S. Deepthi

Injectable Chitin-Poly(ε-caprolactone)/Nanohydroxyapatite Composite Microgels Prepared by Simple Regeneration Technique for Bone Tissue Engineering

Injectable gel systems, for the purpose of bone defect reconstruction, have many advantages, such as controlled flowability, adaptability to the defect site, and increased handling properties when compared to the conventionally used autologous graft, scaffolds, hydroxyapatite blocks, etc. In this work, nanohydroxyapatite (nHAp) incorporated chitin-poly(ε-caprolactone) (PCL) based injectable composite microgels has been developed by a simple regeneration technique for bone defect repair. The prepared microgel systems were characterized using scanning electron microscope (SEM), Fourier transformed infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The composite microgel, with the incorporation of nHAp, showed an increased elastic modulus and thermal stability and had shear-thinning behavior proving the injectability of the system. The protein adsorption, cytocompatibility, and migration of rabbit adipose derived mesenchymal stem cells (rASCs) were also studied. Chitin-PCL-nHAp microgel elicited an early osteogenic differentiation compared to control gel. The immunofluorescence studies confirmed the elevated expression of osteogenic-specific markers such as alkaline phosphatase, osteopontin, and osteocalcin in chitin-PCL-nHAp microgels. Thus, chitin-PCL-nHAp microgel could be a promising injectable system for regeneration of bone defects which are, even in deeper planes, irregularly shaped and complex in nature.

An overview of chitin or chitosan/nano ceramic composite scaffolds for bone tissue engineering.

Chitin and chitosan based nanocomposite scaffolds have been widely used for bone tissue engineering. These chitin and chitosan based scaffolds were reinforced with nanocomponents viz Hydroxyapatite (HAp), Bioglass ceramic (BGC), Silicon dioxide (SiO2), Titanium dioxide (TiO2) and Zirconium oxide (ZrO2) to develop nanocomposite scaffolds. Plenty of works have been reported on the applications and characteristics of the nanoceramic composites however, compiling the work done in this field and presenting it in a single article is a thrust area. This review is written with an aim to fill this gap and focus on the preparations and applications of chitin or chitosan/nHAp, chitin or chitosan/nBGC, chitin or chitosan/nSiO2, chitin or chitosan/nTiO2 and chitin or chitosan/nZrO2 in the field of bone tissue engineering in detail. Many reports so far exemplify the importance of ceramics in bone regeneration. The effect of nanoceramics over native ceramics in developing composites, its role in osteogenesis etc. are the gist of this review.

Layered chitosan-collagen hydrogel/aligned PLLA nanofiber construct for flexor tendon regeneration

The aim of our study was to develop a tendon construct of electrospun aligned poly (l-lactic acid) (PLLA) nanofibers, to mimic the aligned collagen fiber bundles and layering PLLA fibers with chitosan-collagen hydrogel, to mimic the glycosaminoglycans of sheath ECM for tendon regeneration. The hydrogel coated electrospun membrane was rolled and an outer coating of alginate gel was given to prevent peritendinous adhesion. The developed constructs were characterized by SEM, FT-IR and tensile testing. Protein adsorption studies showed lower protein adsorption on coated scaffolds compared to uncoated scaffolds. The samples were proven to be non-toxic to tenocytes. The chitosan-collagen/PLLA uncoated scaffolds and alginate gel coated chitosan-collagen/PLLA scaffolds showed good cell proliferation. The tenocytes showed good attachment and spreading on the scaffolds. This study indicated that the developed chitosan-collagen/PLLA/alginate scaffold would be suitable for flexor tendon regeneration.

