3D mesoporous bioactive glass/silk/chitosan scaffolds and their compatibility with human adipose-derived stromal cells

Human adipose-derived stem/stromal cells (hASCs) have been popularly studied as cell-based therapy in the field of regenerative medicine due to their ability to differentiate into several cell types. In this study, in order to improve the mechanical strength and bioactivity of scaffolds for bone tissue engineering, three types of mesoporous bioactive glasses with different shapes and compositions were dispersed in the silk fibroin/chitosan (SF/CS)-based scaffolds, which were fabricated with a combination of freezing and lyophilization. The characteristic and physical properties of these composite scaffolds were evaluated. The biocompatibility was also assessed through hASCs in vitro tests. Both Alamar Blue® and Live/Dead assay® revealed that the spherical mesoporous bioactive glass doped scaffolds enhanced cell viability and proliferation. Furthermore, the addition of spherical mesoporous bioactive glass into SF/CS scaffolds encouraged hASC osteogenic differentiation as well. These results suggested that this composite scaffold can be applicable material for bone regeneration.     

Metformin-Incorporated Gelatin/Nano-Hydroxyapatite Scaffolds Promotes Bone Regeneration in Critical Size Rat Alveolar Bone Defect Model

In this study, we fabricated gelatin/nano-hydroxyapatite/metformin scaffold (GHMS) and compared its effectiveness in bone regeneration with extraction-only, Sinbone, and Bio-Oss Collagen^® groups in a critical size rat alveolar bone defect model. GHMS was synthesized by co-precipitating calcium hydroxide and orthophosphoric acid within gelatin solution, incorporating metformin, and cross-linked by microbial transglutaminase. The morphology, characterization, and biocompatibility of scaffold were examined. The in vitro effects of GHMS on osteogenic gene and protein expressions were evaluated. In vivo bone formation was assessed in a critical size rat alveolar bone defect model with micro-computed tomography and histological examination by comparing GHMS with extraction-only, Sinbone, and Bio-Oss Collagen^®. The synthesized GHMS had a highly interconnected porous structure with a mean pore size of 81.85 ± 13.8 µm. GHMS exhibited good biocompatibility; promoted ALPL, RUNX2, SP7, BGLAP, SPARC and Col1a1 gene expressions; and upregulated the synthesis of osteogenic proteins, including osteonectin, osteocalcin, and collagen type I. In critical size rat alveolar bone defects, GHMS showed superior bone regeneration compared to extraction-only, Sinbone, and Bio-Oss Collagen^® groups as manifested by greater alveolar ridge preservation, while more bone formation with a lower percentage of connective tissue and residual scaffold at the defect sites grafted with GHMS in histological staining. The GHMS presented in this study may be used as a potential bone substitute to regenerate alveolar bone. The good biocompatibility, relatively fast degradation, interconnected pores allowing vascularization, and higher bioactivity properties of the components of the GHMS (gelatin, nHA, and metformin) may contribute to direct osteogenesis.     

Modified calcium magnesium phosphate bone cement with improved microenvironment

Calcium magnesium phosphate bone cement (MCPC) has been widely used in bone defects restoration and attracted much attention due to excellent mechanical properties and biodegradability. However, excessive MgO tends to cause a local alkaline microenvironment which is adverse for cell growth and differentiation. In this work, we constructed the MCPC composites with improved microenvironment, enhanced osteogenic differentiation and biomineralization by introducing various concentrations of gelatin solutions. Gelatin played important roles in the improvement of physicochemical property, biodegradability, biocompatibility, osteogenic differentiation activity and biomineralization of MCPC. When incorporated with 1% and 5% of gelatin, the MCPC composites exhibited higher compressive strength and osteogenic differentiation ability of BMSCs in vitro. In conclusion, the modified MCPC composite is a potential candidate for bone defects repair and regenerate.     

