Mineralized paper scaffolds for bone tissue engineering.

The effects of collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering

Tissue engineering (TE) is envisaged to play a vital role in improving quality of life by restoring, maintaining or enhancing tissue and organ functions. TE scaffolds that are two-dimensional in structure suffer from undesirable issues, such as pore blockage, and do not closely mimic the native extra-cellular matrix in tissues. Significant efforts have therefore been channeled to fabricate three-dimensional (3D) scaffolds using various techniques, especially electrospinning. In this study, we propose a modified one-step electrospinning process to arrive at a 3D scaffold with highly interconnected pores. Using a blend of poly (L-lactide)/polycaprolactone/poly (ethylene oxide), this mechanically viable, sponge-like 3D scaffold exhibited sufficiently large pores and enabled cell penetration beyond 500 μm. Dexamethasone (Dex) was loaded into the fibers and a sustained drug release was achieved. Further, the potential of this Dex-loaded 3D scaffold was evaluated for upregulation of osteogenic genes with mesenchymal stem cells. The as-produced Dex-loaded 3D scaffold possesses a unique intertwined sub-micron fibrous morphology that can be tailored for use in bone tissue engineering and beyond.

Heat-treated polyacrylonitrile (HT-PAN), also referred to as black orlon (BO), is a promising carbon-based material used for applications in tissue engineering and regenerative medicine. To the best of our knowledge, no such complex bone morphology-mimicking three-dimensional (3D) BO structure has been reported to date. We report that BO can be easily made into 3D cryogel scaffolds with porous structures, using succinonitrile as a porogen. The cryogels possess a porous morphology, similar to bone tissue. The prepared scaffolds showed strong osteoconductive activity, providing excellent support for the adhesion, proliferation, and mitochondrial activity of human bone-derived cells. This effect was more apparent in scaffolds prepared from a matrix with a higher content of PAN (i.e., 10% rather than 5%). The scaffolds with 10% of PAN also showed enhanced mechanical properties, as revealed by higher compressive modulus and higher compressive strength. Therefore, these scaffolds have a robust potential for use in bone tissue engineering.

In the past 20 years, mesoporous materials have been attracted great attention due to their significant feature of large surface area, ordered mesoporous structure, tunable pore size and volume, and well-defined surface property. They have many potential applications, such as catalysis, adsorption/separation, biomedicine, etc. [1]. Recently, the studies of the applications of mesoporous materials have been expanded into the field of biomaterials science. A new class of bioactive glass, referred to as mesoporous bioactive glass (MBG), was first developed in 2004. This material has a highly ordered mesopore channel structure with a pore size ranging from 5–20 nm [1]. Compared to non-mesopore bioactive glass (BG), MBG possesses a more optimal surface area, pore volume and improved in vitro apatite mineralization in simulated body fluids [1,2]. Vallet-Regi et al. has systematically investigated the in vitro apatite formation of different types of mesoporous materials, and they demonstrated that an apatite-like layer can be formed on the surfaces of Mobil Composition of Matters (MCM)-48, hexagonal mesoporous silica (SBA-15), phosphorous-doped MCM-41, bioglass-containing MCM-41 and ordered mesoporous MBG, allowing their use in biomedical engineering for tissue regeneration [2-4]. Chang et al. has found that MBG particles can be used for a bioactive drug-delivery system [5,6]. Our study has shown that MBG powders, when incorporated into a poly (lactide-co-glycolide) (PLGA) film, significantly enhance the apatite-mineralization ability and cell response of PLGA films. compared to BG [7]. These studies suggest that MBG is a very promising bioactive material with respect to bone regeneration. It is known that for bone defect repair, tissue engineering represents an optional method by creating three-dimensional (3D) porous scaffolds which will have more advantages than powders or granules as 3D scaffolds will provide an interconnected macroporous network to allow cell migration, nutrient delivery, bone ingrowth, and eventually vascularization [8]. For this reason, we try to apply MBG for bone tissue engineering by developing MBG scaffolds. However, one of the main disadvantages of MBG scaffolds is their low mechanical strength and high brittleness; the other issue is that they have very quick degradation, which leads to an unstable surface for bone cell growth limiting their applications. Silk fibroin, as a new family of native biomaterials, has been widely studied for bone and cartilage repair applications in the form of pure silk or its composite scaffolds [9-14]. Compared to traditional synthetic polymer materials, such as PLGA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the chief advantage of silk fibroin is its water-soluble nature, which eliminates the need for organic solvents, that tend to be highly cytotoxic in the process of scaffold preparation [15]. Other advantages of silk scaffolds are their excellent mechanical properties, controllable biodegradability and cytocompatibility [15-17]. However, for the purposes of bone tissue engineering, the osteoconductivity of pure silk scaffolds is suboptimal. It is expected that combining MBG with silk to produce MBG/silk composite scaffolds would greatly improve their physiochemical and osteogenic properties for bone tissue engineering application. Therefore, in this chapter, we will introduce the research development of MBG/silk scaffolds for bone tissue engineering.

