dynamic mechanical thermal analysis and thermogravimetric analysis. Finally, the cytotoxic profile of the prepared scaffolds was evaluated in vitro, using mesenchymal stem cells. The results obtained showed that cells adhered to scaffolds and proliferated. This study also confirmed that the degradation by-products of sodium hyaluronate/chitosan scaffold are noncytotoxic, which is fundamental for its application in the biomedical field.     

Porous Chitosan Scaffolds with Embedded Hyaluronic Acid/Chitosan/Plasmid-DNA Nanoparticles Encoding TGF-β1 Induce DNA Controlled Release,Transfected Chondrocytes,and Promoted Cell Proliferation

Cartilage defects resulting from traumatic injury or degenerative diseases have very limited spontaneous healing ability. Recent progress in tissue engineering and local therapeutic gene delivery systems has led to promising new strategies for successful regeneration of hyaline cartilage. In the present study, tissue engineering and local therapeutic gene delivery systems are combined with the design of a novel gene-activated matrix (GAM) embedded with hybrid hyaluronic acid(HA)/chitosan(CS)/plasmid-DNA nanoparticles encoding transforming growth factor (TGF)-β1. A chitosan scaffold functioned as the three-dimensional carrier for the nanoparticles. Results demonstrated that scaffold-entrapped plasmid DNA was released in a sustained and steady manner over 120 days, and was effectively protected in the HA/CS/pDNA nanoparticles. Culture results demonstrated that chondrocytes grown in the novel GAM were highly proliferative and capable of filling scaffold micropores with cells and extracellular matrix. Confocal laser scanning microscopy indicated that chondrocytes seeded in the GAM expressed exogenous transgenes labeled with green fluorescent protein. ELISA results demonstrated detectable TGF-β1 expression in the supernatant of GAM cultures, which peaked at the sixth day of culture and afterwards showed a moderate decline. Histological results and biochemical assays confirmed promotion of chondrocyte proliferation. Cell culture indicated no affects on phenotypic expression of ECM molecules, such as GAG. The results of this study indicate the suitability of this novel GAM for enhanced in vitro cartilage tissue engineering.     

Inter-connecting pores of chitosan scaffold with basic fibroblast growth factor modulate biological activity on human mesenchymal stem cells

In this study, we developed bio-active molecules immobilized chitosan scaffolds with controlled pore architectures for enhanced viability of human mesenchymal stem cells (hMSCs). The decreasing in molecular weight of chitosan by ultrasonication of chitosan solution was effective in the formation of porous chitosan scaffolds, resulting in an increase of inter-connecting micropores (∼10 μm) between macropores. Using a layer-by-layer method, we then prepared heparin-coated chitosan scaffolds as depots for basic fibroblast growth factors (bFGF). Enzyme-linked immunosorbent assays confirmed that heparin-coated chitosan scaffolds could bind higher amount of bFGF (24.21 ng/mg) compared to 2.53 ng/mg of non-coated scaffold. Moreover, we were able to manipulate the release profiles of bFGF from the scaffolds for 7 days. In vitro studies showed that chitosan scaffolds induced the improved viability and proliferation of hMSCs through their synergetic effects of the inter-connecting micropores and the sustained released of bFGF. Our results suggest that bFGF immobilized chitosan scaffolds with controlled inter-connecting pores, in combination with other heparin-binding growth factors, have potential implants for controlling biological functions in regenerative medicine.     

3D Porous Chitosan Scaffolds Suit Survival and Neural Differentiation of Dental Pulp Stem Cells

A key aspect of cell replacement therapy in brain injury treatment is construction of a suitable biomaterial scaffold that can effectively carry and transport the therapeutic cells to the target area. In the present study, we created small 3D porous chitosan scaffolds through freeze-drying, and showed that these can support and enhance the differentiation of dental pulp stem cells (DPSCs) to nerve cells in vitro. The DPSCs were collected from the dental pulp of adult human third molars. At a swelling rate of ~84.33 ± 10.92 %, the scaffold displayed high porosity and interconnectivity of pores, as revealed by SEM. Cell counting kit-8 assay established the biocompatibility of the chitosan scaffold, supporting the growth and survival of DPSCs. The successful neural differentiation of DPSCs was assayed by RT-PCR, western blotting, and immunofluorescence. We found that the scaffold-attached DPSCs showed high expression of Nestin that decreased sharply following induction of differentiation. Exposure to the differentiation media also increased the expression of neural molecular markers Microtubule-associated protein 2, glial fibrillary acidic protein, and 2′,3′-cyclic nucleotide phosphodiesterase. This study demonstrates that the granular 3D chitosan scaffolds are non-cytotoxic, biocompatible, and provide a conducive and favorable micro-environment for attachment, survival, and neural differentiation of DPSCs. These scaffolds have enormous potential to facilitate future advances in treatment of brain injury.     

