Bone Tissue Engineering Approaches Research Articles

ALKBH5 Regulates Osteogenic Differentiation via the lncRNA/mRNA Complex

Human adipose-derived stem cells (hASCs) are commonly used in bone tissue regeneration. The N6-methyladenosine (m6A) modification has emerged as a novel regulatory mechanism for gene expression, playing a critical role in osteogenic differentiation of stem cells. However, the precise role and mechanism of alkylation repair homolog 5 (ALKBH5) in hASC osteogenesis remain incompletely elucidated and warrant further investigation. Herein, we employed methylated RNA immunoprecipitation sequencing, RNA sequencing, and weighted gene coexpression network analysis to identify a key long noncoding RNA (lncRNA) in hASCs: lncRNA AK311120. Functional experiments demonstrated that lnc-AK311120 promoted the osteogenic differentiation of hASCs, while a mutation at the m6A central site A of lnc-AK311120 was found to decrease the level of m6A modification. The osteogenic effect of ALKBH5 was confirmed both in vitro and in vivo using a mandibular defect model in nude mice. Subsequent investigations revealed that knockdown of ALKBH5 resulted in a significant increase in the m6A modification level of lnc-AK311120, accompanied by a downregulation in the expression level of lnc-AK311120. Additional rescue experiments demonstrated that overexpression of lnc-AK311120 could restore the phenotype after ALKBH5 knockdown. We observed that AK311120 interacted with the RNA-binding proteins DExH-Box helicase 9 (DHX9) and YTH domain containing 2 (YTHDC2) to form a ternary complex, while mitogen-activated protein kinase kinase 7 (MAP2K7) served as the shared downstream target gene of DHX9 and YTHDC2. Knockdown of AK311120 led to a reduction in the binding affinity between DHX9/YTHDC2 and the target gene MAP2K7. Furthermore, ALKBH5 facilitated the translation of MAP2K7 and activated the downstream JNK signaling pathway through the AK311120–DHX9–YTHDC2 complex, without affecting its messenger RNA level. Collectively, we have investigated the regulatory effect and mechanism of ALKBH5-mediated demethylation of lncRNA in hASC osteogenesis for the first time, offering a promising approach for bone tissue engineering.

Application of polydopamine-modified triphasic PLA/PCL-PLGA/Mg(OH)2-velvet antler polypeptides scaffold loaded with fibrocartilage stem cells for the repair of osteochondral defects.

Articular cartilage defects often involve damage to both the cartilage and subchondral bone, requiring a scaffold that can meet the unique needs of each tissue type and establish an effective barrier between the bone and cartilage. In this study, we used 3D printing technology to fabricate a tri-phasic scaffold composed of PLA/PCL-PLGA/Mg(OH)₂, which includes a cartilage layer, an osteochondral interface, and a bone layer. The scaffold was filled with Velvet antler polypeptides (VAP), and its characterization was assessed using compression testing, XRD, FTIR, SEM, fluorescence microscopy, and EDS. In vitro investigation demonstrated that the scaffold not only supported osteogenesis but also promoted chondrogenic differentiation of fibrocartilage stem cells (FCSCs). n vivo experiments showed that the tri-phasic PLA/PCL-PLGA/Mg(OH)2-VAP scaffold together with FCSC, when transplanted to animal models, increased the recovery of osteochondral defects. Those results demonstrate the promising future of illustrated tri-phasic PLA/PCL-PLGA/Mg(OH)2-VAP scaffold loaded with FCSCs as a new bone and cartilage tissue engineering approach for osteochondral defects treatment.

Liquefied capsules containing nanogrooved microdiscs and umbilical cord-derived cells for bone tissue engineering.