Bioengineering scaffolds have been improved to achieve efficient regeneration of various damaged tissues. In this study, we attempted to fabricate mechanically and biologically activated 3D printed scaffold in which porous gelatin/hydroxyapatite (G/H) as a matrix material provided outstanding mechanical properties with recoverable behavior, and human placental extracts (hPE) embedded in the scaffold were used as bioactive components.Methods: Various cell types (human adipose-derived stem cells; hASCs, pre-osteoblast; MC3T3-E1, human endothelial cell line; EA.hy926, and human dermal fibroblast; hDFs) were used to assess the effect of the hPE on cellular responses. High weight fraction (~ 70 wt%) of hydroxyapatite (HA) in a gelatin solution supplemented with glycerol was used for the G/H scaffold fabrication, and the scaffolds were immersed in hPE for the embedding (G/H/hPE scaffold). The osteogenic abilities of the scaffolds were investigated in cultured cells (hASCs) assaying for ALP activity and expression of osteogenic genes. For the in vivo test, the G/H and G/H/hPE scaffolds were implanted in the rat mastoid obliteration model.Results: The G/H/hPE scaffold presented unique elastic recoverable properties, which are important for efficient usage of implantable scaffolds. The effects of G/H and G/H/hPE scaffold on various in vitro cell-activities including non-toxicity, biocompatibility, and cell proliferation were investigated. The in vitro results indicated that proliferation (G/H = 351.1 ± 13.3%, G/H/hPE = 430.9 ± 8.7% at day 14) and expression of osteogenic markers (ALP: 3.4-fold, Runx2: 3.9-fold, BMP2: 1.7-fold, OPN: 2.4-fold, and OCN: 4.8-fold at day 21) of hASCs grown in the G/H/hPE scaffold were significantly enhanced compared with that in cells grown in the G/H scaffold. In addition, bone formation was also observed in an in vivo model using rat mastoid obliteration.Conclusion: In vitro and in vivo results suggested that the G/H/hPE scaffold is a potential candidate for use in bone tissue engineering.

Electrospinning is a promising and versatile technique that is used to fabricate polymeric nanofibrous scaffolds for bone tissue engineering. Ideal scaffolds should be biocompatible and bioactive with appropriate surface chemistry, good mechanical properties and should mimic the natural extracellular matrix (ECM) of bone. Selection of the most appropriate material to produce a scaffold is an important step towards the construction of a tissue engineered product. Bone tissue engineering is an interdisciplinary field, where the principles of engineering are applied on bone-related biochemical reactions. Scaffolds, cells, growth factors, and their interrelation in microenvironment are the major concerns in bone tissue engineering. This review covers the latest development of biomimetic electrospun polymeric biomaterials for bone tissue engineering. It includes the brief details to bone tissue engineering along with bone structure and ideal bone scaffolds requirements. Details about various engineered materials and methodologies used for bone scaffolds development were discussed. Description of electrospinning technique and its parameters relating their fabrication, advantages, and applications in bone tissue engineering were also presented. The use of synthetic and natural polymers based electrospun nanofibrous scaffolds for bone tissue engineering and their biomineralization processes were discussed and reviewed comprehensively. Finally, we give conclusion along with perspectives and challenges of biomimetic scaffolds for bone tissue engineering based on electrospun nanofibers.

