Tunable mechanical properties of chitosan-based biocomposite scaffolds for bone tissue engineering applications: A review.

Bone tissue engineering (BTE) aims to develop implantable bone replacements for severe skeletal abnormalities that do not heal. In the field of BTE, chitosan (CS) has become a leading polysaccharide in the development of bone scaffolds. Although CS has several excellent properties, such as biodegradability, biocompatibility, and antibacterial properties, it has limitations for use in BTE because of its poor mechanical properties, increased degradation, and minimal bioactivity. To address these issues, researchers have explored other biomaterials, such as synthetic polymers, ceramics, and CS coatings on metals, to produce CS-based biocomposite scaffolds for BTE applications. These CS-based biocomposite scaffolds demonstrate superior properties, including mechanical characteristics, such as compressive strength, Young's modulus, and tensile strength. In addition, they are compatible with neighboring tissues, exhibit a controlled rate of degradation, and promote cell adhesion, proliferation, and osteoblast differentiation. This review provides a brief outline of the recent progress in making different CS-based biocomposite scaffolds and how to characterize them so that their mechanical properties can be tuned using crosslinkers for bone regeneration.

Nuciferine-loaded chitosan hydrogel-integrated 3D-printed polylactic acid scaffolds for bone tissue engineering: A combinatorial approach.

Critical-sized bone defects resulting from severe trauma and open fractures cannot spontaneously heal and require surgical intervention. Limitations of traditional bone grafting include immune rejection and demand-over-supply issues leading to the development of novel tissue-engineered scaffolds. Nuciferine (NF), a plant-derived alkaloid, has excellent therapeutic properties, but its osteogenic potential is yet to be reported. Furthermore, the bioavailability of NF is obstructed due to its hydrophobicity, requiring an efficient drug delivery system, such as chitosan (CS) hydrogel. We designed and fabricated polylactic acid (PLA) scaffolds via 3D printing and integrated them with NF-containing CS hydrogel to obtain the porous biocomposite scaffolds (PLA/CS-NF). The fabricated scaffolds were subjected to in vitro physicochemical characterization, cytotoxicity assays, and osteogenic evaluation studies. Scanning electron microscopic studies revealed uniform pore size distribution on PLA/CS-NF scaffolds. An in vitro drug release study showed a sustained and prolonged release of NF. The cyto-friendly nature of NF in PLA/CS-NF scaffolds towards mouse mesenchymal stem cells (mMSCs) was observed. Also, cellular and molecular level studies signified the osteogenic potential of NF in PLA/CS-NF scaffolds on mMSCs. These results indicate that the PLA/CS-NF scaffolds could promote new bone formation and have potential applications in bone tissue engineering.

Nanogels for bone tissue engineering - from synthesis to application.

Nanogels are cross-linked hydrogel nanoparticles with a three-dimensional, tunable porous structure that merges the best features of hydrogels and nanoparticles, including the ability to retain their hydrated nature and to swell and shrink in response to environmental changes. Nanogels have attracted increasing attention for use in bone tissue engineering as scaffolds for growth factor transport and cell adhesion. Their three-dimensional structures allow the encapsulation of a wide range of hydrophobic and hydrophilic drugs, enhance their half-life, and impede their enzymatic breakdown in vivo. Nanogel-based scaffolds are a viable treatment modality for enhanced bone regeneration. They act as carriers for cells and active ingredients capable of controlled release, enhanced mechanical support, and osteogenesis for enhanced bone tissue regeneration. However, the development of such nanogel constructs might involve combinations of several biomaterials to fabricate active ingredients that can control release, enhance mechanical support, and facilitate osteogenesis for more effective bone tissue regeneration. Hence, this review aims to highlight the potential of nanogel-based scaffolds to address the needs of bone tissue engineering.

Bioactive Molecule-incorporated Polymeric Electrospun Fibers for Bone Tissue Engineering.

Bone tissue engineering (BTE) is based on the participation and combination of different biomaterials, cells, and bioactive molecules to generate biosynthetic grafts for bone regeneration. Electrospinning has been used to fabricate fibrous scaffolds, which provide nanoscale architecture comprising interconnecting pores, resembling the natural hierarchy of tissues and enabling the formation of artificial functional tissues. Electrospun fibers for BTE applications have been mostly produced from polymers (chitosan, alginate, polycaprolactone, polylactic acid) and bioceramics (hydroxyapatite). Stem cells are among the most prolific cell types employed in regenerative medicine owing to their self-renewal and differentiation capacity. Most importantly, bioactive molecules, such as synthetic drugs, growth factors, and phytocompounds, are consistently used to regulate cell behavior inducing differentiation towards the osteoblast lineage. An expanding body of literature has provided evidence that these electrospun fibers loaded with bioactive molecules support the differentiation of stem cells towards osteoblasts. Thus, this review briefly describes the current development of polymers and bioceramic-based electrospun fibers and the influence of bioactive molecules in these electrospun fibers on bone tissue regeneration.