Surface topography has been shown to influence cell behavior and direct stromal cell differentiation into distinct lineages. Whereas this phenomenon has been verified in two-dimensional cultures, there is an urgent need for a thorough investigation of topography's role within a three-dimensional (3D) environment, as it better replicates the natural cellular environment. A co-culture of Wharton's jelly-derived mesenchymal stem/stromal cells (WJ-MSCs) and human umbilical vein endothelial cells (HUVECs) was encapsulated in a 3D system consisting of a permselective liquefied environment containing freely dispersed spherical microparticles (spheres) or nanogrooved microdiscs (microdiscs). Microdiscs presenting 358 ± 23 nm grooves and 944 ± 49 nm ridges were produced via nanoimprinting of spherical polycaprolactone microparticles between water-soluble polyvinyl alcohol counter molds of nanogrooved templates. Spheres and microdiscs were cultured in vitro with umbilical cord-derived cells in a basal or osteogenic medium within liquefied capsules for 21 days. WJ-MSCs and HUVECs were successfully encapsulated within liquefied capsules containing spheres and microdiscs, ensuring high cellular viability. Results show an enhanced osteogenic differentiation in microdiscs compared to spheres, even in basal medium, evidenced by alkaline phosphatase activity and osteopontin expression. This work suggests that the topographical features present in microdiscs induce the osteogenic differentiation of adhered WJ-MSCs along the contact guidance, without additional differentiation factors. The developed 3D bioencapsulation system comprising topographical features might be suitable for bone tissue engineering approaches with minimum in vitro manipulation.

The balance between helper T 17 and regulatory T cells in osteoimmunology and relevant research progress on bone tissue engineering.

Bone regeneration is a well-regulated dynamic process, of which the prominent role of the immune system on bone homeostasis is more and more revealed by recent research. Before fully activation of the bone remodeling cells, the immune system needs to clean up the microenvironment in facilitating the bone repair initiation. Furthermore, this microenvironment must be maintained properly by various mechanisms over the entire bone regeneration process. This review aims to summarize the role of the T-helper 17/Regulatory T cell (Th17/Treg) balance in bone cell remodeling and discuss the relevant progress in bone tissue engineering. The role of the immune response in the early stages of bone regeneration is crucial, especially the impact of the Th17/Treg balance on osteoclasts, mesenchymal stem cells (MSCs), and osteoblasts activity. By virtue of these knowledge advancements, innovative approaches in bone tissue engineering, such as nano-structures, hydrogel, and exosomes, are designed to influence the Th17/Treg balance and thereby augment bone repair and regeneration. Targeting the Th17/Treg balance is a promising innovative strategy for developing new treatments to enhance bone regeneration, thus offering potential breakthroughs in bone injury clinics.

Production of platelet-rich plasma (PRP)-enriched scaffolds for bone tissue regeneration with 3D printing technology

Bone disorders signify diverse abnormalities in the structure, development, and functions of bone tissue in the human body, with a significant correlation to ageing, insufficient physical activity, and escalating obesity. Recent advancements in bone tissue engineering aim to enhance bone tissue formation through the use of biomaterials, growth factors, and cells. The present study focuses on the fabrication and characterisation of scaffolds with a composition of gelatin (GEL) / sodium alginate (SA) / hydroxyapatite (HA) / platelet-rich plasma (PRP) using the 3D printing process. The inclusion of PRP, derived from blood, is of particular interest due to its potential to enhance bone regeneration through various growth factors. Scanning electron microscope (SEM) analysis revealed average pore sizes ranging from 481.50 ± 7.65 to 623.96 ± 11.54 µm. SEM images also showed that scaffold surfaces became smooth as the concentration of PRP increased. The mechanical test results demonstrated that as the PRP increased, the compressive strength decreased. When the swelling and degradation behaviours of scaffolds were examined, it was observed that GEL/SA/HA/3PRP scaffolds exhibited approximately 200 % swelling capability until the 4th day. GEL/SA/HA scaffolds showed a degradation behaviour about 70 % higher compared to other groups. A controlled release profile of PRP was maintained up to the 144th, 216th, and 240th hours from the scaffolds. According to the highest correlation coefficients (R2) in the release kinetics of scaffolds, GEL/SA/HA/0.5PRP and GEL/SA/HA/1PRP scaffolds were explained by the first-order model. In contrast, the GEL/SA/HA/3PRP scaffold was described using the Korsmeyer–Peppas model. The MTT analysis conducted with osteoblast cells showed that scaffolds did not demonstrate any toxic effects and facilitated cell adhesion by inducing the formation of extensions. These findings underscore the potential of PRP-incorporated GEL/SA/HA composites as a promising approach for bone tissue engineering, offering significant advancements in the treatment of bone disorders. This could lead to more effective treatments for bone disorders and injuries, reducing the need for more invasive procedures and improving patient recovery times.