Currently available bone grafts present certain limitations such as donor-site morbidity for autografts and risk of disease transmission for allografts. These limitations suggest a need for alternative strategies. The use of polymer/ceramic composites as scaffolds for trabecular bone tissue engineering capitalizes on the benefits of both materials. Polymers are easily formed and shaped and add structural rigidity while calcium phosphates impart osteoconductivity and osteointegration. We have developed a biodegradable, microsphere-based scaffold for bone tissue engineering based on poly(lactide-co-glycolide)/calcium phosphate composite microspheres in which a nanocrystalline hydroxyapatite is synthesized within the forming microspheres.

Macroporous calcium phosphate-chitosan composite scaffolds were fabricated and evaluated for use in bone tissue engineering. Human osteoblast-like MG63 cells were cultured on the composite scaffolds, and their response to the materials was studied. Cell morphology, total protein content, and expression of classic markers for osteoblast differentiation were characterized. MG63 cells on the hydroxyapatite scaffolds nesting chitosan sponges (HC1) showed significantly higher alkaline phosphatase (ALP) level and osteocalcin (OC) production during the 11-day culture period, compared with the control culture on tissue culture plates. Cells on the chitosan scaffolds incorporated with hydroxyapatite powders (HC2) exhibited lower ALP activity during the 11-day culture period and OC secretion during the first 7 days, in comparison with that on HC1. The addition of calcium phosphate glass as in HC3 scaffolds increased the ALP and OC levels of MG63 cells. Our study indicated that the hydroxyapatite-matrix composite scaffolds might enhance the phenotype expression of MG63 cells, in comparison with chitosan-matrix scaffolds. Soluble calcium phosphate glasses should be added to the scaffolds to prevent chitosan from fast degradation that may affect the differentiation of osteoblast cells.

Fabrication of a three-dimensional (3D) scaffold with increased mechanical strength may be an essential requirement for more advanced bone tissue engineering scaffolds. Various material- and chemical-based approaches have been explored to enhance the mechanical properties of engineered bone tissue scaffolds. In this study, the effects of pore architecture and stacking direction on the mechanical and cell proliferation properties of a scaffold were investigated. The 3D scaffold was prepared using solid freeform fabrication technology with a multihead deposition system. Various types of scaffolds with different pore architectures (lattice, stagger, and triangle types) and stacking directions (horizontal and vertical directions) were fabricated with a blend of polycaprolactone and poly lactic-co-glycolic acid. In compression tests, the triangle-type scaffold was the strongest among the experimental groups. Stacking direction affected the mechanical properties of scaffolds. An in vitro cell counting kit-8 assay showed no significant differences in optical density depending on the different pore architectures and stacking directions. In conclusion, mechanical properties of scaffolds can be enhanced by controlling pore architecture and stacking direction.

The potential of melt-electrowriting (MEW) to fabricate highly porous 3D composite scaffolds for bone tissue engineering (BTE) with antibacterial properties was explored. Zinc oxide (ZnO) nanoparticles (NPs) were loaded into biodegradable medical grade Polycaprolactone (PCL) loaded with 10 wt% hydroxyapatite (HA) NPs to create antibacterial constructs by the MEW technique. The constructs’ morphology, chemical, thermal and mechanical properties, osteoblasts cell viability and bacterial attachment were investigated. Our results demonstrate a unique homogeneous dispersion of ZnO-HA NPs in the outer shell of the fabricated composite fibres by MEW, leading to significant inhibition of bacterial attachment against microorganisms while retaining osteoiblast viability. This novel composite MEW 3D scaffolds yielded promising multifunctional constructs with high potential in BTE.