Injectable osteogenic and angiogenic nanocomposite hydrogels for irregular bone defects

Injectable hydrogels with their 3D structure and good moldability serve as excellent scaffolding material for regenerating irregular non load-bearing bone defects. Most of the bone defects do not heal completely due to the lack of vasculature required for the transport of nutrients and oxygen to the regenerating tissues. To enhance vasculature, we developed an injectable hydrogel system made of chitin and poly (butylene succinate) (PBSu) loaded with 250  ±  20 nm sized fibrin nanoparticles (FNPs) and magnesium-doped bioglass (MBG). FNPs were expected to enhance vascularisation and MBG was expected to help induce early osteogenesis. Composite hydrogels were analysed using Fourier transform infra-red spectroscopy, scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy, and rheology. Hydrogels with MBG showed a slightly rougher morphology upon SEM analysis. Composites containing 5% MBG and 2% FNPs showed good rheological properties, injectability, temperature stability, biomineralization and protein adsorption. Human umbilical vein endothelial cells (HUVECs) and rabbit-adipose derived mesenchymal stem cells (rASCs) were used for cyto-compatibility studies. Composite gels with 2% FNPs and 2% MBG (composite 1) were considered to be non-toxic to both the cells and were taken for further in vitro studies. Aortic ring assay was carried out to study the angiogenic potential of the hydrogels. The aorta placed with composite hydrogels showed enhanced sprouting of blood vessels. rASCs too showed good spreading on the composite hydrogels. Hydrogels containing MBG showed early initiation of differentiation and higher expression of alkaline phosphatase and osteocalcin confirming the osteoinductive property of MBG. These studies indicate that this composite hydrogel can be used for regenerating irregular bone defects.

Bilayered construct for simultaneous regeneration of alveolar bone and periodontal ligament

Periodontitis is an inflammatory disease that causes destruction of tooth-supporting tissues and if left untreated leads to tooth loss. Current treatments have shown limited potential for simultaneous regeneration of the tooth-supporting tissues. To recreate the complex architecture of the periodontium, we developed a bilayered construct consisting of poly(caprolactone) (PCL) multiscale electrospun membrane (to mimic and regenerate periodontal ligament, PDL) and a chitosan/2wt % CaSO4 scaffold (to mimic and regenerate alveolar bone). Scanning electron microscopy results showed the porous nature of the scaffold and formation of beadless electrospun multiscale fibers. The fiber diameter of microfiber and nanofibers was in the range of 10 ± 3 µm and 377 ± 3 nm, respectively. The bilayered construct showed better protein adsorption compared to the control. Osteoblastic differentiation of human dental follicle stem cells (hDFCs) on chitosan/2wt % CaSO4 scaffold showed maximum alkaline phosphatase at seventh day followed by a decline thereafter when compared to chitosan control scaffold. Fibroblastic differentiation of hDFCs was confirmed by the expression of PLAP-1 and COL-1 proteins which were more prominent on PCL multiscale membrane in comparison to control membranes. Overall these results show that the developed bilayered construct might serve as a good candidate for the simultaneous regeneration of the alveolar bone and PDL.

Nano-fibrin stabilized CaSO4 crystals incorporated injectable chitin composite hydrogel for enhanced angiogenesis & osteogenesis.

Calcium sulfate (CaSO4), an excellent biodegradable bone forming agent that is an ideal choice as additive in gels, however, its disadvantage being poor gel rheology and angiogenesis. Here, we have synthesized chitin-CaSO4-nano-fibrin based injectable gel system which shows improved rheology and angiogenic potential. Rheological studies showed that the composite gel was a shear thinning gel with elastic modulus of 15.4±0.275kPa; a 1.67 fold increase over chitin control. SEM and XRD analyses revealed the effect of nano-fibrin (nFibrin) in transforming CaSO4 crystal shape from needle to hexagonal. It also masked the retarding effect of CaSO4 towards in vitro early cell attachment and angiogenesis using rabbit adipose derived mesenchymal stem cells (rASCs) and HUVECs, respectively. rASCs osteogenesis was confirmed by spectrophotometric endpoint assay, which showed 6-fold early increase in alkaline phosphatase levels and immuno-cytochemistry analysis. These in vitro results highlight the potential of injectable chitin-CaSO4-nFibrin gel for osteo-regeneration via enhanced angiogenesis.

Prolonged release of TGF-β from polyelectrolyte nanoparticle loaded macroporous chitin-poly(caprolactone) scaffold for chondrogenesis.