Raman spectroscopic investigation of polymer based magnetic multicomponent scaffolds

Scaffolds acting as an artificial matrix for cell proliferation are one of the bone tissue engineering approaches to the treatment of bone tissue defects. In the presented study, novel multicomponent scaffolds composed of a poly(ε-caprolactone) (PCL), phenolic compounds such as tannic (TA) and gallic acids (GA), and nanocomponents such as silica-coated magnetic iron oxide nanoparticles (MNPs-c) and functionalized multi-walled carbon nanotubes (CNTs) have been produced as candidates for such artificial substitutes. Well-developed interconnected porous structures were observed using scanning electron microscopy (SEM). Raman spectra showed that the highly crystalline nature of PCL was reduced by the addition of nanoadditives. In the case of scaffolds containing MNPs-c and TA, the formation of a Fe-TA complex was concluded because characteristic bands of chelation of the Fe3+ ion by phenolic catechol oxygen appeared. It was found that the necessary conditions for the crystallization of the PCL/MNPs-c/TA are for the catechol groups to be able to penetrate the porous silica shell of MNPs-c, as during experiment with MNPs-c and TA without polymer, no such complexation was observed. Moreover, the number of catechol groups, the spatial structure and molecular size of this phenolic compound are also crucial for complexation process because GA does not form complexes. Therefore, the PCL/CNTs/MNPs-c/TA scaffolds are interesting candidates to consider for their possible medical applications.

T. gondii excretory proteins promote the osteogenic differentiation of human bone mesenchymal stem cells via the BMP/Smad signaling pathway

BackgroundBone defects, resulting from substantial bone loss that exceeds the natural self-healing capacity, pose significant challenges to current therapeutic approaches due to various limitations. In the quest for alternative therapeutic strategies, bone tissue engineering has emerged as a promising avenue. Notably, excretory proteins from Toxoplasma gondii (TgEP), recognized for their immunogenicity and broad spectrum of biological activities secreted or excreted during the parasite’s lifecycle, have been identified as potential facilitators of osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs). Building on our previous findings that TgEP can enhance osteogenic differentiation, this study investigated the molecular mechanisms underlying this effect and assessed its therapeutic potential in vivo.MethodsWe determined the optimum concentration of TgEP through cell cytotoxicity and cell proliferation assays. Subsequently, hBMSCs were treated with the appropriate concentration of TgEP. We assessed osteogenic protein markers, including alkaline phosphatase (ALP), Runx2, and Osx, as well as components of the BMP/Smad signaling pathway using quantitative real-time PCR (qRT-PCR), siRNA interference of hBMSCs, Western blot analysis, and other methods. Furthermore, we created a bone defect model in Sprague-Dawley (SD) male rats and filled the defect areas with the GelMa hydrogel, with or without TgEP. Microcomputed tomography (micro-CT) was employed to analyze the bone parameters of defect sites. H&E, Masson and immunohistochemical staining were used to assess the repair conditions of the defect area.ResultsOur results indicate that TgEP promotes the expression of key osteogenic markers, including ALP, Runx2, and Osx, as well as the activation of Smad1, BMP2, and phosphorylated Smad1/5—crucial elements of the BMP/Smad signaling pathway. Furthermore, in vivo experiments using a bone defect model in rats demonstrated that TgEP markedly promoted bone defect repair.ConclusionOur results provide compelling evidence that TgEP facilitates hBMSC osteogenic differentiation through the BMP/Smad signaling pathway, highlighting its potential as a therapeutic approach for bone tissue engineering for bone defect healing.

