Progranulin-loaded silk fibroin/chitosan scaffold with polydopamine modification promotes bone regeneration via PI3K/Akt pathway activation.

Endothelialized microvessels fabricated by microfluidics facilitate osteogenic differentiation and promote bone repair

Gene therapy provides a promising approach for regeneration and repair of injured bone. Application of gene therapy has displayed increased efficiency in various animal models and preclinical trials in comparison with traditional bone grafting methods. The objective of this review is to highlight fundamental principles of gene therapy strategies in bone tissue engineering and solutions of their current limitations for the healing of bone injury. Vector types are debated for the repair of defected site due to demonstration of constraints and applications of the protocols. In recent years, the combination of gene therapy strategies and bone tissue engineering has highly gained attention. We discussed viral and non-viral mediated delivery of therapeutic protein by using scaffolds for bone tissue engineering. Although pre-clinical studies have showed that gene therapy has very promising results to heal injured bone, there are several limitations regarding with the usage of gene delivery methods into clinical applications. Choice of suitable vector, selection of transgene and gene delivery protocols are the most outstanding questions. This article also addresses current state of gene delivery strategies in bone tissue engineering for their potential applications in clinical considerations.

For decades, researchers have been developing a range of promising strategies in bone tissue engineering with the aim of producing a significant clinical benefit over existing therapies. However, a major problem concerns the traditional use of xenogeneic substances for the expansion of cells, which complicates direct clinical transfer. The study's aim was to establish a totally autologous sheep model as a basis for further preclinical studies and future clinical application. Ovine mesenchymal stromal cells (MSC) were cultivated in different concentrations (0%, 2%, 5%, 10%, and 25%) of either autologous serum (AS) or fetal calf serum (FCS). With an increase of serum concentration, enhanced metabolic activity and proliferation could be observed. There were minor differences between MSC cultivated in AS or FCS, comparing gene and protein expression of osteogenic and stem cell markers, morphology, and osteogenic differentiation. MSC implanted subcutaneously in the sheep model, together with a nanostructured bone substitute, either in stable block or moldable putty form, induced similar vascularization and remodeling of the bone substitute irrespective of cultivation of MSC in AS or FCS and osteogenic differentiation. The bone substitute in block form together with MSC proved particularly advantageous in the induction of ectopic bone formation compared to the cell-free control and putty form. It could be demonstrated that AS is suitable for replacement of FCS for cultivation of ovine MSC for bone tissue engineering purposes. Substantial progress has been made in the development of a strictly xenogeneic-free preclinical animal model to bring future clinical application of bone tissue engineering strategies within reach.

The growing number of non-union fractures in an aging population has increased the clinical demand for tissue-engineered bone. Electrical stimulation (ES) has been described as a promising strategy for bone regeneration treatments in several clinical studies. However the underlying mechanism by which ES augments bone formation is still poorly understood and its use in bone tissue engineering (BTE) strategies is currently underexplored. Additive manufacturing (AM) technologies (Fused Deposition Modeling/3D Printing) have been widely used in BTE due to their ability to fabricate scaffolds with a high control over their structural and mechanical properties in a reproducible and scalable manner. Thus, in this work, we combined AM methods with conductive biomaterials and ES to enhance the osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) envisaging improved BTE strategies.First, we started by developing AM-based electro-bioreactor devices containing medical-grade electrodes (stainless steel and Ti6Al4V) to apply ES to monolayer 2D cultures and 3D cell-seeded scaffolds. Computer modeling(Finite Element Analysis-FEA) was employed to predict the magnitude/distribution of electrical fields within the ES devices and along the different conductive scaffolds. Prior to scaffold culture, 5 different ES protocols were tested in terms of their ability to promote hBMSCs proliferation and osteogenic differentiation in 2D cultures. The best performance ES protocol was then used in two different AM-based BTE strategies: 1) Two different conductive scaffolds (conductive poly lactic acid (PLA) and titanium) were seeded with hBMSCs and cultured for 21 days under osteogenic medium conditions with and without ES and their biological performance was evaluated in comparison to non-conductive standard PLA scaffolds; 2) Different PEDOT:PSS-based coating solutions were screened to obtain PEDOT:PSS/Gelatin-coated 3D polycaprolactone (PCL) scaffolds with a high(11 S.cm-1) and stable electroconductivity. When cultured under ES, PEDOT:PSS/Gelatin-PCL scaffolds enhanced significantly hBMSCs osteogenic differentiation and mineralization(calcium deposition), highlighting their potential for BTE applications.Acknowledgements: Funding received from FCT through projects InSilico4OCReg (PTDC/EME-SIS/0838/2021), OptiBioScaffold (PTDC/EME-SIS/4446/2020) and BioMaterARISES (EXPL/CTM-CTM/0995/2021), and to the institutions iBB (UIDB/04565/2020), CDRSP (UIDB/04044/2020) and Associate Laboratory i4HB (LA/P/0140/2020).