Three-dimensional-poly(lactic acid) scaffolds coated with gelatin/magnesium-doped nano-hydroxyapatite for bone tissue engineering.

BACKGROUND: Treatment of critical-sized bone defects has progressively evolved over the years from metallic implants to more ingenious three-dimensional-based scaffolds. The use of three-dimensional scaffolds for bone regeneration from biodegradable polymers like poly(lactic acid) (PLA) is gaining popularity. Scaffolds with surface functionalization using gelatin (Gel) have the advantages of biocompatibility and cell adhesion. Nano-hydroxyapatite (nHAp) is one of the most promising implant materials utilized in orthopaedics. The osteogenic potential of the nHAp can be improved by the substitution of magnesium (Mg) ions onto the crystal lattice of nHAp. Thus, the goal of this work was to make three-dimensional-PLA scaffolds covered with Gel/Mg-nHAp for osteogenic effect. METHODS AND RESULTS: The designed three-dimensional-PLA/Gel/Mg-nHAp scaffolds were attributed to various characterizations for the examination of their physicochemical, mechanical properties, cyto-compatibility, and biodegradability as well as their ability to promote osteogenesis in vitro. Mouse mesenchymal stem cells (mMSCs) were cytocompatible with these scaffolds. The osteogenic potential of three-dimensional-PLA/Gel/Mg-nHAp scaffolds employing mMSCs was validated at the cellular and molecular levels. The three-dimensional-PLA/Gel/Mg-nHAp scaffolds stimulated the differentiation of mMSCs towards osteoblastic lineage. CONCLUSION: Based on these findings, we suggest that the three-dimensional-PLA/Gel/Mg-nHAp scaffolds' osteogenic capability may be advantageous in the mending of bone defects in orthopedic applications.

Polycaprolactone fibrous electrospun scaffolds reinforced with copper doped wollastonite for bone tissue engineering applications.

The bone defects healing are always associated with post implantation infections; hence biomaterials rules significant role for orchestration of defective bone. In this study, we synthesized biocomposite scaffold by combining polycaprolactone (PCL), wollastonite (Ws) and metal ions (Cu) by electrospinning technique. The manufactured scaffolds (PCL/Ws andPCL/Cu-Ws) were subjected to physio-chemical characterization by scanning electron microscopy, energy dispersive X-ray spectroscopy, Fourier Transform Infra Red Spectroscopy (FTIR) and XRD. The surface topography of the scaffolds was found to be micro-fibrous in nature and each fiber was cylindrical in structure. The exogenous biomineralization and protein adsorption capacity of these scaffolds were studied. Enhanced amount of protein was adsorbed on PCL/Cu-Ws than PCL/Ws scaffold after incubating for 48 hr in foetal bovine serum (FBS) also the biomineralization shown to be promoted the apatite formation in vitro. The synthesized PCL/Cu-Ws scaffold was biocompatible to mouse mesenchymal stem cells and enhanced the mRNA expressionof osteoblastic specific marker genes including alkaline phosphatase and type I collagen and major transcription factor Runx2 in the presence of osteogenic medium indicates the osteoconductive nature of the scaffolds. The amount of calcium deposition and promotion of osteoblast differentiation and mineralization on human osteoblast cells was confirmed by alizarin red staining. The fabricated scaffolds possess potent antibacterial effect against Staphylococcu aureus and Escherichia coli. Hence, our outcomes confirmed that the PCL/Ws and PCL/Cu-Ws scaffolds promote bonesynthesis by cell proliferation and differentiation suitable for applications in bone regeneration orbone defects.

Chitosan and gelatin-based electrospun fibers for bone tissue engineering.

Fractures and injuries pertaining to bone tissue usually take prolonged periods for its natural healing. To overcome this problem, the field of bone tissue engineering (BTE) has acquired an efficient designing mechanism that incorporates cells, biomaterials and the corresponding growth factors to promote both osteogenesis as well as mineralization of the bone. Amidst the various techniques available for scaffold creation, electrospinning is considered superior as it paves the way for the creation of nanostructured scaffolds using biopolymers. Chitosan (CS) and Gelatin (Gel) are two of the cardinal natural biopolymers used in the field of biomaterials and tissue engineering. They can be used either exclusively or in combination with other biopolymers for the enhancement of bone regeneration. Hence, this review aims to render an elaborate study on the CS and Gel-based nanofibrous scaffolds with and without additional composites and the properties that they portray in terms of BTE.

