Calcium phosphate-chitosan composite 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

Human bone cells used for in vitro studies are mainly derived from bone marrow (BM) or trabecular bone (TB). There are no specific markers or procedures for isolation and growth of these cells. To validate the potentiality of these cells, we isolated human mesenchymal stromal cells (MSCs) and osteoblasts (OBs) from the tibial plateau of the same subject, grown in two different media (α-MEM and DMEM/F12) and analyzed for cell growth, proliferation, phenotype and osteogenic potential. We found that OBs grew well in both media tested, but MSCs were able to grow only in α-MEM medium. OBs in DMEM/F12 showed reduced proliferation capability and expressed a low level of alkaline phosphatase (AP), RUNX-2, osteocalcin (OC), bone sialoprotein (BSP), collagen type I (Col.I) compared with OBs in α-MEM but high level of collagen type XV (Col.XV). Compared with MSCs in α-MEM, OBs have an increased ability to proliferate and express more OC and BSP at molecular level but less AP, RUNX-2 and Col.I than MSCs. Time-course experiments to analyze the osteogenic potential of these cells showed that OBs were more efficient than MSCs. However, these cells obtained from tibial plateau showed a different trend of AP, OC and Col.I osteogenic markers compared to control MSCs from the iliac crest. This study shows that bone-adherent OBs grown in α-MEM medium are more efficient for osteogenic differentiation than BM MSCs and contribute to defining their phenotypic and functional characteristics, so providing a rationale for their use in bone tissue engineering or therapeutic purposes.

The application of a three-dimensional (3D)-printed biological functional scaffold in the repair of bone defects is a promising strategy. In this study, strontium-containing hydroxyapatite (SrHA) powder was synthesized by the hydrothermal method, and then poly(ε-caprolactone) (PCL)/HA and PCL/SrHA composite scaffolds were prepared by the high-temperature melt extrusion 3D printing technology. The basic physical and chemical properties, in vitro biological properties, osteogenesis, and angiogenesis abilities of the scaffold were studied. The results showed that HA and SrHA were uniformly embedded in the composite scaffold, and the scaffold exhibited a 3D interconnected porous structure and rough microsurface. The in vitro release curve showed that Sr2+ and Ca2+ were continuously released from the PCL/SrHA scaffold. In order to verify the performance of the composite scaffold in bone regeneration, the proliferation and osteogenic differentiation of mouse embryonic osteoblasts (MC3T3E1) grown on the scaffold were evaluated. The experimental results showed that the incorporation of SrHA significantly promoted cell proliferation. Compared with the PCL/HA scaffold, the PCL/SrHA scaffold could better promote cell osteogenic differentiation. Deferoxamine (DFO) was loaded on the surface of the PCL/SrHA scaffold. By studying the proliferation, angiogenesis, and expression of osteogenesis and angiogenesis-related genes of human umbilical vein endothelial cells (HUVECs) on PCL/SrHA@DFO scaffold, it was verified that DFO had the ability to promote angiogenesis. It could induce angiogenesis in vitro in combination with Sr2+. Therefore, we believe that the composite scaffold has potential application prospects in the field of bone tissue engineering.

Mineralized polymer scaffolds have proven to be effective biomaterials for inducing osteoinductivity in bone tissue engineering. Sequential mineralization is a promising technique for depositing minerals in three-dimensional (3D) scaffolds. Paper, which is made of cellulose fibers, can be used as a tissue scaffold due to its highly porous structure and flexibility, as well as its excellent ability to wick fluids and support the growth of bone cells. In this study, paper-based, mineralized scaffolds were fabricated using sequential mineralization. We conducted experiments with two groups of scaffolds based on different incubation times in the mineralization solutions (30 min and 24 h). Ten cycles of mineralization were performed for each group. We found that the mineral content increased as the cycle number increased and that the 24-h group scaffolds consistently had more mineralization than did the 30-min group scaffolds when measured at the same cycle number. A quantitative reverse transcription-polymerase chain reaction was performed for two osteogenic differentiation markers of the preosteoblasts that were grown on the mineralized paper scaffolds. The gene expression results for bone-specific markers revealed that the mineralized scaffolds were osteoinductive. Subcutaneous implantation of the scaffolds in rats demonstrated favorable biocompatibility, high vascularization, and non-immunogenicity in vivo. The overall results suggest that the sequentially mineralized paper scaffolds are promising materials for use in bone tissue engineering.

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.

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.