Polydopamine-mediated immobilization of BMP-2 onto electrospun nanofibers enhances bone regeneration

Dealing with bone defects is a significant challenge to global health. Electrospinning in bone tissue engineering has emerged as a solution to this problem. In this study, we designed a PVDF-b-PTFE block copolymer by incorporating TFE, which induced a phase shift in PVDF from α to β, thereby enhancing the piezoelectric effect. Utilizing the electrospinning process, we not only converted the material into a film with a significant surface area and high porosity but also intensified the piezoelectric effect. Then we used polydopamine to immobilize BMP-2 onto PVDF-b-PTFE electrospun nanofibrous membranes, achieving a controlled release of BMP-2. The scaffold’s characters were examined using SEM and XRD. To assess its osteogenic effects in vitro, we monitored the proliferation of MC3T3-E1 cells on the fibers, conducted ARS staining, and measured the expression of osteogenic genes. In vivo, bone regeneration effects were analyzed through micro-CT scanning and HE staining. ELISA assays confirmed that the sustained release of BMP-2 can be maintained for at least 28 d. SEM images and CCK-8 results demonstrated enhanced cell viability and improved adhesion in the experimental group. Furthermore, the experimental group exhibited more calcium nodules and higher expression levels of osteogenic genes, including COL-I, OCN, and RUNX2. HE staining and micro-CT scans revealed enhanced bone tissue regeneration in the defective area of the PDB group. Through extensive experimentation, we evaluated the scaffold’s effectiveness in augmenting osteoblast proliferation and differentiation. This study emphasized the potential of piezoelectric PVDF-b-PTFE nanofibrous membranes with controlled BMP-2 release as a promising approach for bone tissue engineering, providing a viable solution for addressing bone defects.

The treatment efficacy of bone tissue engineering strategy for repairing segmental bone defects under diabetic condition.

Diabetes mellitus is a systematic disease which exert detrimental effect on bone tissue. The repair and reconstruction of bone defects in diabetic patients still remain a major clinical challenge. This study aims to investigate the potential of bone tissue engineering approach to improve bone regeneration under diabetic condition. In the present study, decalcified bone matrix (DBM) scaffolds were seeded with allogenic fetal bone marrow-derived mesenchymal stem cells (BMSCs) and cultured in osteogenic induction medium to fabricate BMSC/DBM constructs. Then the BMSC/DBM constructs were implanted in both subcutaneous pouches and large femoral bone defects in diabetic (BMSC/DBM in DM group) and non-diabetic rats (BMSC/DBM in non-DM group), cell-free DBM scaffolds were implanted in diabetic rats to serve as the control group (DBM in DM group). X-ray, micro-CT and histological analyses were carried out to evaluate the bone regenerative potential of BMSC/DBM constructs under diabetic condition. In the rat subcutaneous implantation model, quantitative micro-CT analysis demonstrated that BMSC/DBM in DM group showed impaired bone regeneration activity compared with the BMSC/DBM in non-DM group (bone volume: 46 ± 4.4mm3 vs 58.9 ± 7.15mm3, *p < 0.05). In the rat femoral defect model, X-ray examination demonstrated that bone union was delayed in BMSC/DBM in DM group compared with BMSC/DBM in non-DM group. However, quantitative micro-CT analysis showed that after 6months of implantation, there was no significant difference in bone volume and bone density between the BMSC/DBM in DM group (199 ± 63mm3 and 593 ± 65mg HA/ccm) and the BMSC/DBM in non-DM group (211 ± 39mm3 and 608 ± 53mg HA/ccm). Our data suggested that BMSC/DBM constructs could repair large bone defects in diabetic rats, but with delayed healing process compared with non-diabetic rats. Our study suggest that biomaterial sacffolds seeded with allogenic fetal BMSCs represent a promising strategy to induce and improve bone regeneration under diabetic condition.