Bone Regeneration in rat using a gelatin/bioactive glass nanocomposite scaffold along with endothelial cells (HUVECs)

In our previous study, a three‐dimensional gelatin/bioactive glass nanocomposite scaffold with a total porosity of about 85% and pore sizes ranging from 200 to 500 μm was prepared through layer solvent casting combined with lamination technique. The aim of this study was to evaluate in vitro biocompatibility and in vivo bone regeneration potential of these scaffolds with and without endothelial cells when implanted into a critical‐sized rat calvarial defect. MTT assay, SEM observation, and DAPI staining were used to evaluate cell viability and adhesion in macroporous scaffolds and results demonstrated that the scaffolds were biocompatible enough to support cell attachment and proliferation. To investigate the in vivo osteogenesis of the scaffold, blank scaffolds and endothelial/scaffold constructs were implanted in critical‐sized defects, whereas in control group defects were left untreated. Bone regeneration and vascularization were evaluated at 1, 4, and 12 weeks postsurgery by histological, immunohistochemical, and histomorphometric analysis. It was shown that both groups facilitated bone growth into the defect area but improved bone regeneration was seen with the incorporation of endothelial cells. The data showed that the porous Gel/BaG nanocomposite scaffolds could well support new bone formation, indicating that the proposed strategy is a promising alternative for tissue‐engineered bone defects.     

Biomimetic mineralization of chitosan/gelatin cryogels and in vivo biocompatibility assessments for bone tissue engineering

The objective of this study is to develop biomimetic chitosan: gelatin (CH:Gel) cryogels for bone tissue engineering, combining the biological recognition of natural polymers with the distinguished interconnected porosity of cryogels, and biomimicking properties of bone like hydroxyapatite. The control of the biomineralization process onto biomaterials should be evaluated before clinical application. Therefore, the effect of chitosan and gelatin ratios on the final properties of the cryogels were investigated. FTIR, XRD, and SEM analysis indicated that the SBF coating exhibited similar characteristics to hydroxyapatite. The cryogels showed good biocompatibility with L929 mouse fibroblasts. Clinical outcomes and gross pathological examination showed that neither necrosis nor foreign body reaction was noted at the end of implantation. The biomimetically mineralized scaffold was found to be non-irritant and non-toxic for bone tissue. The biological performance and favorable properties demonstrated that the SBF coated CH:Gel cryogel can be a promising biomimetic scaffold for bone tissue engineering applications.     

The Osteogenesis Effect and Underlying Mechanisms of Local Delivery of gAPN in Extraction Sockets of Beagle Dogs

A plastic and biodegradable bone substitute consists of poly (l-lactic-co-glycolic) acid and 30 wt % β-tricalcium phosphate has been previously fabricated, but its osteogenic capability required further improvement. We investigated the use of globular adiponectin (gAPN) as an anabolic agent for tissue-engineered bone using this scaffold. A qualitative analysis of the bone regeneration process was carried out using μCT and histological analysis 12 weeks after implantation. CBCT (Cone Beam Computed Tomography) superimposition was used to characterise the effect of the different treatments on bone formation. In this study, we also explored adiponectin’s (APN) influence on primary cultured human jaw bone marrow mesenchymal stem cells gene expressions involved in the osteogenesis. We found that composite scaffolds loaded with gAPN or bone morphogenetic protein 2 (BMP2) exhibited significantly increased bone formation and mineralisation following 12 weeks in the extraction sockets of beagle dogs, as well as enhanced expression of osteogenic markers. In vitro investigation revealed that APN also promoted osteoblast differentiation of primary cultured human jaw bone marrow mesenchymal stem cells (h-JBMMSCs), accompanied by increased activity of alkaline phosphatase, greater mineralisation, and production of the osteoblast-differentiated genes osteocalcin, bone sialoprotein and collagen type I, which was reversed by APPL1 siRNA. Therefore, the composite scaffold loaded with APN exhibited superior activity for guided bone regeneration compared with blank control or Bio-Oss^® (a commercially available product). The composite scaffold with APN has significant potential for clinical applications in bone tissue engineering.     

