Preparation and characterization of 3D printed polycaprolactone/pluronic F-127 scaffold incorporating ciprofloxacin-loaded halloysite nanotubes to promote in vivo bone regeneration

Bone regeneration remains a clinical challenge with limited bone substitutes, urging for effective alternative strategies. Nanotubes, especially carbon nanotubes and titanium dioxide nanotubes, have been widely utilized for bone regeneration; however, their further applications were limited by the composition and degradability. As naturally occurring aluminosilicate nanoclay, halloysite nanotubes (HNTs), with good biocompatibility, functionality, and nanotubular structures, may be a promising platform for promoting bone regeneration. Herein, we presented a HNTs incorporated hydrogel and explored the potential bone tissue engineering applications of HNTs. The HNTs encapsulated hydrogel was simply fabricated by using the photopolymerization method with gelatin methacrylate (GelMA) and HNTs. The incorporation of HNTs led to an enhanced mechanical performance while maintaining a good cytocompatibility in vitro. The osteogenic activities of the HNTs incorporated platform have also been studied in vitro and in vivo. Remarkably, the addition of HNTs obviously upregulated the expression of osteogenic differentiation-related genes and concomitant protein of human dental pulp stem cells (hDPSCs) and therefore facilitated subsequent bone regeneration in calvarial defects of rats. Overall, the results obtained in this study highlight the bone regeneration capacity of HNTs, which may enhance current understanding of HNTs, and present a promising alternative strategy for bone regeneration.

Chapter 11 - Nosocomial Bacterial Infection of Orthopedic Implants and Antibiotic Hydroxyapatite/Silver-Coated Halloysite Nanotube With Improved Structural Integrity as Potential Prophylaxis

Tissue engineering strives to integrate structure and functionality in bioengineering scaffolds or implants. These materials must be biodegradable, immunocompatible, and integrate well with the natural tissue. The bioengineered scaffolds and implants should also be eco‐friendly to manufacture. The aim was to design an implant that enhances inherent bone regeneration y, mimics the natural internal environment and is composed of eco‐friendly materials. We used halloysite nanotubes (HNTs), naturally occurring nanoclay, similar to the kaolin chemically and structurally, and composed of oppositely charged concentric layers of hollow tubules. This structure enables HNTs to acts as a nanocontainer and release bioactive molecules periodically and in a sustained manner. We combined HNTs with alginate hydrogels to release BMPs 2, 4, and 6 and are known to induce osteogenic expression in osteoblasts. We obtained the sustained release of BMPs over a seven‐day period. The amount released was in the range of picograms per milliliter. Osteoblasts encapsulated in these nanoenhanced hydrogels showed enhanced mineralization and ECM secretion as compared with controls over a period of 28 days. Cellular response varied depending on BMP treatment. This nanoenhanced hydrogel system may be a viable solution for in situ bone regeneration for use in degenerative joint disease, small fractures and other bone defects.

In-situ forming hydrogels have gained noticeable attention to encapsulate osteogenic agents and regenerate irregular-shape bone defects. In this study, a novel thermosensitive hydrogel based on blended methylcellulose (MC) with Persian gum (PG) was fabricated and enriched with taxifolin (TAX) loaded halloysite nanotubes (HNTs) to enhance mechanical and biological characteristics of the hydrogel in bone tissue engineering. The injectability, mechanical and rheological tests were performed for different hydrogel formulations containing 0.25–1.5 w/v% PG and 1–7 w/v% HNTs. Also, to evaluate the impact of PG and HNTs on hydrogel behavior, the degradation rate and swelling degree of hydrogels were assessed. The optimized MC/PG/HNTs hydrogel containing 1% PG and 3% HNTs (MC/PG-1/HNTs 3%) was easily injectable and gelled rapidly at physiological temperature, while it had the highest mechanical strength due to the existence of PG and HNTs. In vitro release study of TAX from this system also revealed more sustained release compared to HNTs-TAX nanoparticles. Furthermore, the interaction of cells with hydrogel and osteo-conductivity was studied using osteoblast-like cells (MG-63). Results showed higher cell adhesion, proliferation, and gene expression for MC/PG-1/HNTs-TAX hydrogel compared to MC/PG-1 and MC/PG-1/HNTs 3% possibly due to the synergic effect of HNTs and TAX. In addition, Alizarin Red S staining and alkaline phosphatase measurements indicated that the existence of HNTs-TAX promoted osteogenic differentiation. Eventually, animal studies on the femoral defects indicated improved remedy when using the MC/PG-1/HNTs-TAX hydrogel carrying MG-63 cells.