Sinapic acid-loaded chitosan nanoparticles in polycaprolactone electrospun fibers for bone regeneration in vitro and in vivo.

Sinapic acid (SA) is a plant-derived phenolic compound known for its multiple biological properties, but its role in the promotion of bone formation is not yet well-studied. Moreover, the delivery of SA is hindered by its complex hydrophobic nature, limiting its bioavailability. In this study, we fabricated a drug delivery system using chitosan nanoparticles (nCS) loaded with SA at different concentrations. These were incorporated into polycaprolactone (PCL) fibers via an electrospinning method. nCS loaded with 50 µM SA in PCL fibers promoted osteoblast differentiation. Furthermore, SA treatment activated the osteogenesis signaling pathways in mouse mesenchymal stem cells. A critical-sized rat calvarial bone defect model system identified that the inclusion of SA into PCL/nCS fibers accelerated bone formation. Collectively, these data suggest that SA promoted osteoblast differentiation in vitro and bone formation in vivo, possibly by activating the TGF-ß1/BMP/Smads/Runx2 signaling pathways, suggesting SA might have therapeutic benefits in bone regeneration.

Chitosan in Surface Modification for Bone Tissue Engineering Applications.

Traditional methods of bone defect repair include autografts, allografts, surgical reconstruction, and metal implants that have several disadvantages such as donor site morbidity, rejection, risk of disease transmission, and repetitive surgery. Biomaterial-based bone reconstructions can, therefore, be an efficient alternative due to the inherent properties of the materials. Chitosan (CS), the deacetylated form of chitin, is a biopolymer having a wide array of applicability in regenerative tissue applications owing to its biocompatible, in vitro degradative and bioresorbable nature. Extensive studies are being carried out using CS to augment the properties of the already existing methods and to also improve the applicability of CS-based biocomposites in bone tissue repair. In this review, the suitability of CS as a surface modifier has been discussed in detail for the already existing implants, surface modifications of CS-based natural biocomposites for bone tissue regeneration, and the wide range of techniques that can introduce these modifications. CS, being a natural polymer, possesses advantageous properties including surface modifier that makes it a suitable candidate for bone regeneration, and further research to investigate its osteogenic potential in vivo along with the molecular and signaling mechanisms involved in bone regeneration can aid in expanding its applicability in clinical trials.

Bioactive mesoporous wollastonite particles for bone tissue engineering.

The current investigation was aimed at identifying the role of mesoporous wollastonite particles on the healing of rat tibial bone defect. The bone defect was created with a 3-mm-diameter dental drill, and it was filled with mesoporous wollastonite particles. After second and fourth weeks of filling treatments, it was found that mesoporous wollastonite particles promoted bone formation as evidenced by X-ray, histological, scanning electron microscope, and energy-dispersive spectra studies. X-ray study showed the closure of drill hole as seen by high-dense radio-opacity image. Histological analysis depicted the deposition of collagen in the bone defect area in response to mesoporous wollastonite particles' treatment. Scanning electron microscope-energy-dispersive spectra analyses of the sectioned implants also identified the deposition of apatite by these particles. Thus, our results suggested that mesoporous wollastonite particles have bioactive properties, and they can be used as a suitable filling material for promotion of bone formation in vivo.

A novel injectable temperature-sensitive zinc doped chitosan/ß-glycerophosphate hydrogel for bone tissue engineering.

Hydrogels are hydrophilic polymers that have a wide range of biomedical applications including bone tissue engineering. In this study we report preparation and characterization of a thermosensitive hydrogel (Zn-CS/ß-GP) containing zinc (Zn), chitosan (CS) and beta-glycerophosphate (ß-GP) for bone tissue engineering. The prepared hydrogel exhibited a liquid state at room temperature and turned into a gel at body temperature. The hydrogel was characterized by SEM, EDX, XRD, FT-IR and swelling studies. The hydrogel enhanced antibacterial activity and promoted osteoblast differentiation. Thus, we suggest that the Zn-CS/ß-GP hydrogel could have potential impact as an injectable in situ forming scaffold for bone tissue engineering applications.

Chitosan scaffolds containing silicon dioxide and zirconia nano particles for bone tissue engineering.

A scaffold harboring the desired features such as biodegradation, biocompatibility, porous structure could serve as template for bone tissue engineering. In the present study, chitosan (CS), nano-scaled silicon dioxide (Si) and zirconia (Zr) were combined by freeze drying technique to fabricate a bio-composite scaffold. The bio-composite scaffold (CS/Si/Zr) was characterized by SEM, XRD and FT-IR studies. The scaffold possessed a porous nature with pore dimensions suitable for cell infiltration and colonization. The presence of zirconia in the CS/Si/Zr scaffold decreased swelling and increased biodegradation, protein adsorption and bio-mineralization properties. The CS/Si/Zr scaffold was also found to be non-toxic to rat osteoprogenitor cells. Thus, we suggest that CS/Si/Zr bio-composite scaffold is a potential candidate to be used for bone tissue engineering.