Chitosan stabilizes platelet growth factors and modulates stem cell differentiation toward tissue regeneration

The idea of using chitosan as a functional delivery aid to support simultaneously PRP, stem cells and growth factors (GF) is associated with the intention to use morphogenic biomaterials to modulate the natural healing sequence in bone and other tissues. For example, chitosan–chondroitin sulfate loaded with platelet lysate was included in a poly(d,l-lactate) foam that was then seeded with human adipose-derived stem cells and cultured in vitro under osteogenic stimulus: the platelet lysate provided to the bone tissue the most suitable assortment of GF which induces the osteogenic differentiation of the mesenchymal stem cells. PDGF, FGF, IGF and TGF-β were protagonists in the repair of callus fractures. The release of GF from the composites of chitosan–PRP and either nano-hydroxyapatite or tricalcium phosphate was highly beneficial for enhancing MSC proliferation and differentiation, thus qualifying chitosan as an excellent vehicle. A number of biochemical characteristics of chitosan exert synergism with stem cells in the regeneration of soft tissues.     

In Vitro Insulin Release from Thermosensitive Chitosan Hydrogel

Recently, great attention has been paid to in situ gel-forming chitosan/glycerol-phosphate (chitosan/Gp) solution due to their good biodegradability and thermosensitivity. This in situ gel-forming system is injectable fluid that can be introduced into the body in a minimally invasive manner prior to solidifying within the desired tissue. At the present study, insulin release from chitosan/Gp solution has been investigated. Insulin in different concentrations was loaded in two formulations of chitosan/Gp solution and in vitro drug release was studied over a period of 3 weeks. Results indicated that the release of insulin from chitosan/Gp gel decreases by increasing in Gp salt and initial insulin concentration. Stability of released insulin was investigated by 8-anilino-1-naphthalenesulfonate probe. Results proved that insulin have been released in its native form. Because of simple preparation and administration, prolonged release of insulin and stability of released insulin, this in situ gel-forming system could be used as a controlled release delivery system for insulin.KEY WORDS: biodegradable, chitosan, controlled release, in situ forming, insulin     

聚己内酯静电纺丝纳米纤维支架用于小鼠诱导多能干细胞培养

干细胞联合生物支架材料体外构建功能性组织与器官,成为当前组织再生研究的重要策略,而探求具有良好生物相容性的支架材料是其关键.本研究采用扫描电镜、噻唑蓝（MTT）法、荧光显微染色等方法检测小鼠诱导多能干细胞（murine induced pluripotent stem cells, miPSCs）在聚己内酯（poly ε-caprolactone, PCL）静电纺丝纳米纤维支架上的粘附、增殖等生物学特性,探究聚己内酯纳米纤维支架与miPSCs的生物相容性. 结果显示,miPSC在PCL纳米纤维支架上具有良好粘附性并呈集落样生长,其增殖能力及干性标记物（Oct4-GFP+）的表达均不亚于标准对照组;扫描电镜显示,miPSC在PCL纳米纤维支架材料上呈现出绒毛状突起的表面结构.上述结果表明,PCL纳米纤维支架可促进miPSCs的粘附、自我增殖以及干性维持,两者具有良好的生物相容性,为下一步联合生物支架材料与干细胞构建功能性组织奠定了基础.     

Proliferation and Differentiation Potential of Human Adipose-Derived Stem Cells Grown on Chitosan Hydrogel

Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.     