Bone Tissue Engineering in Rat Calvarial Defects Using Induced Bone-like Tissue by rhBMPs from Immature Muscular Tissues In Vitro

This study aimed to induce bone-like tissue from immature muscular tissue (IMT) in vitro using commercially available recombinant human bone morphogenetic protein (rhBMP)-2, rhBMP-4, and rhBMP-7, and then implanting this tissue into a calvarial defect in rats to assess healing. IMTs were extracted from 20-day-old Sprague-Dawley (SD) fetal rats, placed on expanded polytetrafluoroethylene (ePTFE) with 10 ng/μL each of rhBMP-2, BMP-4, and BMP-7, and cultured for two weeks. The specimens were implanted into calvarial defects in 3-week-old SD rats for up to three weeks. Relatively strong radiopacity was observed on micro-CT two weeks after culture, and bone-like tissue, comprising osteoblastic cells and osteoids, was partially observed by H&E staining. Calcium, phosphorus, and oxygen were detected in the extracellular matrix using an electron probe micro analyzer, and X-ray diffraction patterns and Fourier transform infrared spectroscopy spectra of the specimen were found to have typical apatite crystal peaks and spectra, respectively. Furthermore, partial strong radiopacity and ossification were confirmed one week after implantation, and a dominant novel bone was observed after two weeks in the defect site. Thus, rhBMP-2, BMP-4, and BMP-7 differentiated IMT into bone-like tissue in vitro, and this induced bone-like tissue has ossification potential and promotes the healing of calvarial defects. Our results suggest that IMT is an effective tissue source for bone tissue engineering.     

Application of strontium doped calcium polyphosphate bioceramic as scaffolds for bone tissue engineering

The critical success factors for bone tissue engineering in clinical applications are scaffolds. Ion doping is one of the most important methods to modify the properties of bioceramics for better angiogenesis abilities, biomechanical properties, and biocompatibility. This paper presents a novel ion doping method applied in calcium polyphosphate (CPP)-based bioceramic scaffolds substituted by strontium ions to form (SCPP) scaffolds for bone tissue regeneration. The microstructure and crystallization of the scaffolds were detected by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Degradation tests were assessed to evaluate the mechanical and chemical stabilities of SCPP in vitro. The cell biocompatibility was measured with respect to the cytotoxicity of the extractions of scaffolds. Bone implantation was performed to evaluate the biodegradability and osteoconductivity of the scaffolds, and the bone formation examined by using X-ray radiography. The results indicated that the obtained SCPP scaffolds had a single CPP phase. The SCPP scaffolds yielded a better degradation property than the pure CPP scaffold. The MTT assay and in vivo results reveal that the SCPP scaffolds exhibited a better cell biocompatibility and tissue biocompatibility than CPP and hydroxyapatite (HA) scaffolds. The in vivo immunohistochemistry staining for VEGF also showed that SCPP had a potential to promote the formation of angiogenesis and the regeneration of bone. SCPP scaffold could serve as a potential biomaterial with stimulating angiogenesis in bone tissue engineering and bone repair.     

Application of polymeric nanofibers in medical designs,part III: Musculoskeletal and urological tissues

Nanotechnology has potential applications in different sciences, especially in the biological sciences and medicine. The nanomaterials are applicable materials with different morphologies such as nanoparticles, nanotubes, nanowires, nanorods, and nanofibers. The development of nanofibers has greatly enhanced the scope for fabricating designs that can be potentially used in medical sciences. In part III the author summarizes the currently available applications of nanofibers in musculoskeletal and urologic tissues. The graphical abstract shows computed tomography analysis and macroscopic images of calvarial defects in rat, with the regeneration result, after four weeks, of bone healing upon implantation of scaffolds in the defect. (A and D) Control group; (B and E) poly (3-hydroxybutyrate-co-3-hydroxyvalerate)/nanohydroxyapatite (PHBV/nHAp) scaffold; and (C and F) PHBV/nHAp scaffold with unrestricted somatic stem cells precultured in vitro.     

Development of a Novel Polygalacturonic Acid-Gelatin Blend Scaffold Fabrication and Biocompatibility Studies for Tissue-Engineering Applications

In this study, we report the enhanced osteoblast differentiation of mouse embryonic stem cell (mESC) on a novel polygalacturonic acid (PGA)/gelatin scaffold. The matrices of various PGA/geletin ratios were fabricated, biophysically characterized, and optimized for cell culture applications. Blended 2% PGA/gelatin scaffolds were highly porous and were robust with enhanced mechanical strength. Swelling studies showed they had high swelling capacity (six-fold higher than only gelatin scaffold) along with complete degradation in the presence of phosphate-buffered saline. Cytocompatibility of the matrices was evaluated using mouse fibroblast 3T3-L1 cell line showing normal spreading and proliferation as assessed by scanning electron microscopy (SEM) and MTT assay. Among different blends of PGA/gelatin, 2% PGA/gelatin (2PG) scaffold showed the optimum physical and biological compatibility for use in cell culture and differentiation of mESC, especially for osteoblast differentiation. The scaffold, as reported in this study, presents a promising tool for tissue engineering applications.     