Composite 3D scaffolds combining natural polymers and bioceramics are promising candidates for bone tissue engineering (BTE). Zein, as a natural plant protein, offers several advantages, including biocompatibility, adequate strength properties, and low/no immunogenicity; however, it lacks bioactivity. Thus, composite zein: bioactive glass (BG) scaffolds are proposed as promising candidate for BTE applications, with silver-doping of bioactive glass providing an antibacterial effect against possible post-implantation infection. Therefore, the aim of this study was to investigate the in vitro antibacterial properties, biocompatibility, bioactivity and compressive strength of zein scaffolds containing silver-doped bioactive glass. BG nanoparticles, undoped and Ag-doped, were fabricated using the sol-gel method. 3D composite zein:BG scaffolds, containing 20 wt% BG, were prepared and their antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was assessed using the disc diffusion assay. Human osteoblast-like MG-63 cells were used to evaluate the in vitro biocompatibility of the prepared scaffold groups. In addition, the compressive strength of the scaffolds was determined using uniaxial compression strength testing and the scaffold interconnected porosity was measured using helium pycnometer. Disc diffusion assay showed that only zein scaffolds containing Ag-doped sol-gel BG are antibacterially positive against E. coli and S. aureus. Pure zein scaffolds and zein scaffolds containing sol-gel-derived BG showed no negative influence on the growth of MG-63 cells, as evident by the cells’ ability to survive, proliferate, and function on these scaffolds. Moreover, incorporating sol-gel-derived BG into zein scaffolds at zein:BG of 80:20 ratio showed bioactive properties with adequate porosity without affecting the scaffolds’ compressive strengths, which was similar to that of trabecular bone, suggesting that the new composites have potential for BTE applications in non-loaded bearing areas.

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.

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.

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.

The aim of this work was to synthesize non-toxic, biodegradable polyesters of aconitic acid, cinnamic acid and glycerol from by-products of the sugarcane industry as scaffolds for skin and bone tissue engineering. Utilizing the by-products, molasses and sugarcane bagasse, not only add value to the cane industry, but also paves path for synthesizing novel bio-based materials from the isolated specialty chemicals. Molasses contain an economically recoverable quantity of aconitic acid and its extraction was studied in detail as a part of this work. The yields of recovered aconitic acid varied from 25–69% depending on the extraction conditions. Under all the conditions, the purity values of extracted aconitic acid were higher than 99%. In the next step of the research, polyesters of aconitic acid, glycerol and cinnamic acid were synthesized. Different compositions of polyesters were characterized for their mechanical properties, porosity, mass loss in stromal medium, ability to support growth and proliferation of human adipose derived mesenchymal stem cells (hASC). Several biocompatibility tests such as mass loss over a period of time, alamar blue to analyze growth and viability of hASC on polyester scaffolds, picogreen for total DNA content synthesized indicated that these polyesters hold promising potential as tissue engineering scaffolds. The final step of this dissertation involved evaluating these polyesters as skin and bone tissue engineering scaffolds. For skin tissue engineering, especially wound repair, thin film polyester scaffolds laden with hASC were grown in stromal medium supplemented with basic fibroblast growth factor (bFGF). Based on the amount of collagen synthesized and DNA quantification data it was concluded that the polyesters can be used as scaffolds for wound repair by the addition of bFGF. The last step dealt with utilizing these as bone scaffolds where hASC were induced to undergo osteogenesis, and analyzed for mineralization and osteogenic target gene expression over a 21 days period. Based on calcium deposition results, and alkaline phosphatase (ALP) and osteocalcin (OCN) expression data, it was concluded that these scaffolds hold great potential for bone tissue engineering.

The synergetic effect of bioactive ceramic and nanoclay on the properties of chitosan–gelatin/nanohydroxyapatite–montmorillonite scaffold for bone tissue engineering

Preparation and characterization of nano-sized hydroxyapatite/alginate/chitosan composite scaffolds for bone tissue engineering.

Recent advances in three-dimensional printing technology enable facile and on-demand fabrication of patient-specific bone scaffolds. However, there is still an urgent need for printable biomaterials with osteoinductivity. In the present study, we propose an approach to synthesize fibroblast growth factor-2 loaded-mesoporous calcium silicate nanoparticles. The growth factor loaded-nanoparticles served as fillers of polycaprolactone and then the composite scaffolds with a controlled pore structure were obtained through a fused deposition modeling technique. To evaluate the feasibility of the composite scaffolds in bone tissue engineering, drug release kinetic, bioactivity, cell proliferation, differentiation, and animal study were conducted. Our findings illustrate that utilization of mesoporous calcium silicate allowed the introduction of fibroblast growth factor-2 into the composite scaffolds through a simple soaking process and then gradually released from the scaffold to facilitate proliferation and osteogenesis differentiation of human Wharton’s jelly mesenchymal stem cells. Additionally, the in vivo femur defect experiments also indicate that the co-existence of calcium silicate and fibrous growth factor-2 synergistically accelerated new bone formation. These results demonstrate that the fibroblast growth factor-2-loaded mesoporous calcium silicate nanoparticles/polycaprolactone composite scaffolds may serve as potential bone grafts for facilitating repair of defected bone tissues.

Development of genipin-crosslinked and fucoidan-adsorbed nano-hydroxyapatite/hydroxypropyl chitosan composite scaffolds for bone tissue engineering