Three-dimensional differentiation of adipose-derived mesenchymal stem cells into insulin-producing cells

The aim of this study is to evaluate the collagen/hyaluronic acid (Col/HA) scaffold effect on the differentiation of insulin-producing cells (IPCs) from adipose-derived mesenchymal stem cells (ASCs). In this experimental study, ASCs were cultured and seeded in a Col/HA scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was evaluated using gene expression (PDX-1, GLUT-2 and insulin) analysis and immunocytochemistry, while functional maturity was determined by measuring insulin release in response to low- and high-glucose media. The induced IPCs were morphologically similar to pancreatic islet-like cells. Expression of the islet-associated genes PDX-1, GLUT-2 and insulin genes in 3D-cultured cells was markedly higher than the 2D-cultured cells exposure differentiation media. Compared to the 2D culture of ASCs-derived IPCs, the insulin release from 3D ASCs-derived IPCs showed a nearly 4-fold (p?<?0.05) increase when exposed to a high glucose (25 mmol) medium. The percentage of insulin-positive cells in the 3D experimental group showed an approximately 4-fold increase compared to the 2D experimental culture cells. The results of this study demonstrated that the COL/HA scaffold can enhance the differentiation of IPCs from rat ASCs.     

Three-dimensional fibrous PLGA/HAp composite scaffold for BMP-2 delivery

A protein loaded three-dimensional scaffold can be used for protein delivery and bone tissue regeneration. The main objective of this project was to develop recombinant human bone morphogenetic protein-2 (rhBMP-2) loaded poly(D,L-lactide-co-glycolide)/hydroxylapatite (PLGA/HAp) composite fibrous scaffolds through a promising fabrication technique, electrospinning. In vitro release of BMP-2 from these scaffolds, and the attachment ability and viability of marrow derived messenchymal stem cells (MSCs) in the presence of the scaffolds were investigated. The PLGA/HAp composite scaffolds developed in this study exhibit good morphology and it was observed that HAp nanoparticles were homogeneously dispersed inside PLGA matrix within the scaffold. The composite scaffolds allowed sustained (2-8 weeks) release of BMP-2 whose release rate was accelerated with increasing HAp content. It was also shown that BMP-2 protein successfully maintained its integrity and natural conformations after undergoing the process of electrospinning. Cell culture experiments showed that the encapsulation of HAp could enhance cell attachment to scaffolds and lower cytotoxicity.     

Sulfasalazine release from alginate-N,O-carboxymethyl chitosan gel beads coated by chitosan

In the present study, spherical beads were prepared from a water-soluble chitosan (N,O-carboxymethyl chitosan, NOCC) and alginate with ionic gelation method. Then, swollen calcium–alginate–NOCC beads were coated with chitosan. To prepare drug loaded beads, sulfasalazine (SA) was added to the initial aqueous polymer solution. The effect of coating, as well as drying procedure, on the swelling behavior of unloaded beads and SA release of drug loaded ones were evaluated in simulated gastrointestinal tract fluid. The rate of swelling and drug release were decreased for air-dried and coated beads in comparison with freeze-dried and uncoated ones, respectively. No burst release of drug was observed from whole tested beads. Chitosan coated beads released approximately 40% of encapsulated drug in simulated gastric and small intestine tract fluid. Based on these results, the chitosan coated alginate–NOCC hydrogel may be used as potential polymeric carrier for colon-specific delivery of sulfasalazine.     

Enhanced Antibacterial Effect of Ceftriaxone Sodium-Loaded Chitosan Nanoparticles Against Intracellular Salmonella typhimurium

The aim of the present study was to utilize chitosan (CS) nanoparticles for the intracellular delivery of the poorly cell-penetrating antibiotic, ceftriaxone sodium (CTX). In vitro characterization of (CTX-CS) nanoparticles was conducted leading to an optimized formula that was assessed for its biocompatibility to blood (hemolysis test) and cells (MTT assay). Progressively, confocal laser scanning microscopy (CLSM), cellular uptake (microfluorimetry), and antibacterial activity of the nanoparticles were investigated in two cell lines: Caco-2 and macrophages J774.2 pre-infected with Salmonella typhimurium. Results showed that the optimized formula had size 210 nm, positive zeta potential (+30 mV) and appreciable entrapment efficiency for CTX (45%) and included a biphasic release pattern. The nanoparticles were biocompatible and were internalized by cells as verified by CLSM whereas microfluorimetry indicated substantial cellular uptake. Moreover, the CTX–chitosan nanoparticles showed a significant reduction in the count of intracellular S. typhimurium in Caco-2 and macrophages J774.2. This reduction was significantly higher than that obtained in case of placebo nanoparticles, CTX, and CTX–chitosan solutions and might be attributed to enhanced endocytic uptake of the nanoaprticles and antibacterial effect of the chitosan polymer. In conclusion, the results provide evidence for the potential use of chitosan nanoparticles to enhance the intracellular delivery and antibacterial effect of CTX in enterocytes and macrophages.Key words: ceftriaxone sodium, chitosan nanoparticles, enterocytes, intracellular delivery, macrophages     