Fe3O4-nanoparticle-functionalized 3D-printed injectable microgel

Magnetic Fe₃O₄ nanoparticles have attached attention in bone tissue engineering because of their superior magnetism and great biocompatibility. However, some disadvantages such as the potential risk of agglomeration impair their applications. Here, we proposed a hybrid magnetic nanocomposite microgel by the integration of Fe₃O₄ nanoparticles and digital lighting processing (DLP) three-dimensional (3D) printing technology. The 3D-printed microgels could be precisely customized by printing the mixture of gelatin methacryloyl (GelMA) solution and polydopamine-coated Fe₃O₄ nanoparticles, in which polydopamine decoration improved the hydrophilicity and distribution of the incorporated Fe₃O₄. The degradable microgels could be injected through a 22-G needle while retaining their original shape after injection. Interestingly, the addition of Fe₃O₄ nanoparticles into GelMA solution displayed improved printing accuracy. Moreover, these magnetic microgels were biocompatible in vitro and in vivo. After induction within osteogenic medium, addition of nanoparticles upregulated the osteogenic gene expression of rat bone mesenchymal stem cells (BMSCs). In a word, this work provides a magnetic microplatform, which shows great potential in bone tissue engineering.     

Evaluation of a Novel Thiol–Norbornene-Functionalized Gelatin Hydrogel for Bioprinting of Mesenchymal Stem Cells

Introduction: Three-dimensional bioprinting can be considered as an advancement of the classical tissue engineering concept. For bioprinting, cells have to be dispersed in hydrogels. Recently, a novel semi-synthetic thiolene hydrogel system based on norbornene-functionalized gelatin (GelNB) and thiolated gelatin (GelS) was described that resulted in the photoclick hydrogel GelNB/GelS. In this study, we evaluated the printability and biocompatibility of this hydrogel system towards adipose-tissue-derived mesenchymal stem cells (ASCs). Methods: GelNB/GelS was synthesized with three different crosslinking densities (low, medium and high), resulting in different mechanical properties with moduli of elasticity between 206 Pa and 1383 Pa. These hydrogels were tested for their biocompatibility towards ASCs in terms of their viability, proliferation and differentiation. The extrusion-based bioprinting of ASCs in GelNB/GelS-high was performed to manufacture three-dimensional cubic constructs. Results: All three hydrogels supported the viability, proliferation and chondrogenic differentiation of ASCs to a similar extent. The adipogenic differentiation of ASCs was better supported by the softer hydrogel (GelNB/GelS-low), whereas the osteogenic differentiation was more pronounced in the harder hydrogel (GelNB/GelS-high), indicating that the differentiation fate of ASCs can be influenced via the adaption of the mechanical properties of the GelNB/GelS system. After the ex vivo chondrogenic differentiation and subcutaneous implantation of the bioprinted construct into immunocompromised mice, the production of negatively charged sulfated proteoglycans could be observed with only minimal inflammatory signs in the implanted material. Conclusions: Our results indicate that the GelNB/GelS hydrogels are very well suited for the bioprinting of ASCs and may represent attractive hydrogels for subsequent in vivo tissue engineering applications.     

Graphene–Oxide Porous Biopolymer Hybrids Enhance In Vitro Osteogenic Differentiation and Promote Ectopic Osteogenesis In Vivo

Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.     

The Marine Polysaccharide Ulvan Confers Potent Osteoinductive Capacity to PCL-Based Scaffolds for Bone Tissue Engineering Applications

Hybrid composites of synthetic and natural polymers represent materials of choice for bone tissue engineering. Ulvan, a biologically active marine sulfated polysaccharide, is attracting great interest in the development of novel biomedical scaffolds due to recent reports on its osteoinductive properties. Herein, a series of hybrid polycaprolactone scaffolds containing ulvan either alone or in blends with κ-carrageenan and chondroitin sulfate was prepared and characterized. The impact of the preparation methodology and the polysaccharide composition on their morphology, as well as on their mechanical, thermal, water uptake and porosity properties was determined, while their osteoinductive potential was investigated through the evaluation of cell adhesion, viability, and osteogenic differentiation of seeded human adipose-derived mesenchymal stem cells. The results verified the osteoinductive ability of ulvan, showing that its incorporation into the polycaprolactone matrix efficiently promoted cell attachment and viability, thus confirming its potential in the development of biomedical scaffolds for bone tissue regeneration applications.     