Decellularized periosteum as a potential biologic scaffold for bone tissue engineering

To enhance the understanding and usefulness of decellularised plant tissues as scaffolds for bone tissue engineering, and to review the recent advances in plant-based scaffolds for bone regeneration. The systematic review was conducted from February to June 2022, and comprised literature search on PubMed and Science Direct databases for articles published in the English language between 2013 and June, 2022. The search was conducted using key words 'plant-based scaffolds and osteogenesis' and 'decellularised plant and scaffolds and bone tissue engineering' and 'plant root-based scaffold and bone formation'. Full-text articles and short communication covering both in vitro and in vivo studies focussing on plant-based scaffolds for osteogenesis were included. Additional references that satisfied the inclusion criteria were found on Google Scholar and included in the review. Quality evaluation of the studies included was done independently by two researchers, and the risk of bias was calculated and categorised as low, medium and high risk. Of the 564 articles, 10(1.77 %) were included; 6(60%) purely in vitro studies, and 4(40%) studies having both in vitro and in vivo components. Decellularized plant tissue alone was used as three-dimensional scaffold materials in 5(50%) studies, but in 4(40%) studies, plant-based composite scaffolds were used. The risk of bias was medium in 7(70%) studies. Plant-based scaffolds promote osteogenesis, but more in vivo research on plant-based studies is needed to label it as suitable scaffolds for bone tissue engineering.

Electrospinning technology has been widely used in the past few decades to prepare nanofibrous scaffolds that mimic extracellular matrices. However, traditional two-dimensional (2D) electrospun nanofibrous mats still have some inherent disadvantages for bone tissue engineering, such as limited cell infiltration and lack of three-dimensional (3D) structure. The development of 3D electrospun scaffolds with larger pore sizes and porosity provides new perspectives for electrospinning-based tissue engineering scaffolds. However, there are still some challenges and areas for improvement. In this review, the applications of 3D electrospun nanofibrous scaffolds in the field of bone tissue engineering from its fabrication methods to bio-functionalization are summarized, with the aim of providing new insights into the design of electrospinning-based bone tissue engineering scaffolds.

This paper provides an extensive overview of published studies on the development and applications of three-dimensional bone tissue engineering (TE) scaffolds with potential capability for the controlled delivery of therapeutic drugs. Typical drugs considered include gentamicin and other antibiotics generally used to combat osteomyelitis, as well as anti-inflammatory drugs and bisphosphonates, but delivery of growth factors is not covered in this review. In each case reviewed, special attention has been given to the technology used for controlling the release of the loaded drugs. The possibility of designing multifunctional three-dimensional bone TE scaffolds for the emerging field of bone TE therapeutics is discussed. A detailed summary of drugs included in three-dimensional scaffolds and the several approaches developed to combine bioceramics with various polymeric biomaterials in composites for drug-delivery systems is included. The main results presented in the literature are discussed and the remaining challenges in the field are summarized with suggestions for future research directions.

Recently tremendous progress has been evidenced by the advancements in developing innovative three-dimensional (3D) scaffolds using various techniques for addressing the autogenous grafting of bone. In this work, we demonstrated the fabrication of porous polycaprolactone (PCL) scaffolds for osteogenic differentiation based on supercritical fluid-assisted hybrid processes of phase inversion and foaming. This eco-friendly process resulted in the highly porous biomimetic scaffolds with open and interconnected architectures. Initially, a 23 factorial experiment was designed for investigating the relative significance of various processing parameters and achieving better control over the porosity as well as the compressive mechanical properties of the scaffold. Then, single factor experiment was carried out to understand the effects of various processing parameters on the morphology of scaffolds. On the other hand, we encapsulated a growth factor, i.e., bone morphogenic protein-2 (BMP-2), as a model protein in these porous scaffolds for evaluating their osteogenic differentiation. In vitro investigations of growth factor loaded PCL scaffolds using bone marrow stromal cells (BMSCs) have shown that these growth factor-encumbered scaffolds were capable of differentiating the cells over the control experiments. Furthermore, the osteogenic differentiation was confirmed by measuring the cell proliferation, and alkaline phosphatase (ALP) activity, which were significantly higher demonstrating the active bone growth. Together, these results have suggested that the fabrication of growth factor-loaded porous scaffolds prepared by the eco-friendly hybrid processing efficiently promoted the osteogenic differentiation and may have a significant potential in bone tissue engineering.