Liquefied capsules containing nanogrooved microdiscs and umbilical cord-derived cells for bone tissue engineering

Background Surface topography has been shown to influence cell behavior and direct stromal cell differentiation into distinct lineages. Whereas this phenomenon has been verified in two-dimensional cultures, there is an urgent need for a thorough investigation of topography’s role within a three-dimensional (3D) environment, as it better replicates the natural cellular environment. Methods A co-culture of Wharton’s jelly-derived mesenchymal stem/stromal cells (WJ-MSCs) and human umbilical vein endothelial cells (HUVECs) was encapsulated in a 3D system consisting of a permselective liquefied environment containing freely dispersed spherical microparticles (spheres) or nanogrooved microdiscs (microdiscs). Microdiscs presenting 358 ± 23 nm grooves and 944 ± 49 nm ridges were produced via nanoimprinting of spherical polycaprolactone microparticles between water-soluble polyvinyl alcohol counter molds of nanogrooved templates. Spheres and microdiscs were cultured in vitro with umbilical cord-derived cells in a basal or osteogenic medium within liquefied capsules for 21 days. Results WJ-MSCs and HUVECs were successfully encapsulated within liquefied capsules containing spheres and microdiscs, ensuring high cellular viability. Results show an enhanced osteogenic differentiation in microdiscs compared to spheres, even in basal medium, evidenced by alkaline phosphatase activity and osteopontin expression. Conclusions This work suggests that the topographical features present in microdiscs induce the osteogenic differentiation of adhered WJ-MSCs along the contact guidance, without additional differentiation factors. The developed 3D bioencapsulation system comprising topographical features might be suitable for bone tissue engineering approaches with minimum in vitro manipulation.

Rutin promotes osteogenic differentiation of mesenchymal stem cells (MSCs) by increasing ECM deposition and inhibiting p53 expression.

Mesenchymal stem cells (MSCs) are an important source of cells for bone regeneration. Although the utilization of MSCs along with growth factors and scaffolds is a beneficial clinical approach for bone tissue engineering, there is need for improvement on the effectiveness of MSC osteogenesis and differentiation. Rutin is a natural flavonoid and a major component for cell proliferation and bone development. However, studies on the mechanism through which rutin regulates osteogenesis and MSC differentiation are limited. Therefore, this study aimed to investigate the effect and mechanisms of rutin on osteogenic differentiation of MSCs. MSCs were extracted from umbilical cords and treated with rutin, followed by the examination of osteogenesis-related markers. Rutin treatment promoted the differentiation of MSCs towards the osteogenic lineage rather than the adipogenic lineage and increased the expression of osteogenic markers. RNA sequencing and bioinformatic analysis indicated that rutin regulated p53, a key gene in regulating the osteogenic differentiation of MSCs. Additionally, cellular experiments showed that rutin-induced decrease in p53 expression increased the formation of extracellular matrix (ECM) by promoting p65 phosphorylation and caspase-3 cleavage. Conclusively, this study demonstrates the importance of rutin in osteogenesis and indicates that rutin possesses potential pharmaceutical application for bone regeneration and bone tissue engineering.

Nanoparticle-Based Drug Delivery Systems for Enhancing Bone Regeneration.

The treatment of bone defects has been a long-standing challenge in clinical practice. Among the various bone tissue engineering approaches, there has been substantial progress in the development of drug delivery systems based on functional drugs and appropriate carrier materials owing to technological advances in recent years. A large number of materials based on functional nanocarriers have been developed and applied to improve the complex osteogenic microenvironment, including for promoting osteogenic activity, inhibiting osteoclast activity, and exerting certain antibacterial effects. This Review discusses the physicochemical properties, drug loading mechanisms, advantages and disadvantages of nanoparticles (NPs) used for constructing drug delivery systems. In addition, we provide an overview of the osteogenic microenvironment regulation mechanism of drug delivery systems based on nanoparticle (NP) carriers and the construction strategies of drug delivery systems. Finally, the advantages and disadvantages of NP carriers are summarized along with their prospects and future research trends in bone tissue engineering. This Review thus provides advanced strategies for the design and application of drug delivery systems based on NPs in the treatment of bone defects.