Cartilage degeneration occurs when the catabolic factors overtakes the anabolic factors. The regeneration capability of damaged cartilage is poor due to its hypovascular and hypocellular tissue. Tissue engineering strategies aims in development of a suitable substrate that provide the required physical, chemical and biological cues to the proliferating cells to direct chondrogenesis. A macroporous polymeric blend scaffold of chitin and poly(caprolactone) (PCL) was fabricated by lyophilisation technique and characterized using Scanning Electron Microscope (SEM), Fourier Transform Infrared Spectroscopy (FTIR) and Thermogravimetric/Differential thermal Analysis (TG/DTA). The effect of prolonged release of Transforming growth factor-β (TGF-β) was studied by encapsulating it in chondroitin sulphate nanoparticles (nCS) incorporated in chitin-PCL scaffold. Chondroitin sulphate nanoparticles containing TGF-β (TGF-β-nCS) was developed by polyelectrolyte crosslinking using chitosan. Characterization of TGF-β-nCS by Dynamic Light Scattering particle sizer and SEM showed a 230±20nm sized spherical particles. Swelling and degradation studies of the composite scaffold showed its stability. Protein adsorption was enhanced in nanoparticle containing scaffold. The effect of TGF-β was well addressed by the increased attachment and proliferation of rabbit adipose derived mesenchymal stem cells (rASCs). The chondrogenic potential of rASCs in the presence of TGF-β releasing composite scaffold showed an increased proteoglycan deposition. These studies highlight the positive effects of chitin-PCL-TGF-β-nCS scaffold for cartilage regeneration.

Chitosan based metallic nanocomposite scaffolds as antimicrobial wound dressings

Chitosan based nanocomposite scaffolds have attracted wider applications in medicine, in the area of drug delivery, tissue engineering and wound healing. Chitosan matrix incorporated with nanometallic components has immense potential in the area of wound dressings due to its antimicrobial properties. This review focuses on the different combinations of Chitosan metal nanocomposites such as Chitosan/nAg, Chitosan/nAu, Chitosan/nCu, Chitosan/nZnO and Chitosan/nTiO2 towards enhancement of healing or infection control with special reference to the antimicrobial mechanism of action and toxicity.

Nanostrontium ranelate incorporated injectable hydrogel enhanced matrix production supporting chondrogenesis in vitro

An injectable hydrogel, with the advantage of adaptability to defect sites, patient compliance, controlled flowability and high water uptake capability, was explored as a prototype for cartilage tissue regeneration. Chitosan and fibrin are natural biomaterials that are biocompatible, biodegradable, resemble the ECM of the tissues and contain cell adhesion sites thereby providing a support for cell growth. In this study strontium ranelate, a drug recently studied to enhance cartilage regeneration, was encapsulated in chitosan nanoparticles to provide sustained delivery of the drug content within the composite gel (chitosan/alginate/fibrin hydrogel). The developed nanocomposite gel was characterized using SEM, EDS and FTIR. The particle size of the strontium ranelate loaded chitosan nanoparticles was found to be 160 ± 30 nm. The encapsulation and loading efficiency values of strontium ranelate were found to be 40 ± 10% and 36 ± 2% respectively. Rheological data showed a storage modulus of 5.514 ± 0.102 kPa with thermal stability over the studied temperature range, and the gel properties could be restored within 10 s after the application of a high shear rate. The cytocompatibility and chondrogenic potential was analyzed using human mesenchymal stem cells (hMSCs) to evaluate the applicability of the developed hydrogel for cartilage regeneration. hMSCs were found to be viable in the developed hydrogels and chondrogenic differentiation of hMSCs was observed which was confirmed with enhanced proteoglycan and collagen synthesis. These results indicated that the developed injectable nanocomposite gel would be a suitable system for cartilage regeneration.

Chitosan-Gelatin Composite Scaffolds in Bone Tissue Engineering

Regenerative medicine focuses on repair/replacement of the damaged tissue or organ in our body. This is done by growing cells on scaffold materials which help in its attachment, migration and proliferation. Chitosan being natural polymer has many unique properties such as being biocompatible, biodegradable and also has antibacterial and wound-healing abilities. Gelatin a derivative of collagen, which is widely present in our body, has been used as a composite with chitosan for promoting cell attachment, proliferation, and differentiation. Composite scaffolds have also shown better mechanical and functional properties because these composites are made of polymer and inorganic/organic blenders. Overall this review focuses on the role of chitosan-gelatin-based composite scaffolds in bone tissue engineering.