3D-printed scaffold with halloysite nanotubes laden as a sequential drug delivery system regulates vascularized bone tissue healing

Herein, we report the physicochemical, thermal, mechanical and biological characteristics, including bioactivity, biodegradation and cytocompatibility of additive manufacturing-enabled novel nanocomposite scaffolds. The scaffolds comprise a blend of polylactic acid (PLA) and poly-ε-caprolactone (PCL) reinforced with halloysite nanotubes (HNTs). The nanoengineered filaments were developed by melt blending, and the nanocomposite scaffolds were manufactured by fused filament fabrication. Uniform dispersion of HNTs in the PLA/PCL blend is revealed via scanning electron microscopy. Mechanical property loss due to the addition of PCL to realize a suitable biodegradation rate of PLA was fully recovered by the addition of HNTs. Bioactivity, as revealed by the fraction of apatite growth quantified from XRD analysis, was 5.4, 6.3, 6.8 and 7.1% for PLA, 3, 5 and 7 wt% HNT in PLA/PCL blend, respectively, evidencing enhancement in the bioactivity. The degradation rate, in terms of weight loss, was reduced from 4.6% (PLA) to 1.3% (PLA/PCL) upon addition of PCL, which gradually increased to 4.4% by the addition of HNTs (at 7 wt% HNT). The results suggest that the biodegradation rate, mechanical properties and biological characteristics, including cytocompatibility and cell adhesion, of the 3D printed, microarchitected PLA/PCL/HNT composite scaffolds can be tuned by an appropriate combination of HNT and PCL content in the PLA matrix, demonstrating their promise for bone replacement and regeneration applications.Graphical abstract

Halloysite-alkaline phosphatase system-A potential bioactive component of scaffold for bone tissue engineering.

Effect of SrR delivery in the biomarkers of bone regeneration during the in vitro degradation of HNT/GN coatings prepared by EPD

Exploring the effect of one-dimensional halloysite based bionanocomposite hydrogel for bone regeneration

A bone defect refers to the loss of bone tissue caused by trauma or lesion. Bone defects result in high morbidity and deformity rates worldwide. Autologous bone grafting has been widely applied in clinics as the gold standard of treatment; however, it has limitations. Hence, bone tissue engineering has been proposed and developed as a novel therapeutic strategy for treating bone defects. Rapid and effective vascularization is essential for bone regeneration. In this study, a hierarchical composite scaffold with deferoxamine (DFO) delivery system, DFO@GMs-pDA/PCL-HNTs (DGPN), is developed, focusing on vascularized bone regeneration. The hierarchical structure of DGPN imitates the microstructure of natural bone and interacts with the local extracellular matrix, facilitating cell adhesion and proliferation. The addition of 1 wt% of halloysite nanotubes (HNTs) improves the material properties. Hydrophilic and functional groups conferred by polydopamine (pDA) modifications strengthen the scaffold bioactivity. Gelatin microspheres (GMs) protect the pharmacological activity of DFO, achieving local application and sustained release for 7 days. DFO effectively promotes angiogenesis by activating the signaling pathway of hypoxia inducible factor-1 α. In addition, DFO synergizes with HNTs to promote osteogenic differentiation and matrix mineralization. These results indicate that DGPN promotes bone regeneration and accelerates cranial defect healing.

The current therapeutic strategies for healing bone defects commonly suffer from the occurrence of bacterial contamination on the graft, resulting in nonunion in the segmental bone defects and the requirement for secondary surgery to remove or sterilize the primary graft. A membrane with enhanced anti-bacterial efficacy, mechanical strength and osteoconductivity would represent an improvement in the therapeutic strategy for guided bone regeneration. The present study aims to optimize the content of halloysite nanotubes (HNTs) and TiO2 in the polymer matrix of chitosan (CTS) with a constant amount of nano-hydroxyapatite (5%) with the objective of mimicking the mechanical and biological microenvironment of the natural bone extracellular matrix with enhanced anti-bacterial efficacy. HNTs are a low-cost alternative to MWNCTs for enhancing the mechanical properties and anti-bacterial efficacy of the composite. From the first stage of the study, it was concluded that the membranes possessed enhanced mechanical properties and optimum biological properties at 7.5% (w/w) loading of HNTs in the composite. In the second stage of this investigation, we studied the effect of the addition of TiO2 nanoparticles (NPs) and TiO2 nanotubes (NTs) in small amounts to the CTS/n-HAP/HNT nanocomposite at 7.5% HNT loading, with an aim to augment the anti-bacterial efficacy and osteoconductivity of this mechanically strong membrane. The study revealed a significant enhancement in the anti-bacterial efficacy, osteoblast-like MG-63 cell proliferation and ALP expression with the addition of TiO2 NTs. The CHH-TiT membrane successfully inhibited the S. aureus and E. coli growth within 16 hours and simultaneously assisted the enhanced proliferation of osteoblast-like cells on its surface. The study supports the potential exploitation of CHH-TiT (7.5% HNT & 0.2% TiO2 NT) membranes as a template for guided bone tissue regeneration.