Biocompatible nanocomposite scaffolds based on carrageenan incorporating hydroxyapatite and hesperidin loaded nanoparticles for bone tissue regeneration

AbstractIn this study, injectable and osteoinductive carrageenan (CGN)‐based composite hydrogel integrated with 1% wt/vol hydroxyapatite (HA) and hesperidin (HPN) loaded poly (3‐hydroxybutyrate‐co‐3‐hydroxyvalerate acid) nanoparticles (PHBV NPs) were developed as a scaffold for bone tissue engineering. Accordingly, HPN was encapsulated into PHBV NPs by the oil in water emulsion‐solvent evaporation technique and optimized using the fractional factorial design. Then, the effects of the amount of drug, polymer, surfactant concentration, sonication time, and the water/organic phase volume ratio on the characteristics of NPs, including particle size (PS), polydispersity index (PdI), zeta potential (ZP), drug loading (DL) % and encapsulation efficiency (EE) % were investigated. The crystalline state of HPN and the interaction between the drug and the polymer were also investigated using the x‐ray powder diffraction method and the Fourier transform infrared method, respectively. Further, CGN hydrogel was developed and loaded with optimized NPs and HA (HPN‐PHBV NPs‐CGN/HA hydrogel). The prepared hydrogel was then characterized for drug release, swelling and degradation behavior, mechanical properties, and injectability. In addition, the potential of the composite hydrogel as bone tissue scaffolds was assessed by the MTT assay, alkaline phosphatase (ALP) activity, and alizarin red S staining. The optimized formulation had the size of 353.5 nm, PdI of 0.43, EE% of 88.47%, DL% of 29.58% and the ZP of −17.4. HPN‐PHBV NPs‐CGN/HA hydrogel also showed sustained drug release, as compared to the HPN‐CGN/HA hydrogel. Further, the hydrogel containing HA showed improved mechanical properties, as compared with the HA free one. In addition, HPN‐PHBV NPs‐CGN/HA hydrogel significantly enhanced MG63 cell proliferation, ALP activity and matrix mineralization, as compared with other groups. Overall, the use of this hydrogel with enhanced osteogenic capacity could be considered as an effective approach for bone tissue engineering.

Synthesis of bone biocompatible implants using human adipose-derived mesenchymal stem cells (hADMSCs) and PCL/laminin scaffold substrate.

Bone tissue engineering is considered a new method in the treatment of bone defects and can be an effective alternative to surgery and bone grafting. The use of adipose tissue mesenchymal stem cells (ADMSCs) on synthetic polymer scaffolds is one of the new approaches in bone tissue engineering. In this study, we aimed to investigate the effect of laminin coating on biocompatibility and osteogenic differentiation of ADMSCs seeded on polycaprolactone (PCL) scaffolds. The morphology of the electrospun scaffold was evaluated using a scanning electron microscope (SEM). Cell proliferation and cytotoxicity were determined by MTT assay. The adipogenic and osteogenic differentiation potential of the cells was evaluated. The osteogenic differentiation of ADMSCs cultured on the PCL scaffold coated with laminin was assessed by evaluating the level of alkaline phosphatase (ALP) activity, intracellular calcium content, and expression of bone-specific genes. The results showed that the ADMSCs cultured on PCL/laminin showed enhanced osteogenic differentiation compared to those cultured on non-coated PCL or control medium (P<0.05). It seems that laminin enhances the physicochemical properties and biocompatibility of PCL nanofiber scaffolds; and by modifying the surface of the scaffold, improves the differentiation of ADMSCs into osteogenic cells.

A bilayer hydrogel mimicking the periosteum-bone structure for innervated bone regeneration.