Preparation, characterization and antimicrobial activity of a bio-composite scaffold containing chitosan/nano-hydroxyapatite/nano-silver for bone tissue engineering.

In this study, a bio-composite scaffold containing chitosan/nano-hydroxyapatite/nano-silver particles (CS/nHAp/nAg) was developed by freeze drying technique, followed by introduction of silver ions in controlled amount through reduction phenomenon by functional groups of chitosan. The scaffolds were characterized using SEM, FT-IR, XRD, swelling, and biodegradation studies. The testing of the prepared scaffolds with Gram-positive and Gram-negative bacterial strains showed antibacterial activity. The scaffold materials were also found to be non-toxic to rat osteoprogenitor cells and human osteosarcoma cell line. Thus, these results suggested that CS/nHAp/nAg bio-composite scaffolds have the potential in controlling implant associated bacterial infection during reconstructive surgery of bone.

Biocomposites containing natural polymers and hydroxyapatite for bone tissue engineering.

Bone tissue engineering is an alternative strategy to generate bone utilizing a combination of biomaterials and cells. Biomaterials that mimic the structure and composition of bone tissues at nanoscale are important for the development of bone tissue engineering applications. Natural or biopolymer-based composites containing chitin, chitosan, or collagen have advantages such as biocompatibility, biodegradability that are essential for bone tissue engineering. The inclusion of nanoparticles of hydroxyapatite (one of the most widely used bioceramic materials) into the biopolymer matrix improves the mechanical properties and incorporates the nanotopographic features that mimic the nanostructure of bone. This review summarizes the recent work on the development of biocomposites containing natural polymers with hydroxyapatite particles suitable for use in bone defects/bone regeneration.

Polymeric composites containing carbon nanotubes for bone tissue engineering.

Several natural and synthetic polymers are now available for bone tissue engineering applications but they may lack mechanical integrity. In recent years, there are reports emphasizing the importance of carbon nanotubes (CNTs) in supporting bone growth. CNTs possess exceptional mechanical, thermal, and electrical properties, facilitating their use as reinforcements or additives in various materials to improve the properties of the materials. Biomaterials containing polymers often are placed adjacent to bone. The use of CNTs is anticipated in these biomaterials applied to bone mainly to improve their overall mechanical properties and expected to act as scaffolds to promote and guide bone tissue regeneration. This review paper provides a current state of knowledge available examining the use of the polymeric composites containing CNTs for promoting bone growth.

Overexpression of Runx2 directed by the matrix metalloproteinase-13 promoter containing the AP-1 and Runx/RD/Cbfa sites alters bone remodeling in vivo.

The activator protein-1 (AP-1) and runt domain binding (Runx/RD/Cbfa) sites and their respective binding proteins, c-Fos/c-Jun and Runx2 (Cbfa1), regulate the rat matrix metalloproteinase-13 (MMP-13) promoter in both parathyroid hormone (PTH)-treated and differentiating osteoblastic cells in culture. To determine the importance of these regulatory sites in the expression of MMP-13 in vivo, transgenic mice containing either wild-type (-456 or -148) or AP-1 and Runx/RD/Cbfa sites mutated (-148A3R3) MMP-13 promoters fused with the E. coli lacZ reporter were generated. The wild-type transgenic lines expressed higher levels of bacterial beta-galactosidase in bone, teeth, and skin compared to the mutant and non-transgenic lines. Next, we investigated if overexpression of Runx2 directed by the MMP-13 promoter regulated expression of bone specific genes in vivo, and whether this causes morphological changes in these animals. Real time RT-PCR experiments identified increased mRNA expression of bone forming genes and decreased MMP-13 in the tibiae of transgenic mice (14 days and 6 weeks old). Histomorphometric analyses of the proximal tibiae showed increased bone mineralization surface, mineral apposition rate, and bone formation rate in the transgenic mice which appears to be due to decreased osteoclast number. Since MMP-13 is likely to play a role in recruiting osteoclasts to the bone surface, decreased expression of MMP-13 may cause reduced osteoclast-mediated bone resorption, resulting in greater bone formation in transgenic mice. In summary, we show here that the 148 bp upstream of the MMP-13 transcriptional start site is sufficient and necessary for gene expression in bone, teeth, and skin in vivo and the AP-1 and Runx/RD/Cbfa sites are likely to regulate this. Overexpression of Runx2 by these regulatory elements appears to alter the balance between the bone formation-bone resorption processes in vivo.