Among several ions playing a vital role in the body, Sr2+ and Mg2+ are involved in the mechanism of bone formation, making them especially useful for bone tissue engineering applications. Recently, polylactic acid (PLA)/Mg composites have emerged as a promising family of biomaterials due to their inherent biocompatibility and biodegradability properties. In these composites, polymer and bio-metal have a synergetic effect—while the PLA inhibits the Mg fast reactivity, Mg provides bioactivity to the inert polymer buffering the medium pH during degradation. Meanwhile, the typical form of administrating Sr2+ to patients is through the medication strontium ranelate (SrR), which increases the bone mineral density. Following this interesting research line, a new group of composites, which integrates Mg particles and SrR charged onto halloysite nanotubes (HNT) in a polymeric matrix, was proposed. PLA/Mg/SrR–HNT composites have been processed following a colloidal route, obtaining homogenous composites granulated and film-shaped. The drug delivery profile was evaluated in terms of in vitro lixiviation/dissolution paying special attention to the synergism of both ions release. The combination of two of the most reported ions involved in bone regeneration in the composite biomaterial may generate extra interest in bone healing applications.

Natural halloysite nanotubes (HNTs) show unique hollow structure, high aspect ratio and adsorption ability, good biocompatibility, and low toxicity, which allow for various biomedical applications in the diagnosis and treatment of diseases. Here, advances in self-assembly of halloysite for cell capturing and bacterial proliferation, coating on biological surfaces and related drug delivery, bone regeneration, bioscaffolds, and cell labeling are summarized. The in vivo toxicity of these clay nanotubes is discussed. Halloysite allows for 10-20% drug loading and can extend the delivery time to 10-100h. These drug-loaded nanotubes are doped into the polymer scaffolds to release the loaded drugs. The rough surfaces fabricated by self-assembly of the clay nanotubes enhance the interactions with tumor cells, and the cell capture efficacy is significantly improved. Since halloysite has no toxicity toward microorganisms, the bacteria composed within these nanotubes can be explored in oil/water emulsion for petroleum spilling bioremediation. Coating of living cells with halloysite can control the cell growth and is not harmful to their viability. Quantum dots immobilized on halloysite were employed for cell labeling and imaging. The concluding academic results combined with the abundant availability of these natural nanotubes promise halloysite applications in personal healthcare and environmental remediation.

Desirable bone engineering materials should have a conducive three-dimensional (3D) structure and bioactive mediators for guided bone regeneration. In the present study, hydroxyapatite (HA)/collagen (Col) scaffolds were prepared by an optimized freeze-drying process. The porosity, moisture content, and mechanical properties of the composite have been investigated. The micro-morphology and structure were analyzed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM), confirmed that self-cross-linked HA/Col was evenly distributed and formed a 3D porous scaffold. The physicochemical/mechanical characterization was carried out by Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). Morphological observation and CCK-8 assay of co-culture cells indicated that HA/Col scaffolds were biocompatible. Then HA/Col scaffolds coupled with recombinant human bone morphogenetic proteins 2 (rhBMP-2) were implanted in the mandibular critical size defect in rats, and histological staining was used to evaluate the bone reconstruction. The result showed that HA/Col coupled with rhBMP-2 could significantly improve the formation of new bone and angiogenesis within the scaffolds as well as the proliferation and differentiation of osteoblasts. Thanks to the encouraging osteogenesis effects, the well-defined 3D scaffolds (HA/Col) cooperating with bioactive agents (rhBMP-2) are expected to be a promising candidate for bone tissue engineering applied to regenerative medicine.

Improvement of mechanical and biological properties of Polycaprolactone loaded with Hydroxyapatite and Halloysite nanotubes