In bone tissue, nerves are primarily located in the periosteum and play an indispensable role in bone defect repair. However, most bone tissue engineering approaches ignored the reconstruction of the nerve network. Herein, we aimed to develop a bilayer hydrogel simulating periosteum-bone structure to induce innervated bone regeneration. The bottom "bone" layer consisted of gelatin methacryloyl (GelMA), poly(ethylene glycol) diacrylate (PEGDA), and nano-hydroxyapatite (nHA), whereas the upper "periosteum" layer consisted of GelMA, sodium alginate (SA) and MgCl2. The mechanical properties of the upper and bottom hydrogels were designed to be suitable for neurogenesis and osteogenesis, respectively. Besides, Mg2+ from the "periosteum" layer released at the early stage (within 7 d), which aligned with the optimal time window for nerve regeneration and osteogenic related neuropeptide release. Simultaneously, the prevention of long-term Mg2+ release (after 7 d) could avoid osteogenic inhibition caused by prolonged Mg2+ exposure. Additionally, the incorporation of nHA in the bottom "bone" layer supported the long-term osteogenesis due to its osteoconductivity and slow degradation. In vitro biological experiments revealed that the bilayer hydrogel (GS@Mg/GP@nHA) promoted neurite growth and calcitonin gene-related peptide (CGRP) expression in rat dorsal root ganglion (DRG) neurons, as well as the osteogenesis of rat bone-derived mesenchymal stem cells (BMSCs). Moreover, the in vivo experiments demonstrated that the GS@Mg/GP@nHA hydrogel efficiently promoted nerve network reconstruction and bone regeneration of rat calvarial bone defects. Altogether, the bilayer hydrogel GS@Mg/GP@nHA could promote innervated bone regeneration, providing new insights for biomaterial design for bone tissue engineering.

Synthesis and characterization of B-type carbonated hydroxyapatite materials: Effect of carbonate content on mechanical strength and in vitro degradation

The current approach in bone tissue engineering requires resorbable biomaterials that enhance bone formation while maintaining sufficient mechanical stability. In this work, the influence of three levels of B-type carbonate substitution in hydroxyapatite lattice on mechanical strength and degradation rate is analyzed. The inverse aqueous route has been selected as a synthesis method of four powders with carbonate substitution between 4 and 6wt.%. X-ray fluorescence (XRF), (C-S)-Analysis, FT-Infrared, X-ray diffraction, DTA-TG and TEM were used to investigate chemical composition, type of substitution, thermal behaviour, and morphology of the powders. Disc shaped specimens were processed by uniaxial pressing and sintering in argon/CO2 flow. Maximum temperatures of thermal treatment between 750 and 850°C were selected to obtain similar porosity levels for the different compositions. The highest carbonate substituted material (5.3wt.%) presented higher compressive strength and dissolution rate than the other materials showing the beneficial effect of B-type substitution in hydroxyapatite materials for bone repair.

Hydrogels and Bioprinting in Bone Tissue Engineering: Creating Artificial Stem-Cell Niches for In Vitro Models.

Advances in bioprinting have enabled the fabrication of complex tissue constructs with high speed and resolution. However, there remains significant structural and biological complexity within tissues that bioprinting is unable to recapitulate. Bone, for example, has a hierarchical organization ranging from the molecular to whole organ level. Current bioprinting techniques and the materials employed have imposed limits on the scale, speed, and resolution that can be achieved, rendering the technique unable to reproduce the structural hierarchies and cell-matrix interactions that are observed in bone. The shift toward biomimetic approaches in bone tissue engineering, where hydrogels provide biophysical and biochemical cues to encapsulated cells, is a promising approach to enhancing the biological function and development of tissues for in vitro modeling. A major focus in bioprinting of bone tissue for in vitro modeling is creating dynamic microenvironmental niches to support, stimulate, and direct the cellular processes for bone formation and remodeling. Hydrogels are ideal materials for imitating the extracellular matrix since they can be engineered to present various cues whilst allowing bioprinting. Here, recent advances in hydrogels and 3D bioprinting toward creating a microenvironmental niche that is conducive to tissue engineering of in vitro models of bone are reviewed.

Pretreatment of Mesenchymal Stem Cells with Electrical Stimulation as a Strategy to Improve Bone Tissue Engineering Outcomes.