Hyaluronic Acid and a Short Peptide Improve the Performance of a PCL Electrospun Fibrous Scaffold Designed for Bone Tissue Engineering Applications

Bone tissue engineering is a rapidly developing, minimally invasive technique for regenerating lost bone with the aid of biomaterial scaffolds that mimic the structure and function of the extracellular matrix (ECM). Recently, scaffolds made of electrospun fibers have aroused interest due to their similarity to the ECM, and high porosity. Hyaluronic acid (HA) is an abundant component of the ECM and an attractive material for use in regenerative medicine; however, its processability by electrospinning is poor, and it must be used in combination with another polymer. Here, we used electrospinning to fabricate a composite scaffold with a core/shell morphology composed of polycaprolactone (PCL) polymer and HA and incorporating a short self-assembling peptide. The peptide includes the arginine-glycine-aspartic acid (RGD) motif and supports cellular attachment based on molecular recognition. Electron microscopy imaging demonstrated that the fibrous network of the scaffold resembles the ECM structure. In vitro biocompatibility assays revealed that MC3T3-E1 preosteoblasts adhered well to the scaffold and proliferated, with significant osteogenic differentiation and calcium mineralization. Our work emphasizes the potential of this multi-component approach by which electrospinning, molecular self-assembly, and molecular recognition motifs are combined, to generate a leading candidate to serve as a scaffold for bone tissue engineering.     

Fabrication of Hydroxyapatite with Bioglass Nanocomposite for Human Wharton’s-Jelly-Derived Mesenchymal Stem Cell Growing Substrate

Recently, composite scaffolding has found many applications in hard tissue engineering due to a number of desirable features. In this present study, hydroxyapatite/bioglass (HAp/BG) nanocomposite scaffolds were prepared in different ratios using a hydrothermal approach. The aim of this research was to evaluate the adhesion, growth, viability, and osteoblast differentiation behavior of human Wharton’s-jelly-derived mesenchymal stem cells (hWJMSCs) on HAp/BG in vitro as a scaffold for application in bone tissue engineering. Particle size and morphology were investigated by TEM and bioactivity was assessed and proven using SEM analysis with hWJMSCs in contact with the HAp/BG nanocomposite. Viability was evaluated using PrestoBlue^TM assay and early osteoblast differentiation and mineralization behaviors were investigated by ALP activity and EDX analysis simultaneously. TEM results showed that the prepared HAp/BG nanocomposite had dimensions of less than 40 nm. The morphology of hWJMSCs showed a fibroblast-like shape, with a clear filopodia structure. The viability of hWJMSCs was highest for the HAp/BG nanocomposite with a 70:30 ratio of HAp to BG (HAp70/BG30). The in vitro biological results confirmed that HAp/BG composite was not cytotoxic. It was also observed that the biological performance of HAp70/BG30 was higher than HAp scaffold alone. In summary, HAp/BG scaffold combined with mesenchymal stem cells showed significant potential for bone repair applications in tissue engineering.     

Bioengineering of Dental Pulp Stem Cells in a Microporous PNIPAAm-PLGA Scaffold

In the present article a novel bio absorbable polymeric scaffold using poly(N-isopropyl acrylamide-block-poly(L-lactide-co-glycolide) (PNIPAAm-b-PLGA) copolymers is developed for in vitro culture of human dental pulp stem cells (DPSCs). The processing of porous scaffolds has been carried out by emulsion freeze-drying and salt leaching out methods. DPSCs were cultured on scaffolds for up to 14 days. The morphology of the scaffolds, cell viability and interaction between DPSCs and scaffold was characterized by using SEM. The results of cells implantation indicated that scaffold has good cell biocompatibility. Therefore PNIPAAm-PLGA scaffolds have great potential to be used as cell carrier in tissue engineering.