Studies on the effect of vitamin D on calcium absorption and transport

1. Mucosal cells of the small intestine obtained from rats deprived of vitamin D or given excessive amounts of the vitamin accumulated significantly more calcium than did cells from control animals. 2. Mucosal cells from vitamin D-deficient rats released less calcium than did cells from normal or hypervitaminotic D animals. 3. Studies in vivo showed that the transfer of (45)Ca from the intestine to the blood was delayed in vitamin D deficiency, but was accelerated in hypervitaminosis D. 4. The findings support the thesis that vitamin D is involved in the release of calcium rather than in its uptake by mucosal cells. 5. Further evidence is presented suggesting that uptake of calcium by intestinal mucosal cells at 0 degrees is primarily passive, whereas at 38 degrees uptake and release are effected by an active process that depends on energy derived from glycolytic activity.     

Development of hydrocortisone succinic acid/and 5-fluorouracil/chitosan microcapsules for oral and topical drug deliveries

Recently, we demonstrated the safety use of calendula oil/chitosan microcapsules as a carrier for both oral and topical deliveries. We also reported the improved biological activity towards skin cells and Staphylococcus aureus of phyllanthin containing chitosan microcapsules. However, the possibility of both oral and topical applications was still necessary to be further studied. Here we investigated that both oral and topical applications of chitosan-based microcapsules were tested using hydrocortisone succinic acid (HSA) and 5-fluorouracil (5-FU), respectively. The drug loading efficiency, particle size, surface morphology and chemical compositions of both drug loaded microcapsules were confirmed by UV-vis spectrophotometer, particle size analyzer, scanning electron microscope and Fourier transform infrared spectroscopy. The in vitro release studies revealed that both HSA and 5-FU could be released form chitosan microcapsules. The mean adrenocorticotropic hormone concentration in HSA loaded microcapsule mice plasma was detected to be lower than that of water control. One hundred micrograms per milliliter of 5-FU containing microcapsules exhibited a stronger growth inhibition towards skin keratinocytes than that of free 5-FU. In vitro drug delivery model demonstrated the delivery of 5-FU from microcapsule treated textiles into nude mice skin. Further uses of the drug loaded microcapsules may provide an efficiency deliverable tool for both oral and topical applications.     

Electrospun poly-l-lactic acid/polyvinyl alcohol nanofibers improved insulin-producing cell differentiation potential of human adipose-derived mesenchymal stem cells

Combination of adipose-derived mesenchymal stem cells (ADSCs) and synthetic materials in terms of pancreatic tissue engineering can be considered as a treatment of diabetes. This study aimed to evaluate the differentiation of human ADSCs to pancreatic cells on poly-l -lactic acid/polyvinyl alcohol (PLLA/PVA) nanofibers as a three-dimensional (3D) scaffold. Mesenchymal stem cells (MSCs) were characterized for mesenchymal surface markers by flow cytometry. Then ADSCs were seeded on 3D scaffolds and treated with pancreatic differentiation medium. Immunostaining assay showed that ADSCs were very efficiently differentiated into a relatively homogeneous population of insulin-producing cells. Moreover, real-time RT-PCR results revealed that pancreas-specific markers were highly expressed in 3D scaffolds compared with their expression in tissue culture plates and this difference in expression level was significant. In addition, insulin and C-peptide secreted in response to varying concentrations of glucose in the 3D scaffold group was significantly higher than that in 2D culture. The results of the present study confirmed that PLLA/PVA scaffold seeded with ADSCs could be a suitable option in pancreatic tissue engineering.