Electrical stimulation (EStim), whether used alone or in combination with bone tissue engineering (BTE) approaches, has been shown to promote bone healing. In our previous in vitro studies, mesenchymal stem cells (MSCs) were exposed to EStim and a sustained, long-lasting increase in osteogenic activity was observed. Based on these findings, we hypothesized that pretreating MSC with EStim, in 2D or 3D cultures, before using them to treat large bone defects would improve BTE treatments. Critical size femur defects were created in 120 Sprague-Dawley rats and treated with scaffold granules seeded with MSCs that were pre-exposed or not (control group) to EStim 1 h/day for 7 days in 2D (MSCs alone) or 3D culture (MSCs + scaffolds). Bone healing was assessed at 1, 4, and 8 weeks post-surgery. In all groups, the percentage of new bone increased, while fibrous tissue and CD68+ cell count decreased over time. However, these and other healing features, like mineral density, bending stiffness, the amount of new bone and cartilage, and the gene expression of osteogenic markers, did not significantly differ between groups. Based on these findings, it appears that the bone healing environment could counteract the long-term, pro-osteogenic effects of EStim seen in our in vitro studies. Thus, EStim seems to be more effective when administered directly and continuously at the defect site during bone healing, as indicated by our previous studies.

Novel Electroactive Mineralized Polyacrylonitrile/PEDOT:PSS Electrospun Nanofibers for Bone Repair Applications.

Bone defect repair remains a critical challenge in current orthopedic clinical practice, as the available therapeutic strategies only offer suboptimal outcomes. Therefore, bone tissue engineering (BTE) approaches, involving the development of biomimetic implantable scaffolds combined with osteoprogenitor cells and native-like physical stimuli, are gaining widespread interest. Electrical stimulation (ES)-based therapies have been found to actively promote bone growth and osteogenesis in both in vivo and in vitro settings. Thus, the combination of electroactive scaffolds comprising conductive biomaterials and ES holds significant promise in improving the effectiveness of BTE for clinical applications. The aim of this study was to develop electroconductive polyacrylonitrile/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PAN/PEDOT:PSS) nanofibers via electrospinning, which are capable of emulating the native tissue's fibrous extracellular matrix (ECM) and providing a platform for the delivery of exogenous ES. The resulting nanofibers were successfully functionalized with apatite-like structures to mimic the inorganic phase of the bone ECM. The conductive electrospun scaffolds presented nanoscale fiber diameters akin to those of collagen fibrils and displayed bone-like conductivity. PEDOT:PSS incorporation was shown to significantly promote scaffold mineralization in vitro. The mineralized electroconductive nanofibers demonstrated improved biological performance as observed by the significantly enhanced proliferation of both human osteoblast-like MG-63 cells and human bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs). Moreover, mineralized PAN/PEDOT:PSS nanofibers up-regulated bone marker genes expression levels of hBM-MSCs undergoing osteogenic differentiation, highlighting their potential as electroactive biomimetic BTE scaffolds for innovative bone defect repair strategies.

Fabrication of novel bone substitute alginate – N,O-carboxymethyl chitosan – Aldehyde hyaluronic acid – Biphasic calcium phosphate for bone regeneration

Mineralized hydrogels, synthesized by incorporating mineral particles into the hydrogel crosslinking network, have become an effective approach for bone tissue engineering. In this study, hydrogel composites based on alginate, N,O-carboxymethyl chitosan (NOCC), aldehyde hyaluronic acid (AHA) and loaded with biphasic calcium phosphate (BCP) were fabricated by in situ crosslinking. NOCC-AHA hydrogel, forming crosslinks by Schiff base reaction without requiring any chemical linkers or radial light sources, offers a porous scaffold favorable for incorporating with the mineral phase. The integration of BCP – inorganic phase – and alginate within the NOCC-AHA system can be used to supplement mineral agents and reinforcement. Different amounts of BCP particles were investigated to improve cell proliferation. The formation of hydrogel composites was confirmed via scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). In vitro study evidenced that MC3T3 cells were well-attached to hydrogel composites which did not cause cytotoxicity. Furthermore, in vivo study demonstrated that alginate-NOCC-AHA-BCP hydrogels promoted bone healing in the full-thickness calvarial defect mouse model. The obtained results indicated that the fabricated hydrogel composite could be a potential material for bone regeneration with a suitable degradation rate, appropriate pore size, favorable cytocompatibility, high compressive strength, ability to support cell proliferation, and capacity to enhance bone regeneration.