Biointerface control of electrospun fiber scaffolds for bone regeneration: Engineered protein link to mineralized surface

Bone is a dynamic tissue that is able to sense and adapt to mechanical stimuli by modulating its mass, geometry, and structure. Bone marrow stromal cells (BMSCs) are known to play an integral part in bone formation by providing an osteoprogenitor cell source capable of differentiating into mature osteoblasts in response to mechanical stresses. Characteristics of the in vivo bone environment including the three dimensional (3-D) lacunocanalicular structure and extracellular matrix composition have previously been shown to play major roles in influencing mechanotransduction processes within bone cells. To more accurately model this phenomenon in vitro, we cultured human BMSCs on 3-D, partially demineralized bone scaffolds in the presence of four-point bending loads within a novel bioreactor. The effect of mechanical loading and dexamethasone concentration on BMSC osteogenic differentiation and mineralized matrix production was studied for 8 and 16 days of culture. Mechanical stimulation after 16 days with 10 nM dexamethasone promoted osteogenic differentiation of BMSCs by significantly elevating alkaline phosphatase activity as well as alkaline phosphatase and osteopontin transcript levels over static controls. Mineralized matrix production also increased under these culture conditions. Dexamethasone concentration had a dramatic effect on the ability of mechanical stimulation to modulate these phenotypic and genotypic responses. These results provide increased insight into the role of mechanical stimulation on osteogenic differentiation of human BMSCs in vitro and may lead to improved strategies in bone tissue engineering.

Traditional bone tissue engineering presents several challenges, including difficulties in obtaining seed cells, relatively slow proliferation within scaffolds, and the potential to induce postimplantation immunogenic reactions. A promising direction for bone-tissue regeneration involves the development of cell-free scaffolds with superior physicochemical and biological properties. This study focused on encapsulating bone marrow stem cells (BMSCs) within stromal cell-derived factor-1α (SDF-1α)-loaded silk fibroin-gelatin methacryloyl (SF-GelMA) hydrogel to create a ″membrane-jelly″ culture platform. Within a specific concentration range, SDF-1α positively influenced BMSC induction and promoted osteogenic differentiation. Decellularized extracellular matrix mimics the stem cell microenvironment, enhancing BMSC adhesion and proliferation, while preventing the loss of stemness. Building upon this foundation, the SDF-1α/GelMA-SF hydrogel matrix provides mechanical support for both the recruitment of BMSCs and their subsequent osteogenic differentiation. Furthermore, it activates various signaling pathways, including bile acid, Notch pathway, and G protein-coupled receptor signaling according to the GO and KEGG results of the RNAseq, thereby synergistically promoting elevated expression of osteogenic markers in BMSCs from multiple perspectives. This comprehensive approach harnesses osteoinductive capacity and accelerates bone tissue regeneration. This system is expected to represent an advanced strategy for bone tissue engineering.

Background: A combination of bioceramics and polymeric materials has attracted the research community’s interest in bone tissue engineering. These composites are essential to support cell attachment, proliferation, and osteogenesis differentiation, which are vital as a classic strategy in bone tissue engineering. In this study, NiFe2O4/ZnO-coated poly L-Lactide (PLLA) was employed as a scaffold to evaluate the osteogenic differentiation capability of human adipose tissue derived mesenchymal stem cells (hAMSCs).Material and methods: The electrospun PLLA nanofibers were fabricated, coated with nanocomposite (NiFe2O4/ZnO), and evaluated by the water contact angle (WCA), tensile test, attenuated total reflectance fourier-transform infrared (ATR-FTIR) and scanning electron microscopy (SEM). Then, the osteogenic differentiation potential of hAMSCs was assessed using NiFe2O4/ZnO-coated PLLA compared to tissue culture plastic (TCP) and a simple scaffold (PLLA) in vitro conditions.Results: The adhesion, proliferation, and differentiation of hAMSCs were supported by the mechanical and biological properties of the NiFe2O4/ZnO-coated PLLA scaffold, according to SEM and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining patterns. During bone differentiation, Alkaline phosphatase (ALP) enzyme activity, biomineralization, calcium content, and osteogenic gene expression (ALP, Osteonectin, Osteocalcin, Collagen type I, and Runx2) were higher on NiFe2O4/ZnO-coated PLLA scaffold than on PLLA scaffold and TCP.Conclusion: Based on our results, the osteogenic differentiation of hAMSCs on the improved biological scaffold (PLLA coated with NiFe2O4/ZnO) could accelerate due to the stimulating effect of this nanocomposite.

Since robust osteogenic differentiation and mineralization are integral to the engineering of bone constructs, understanding the impact of the cellular microenvironments on human mesenchymal stem cell (hMSCs) osteogenic differentiation is crucial to optimize bioreactor strategy. Two perfusion flow conditions were utilized in order to understand the impact of the flow configuration on hMSC construct development during both pre-culture (PC) in growth media and its subsequent osteogenic induction (OI). The media in the in-house perfusion bioreactor was controlled to perfuse either around (termed parallel flow [PF]) the construct surfaces or penetrate through the construct (termed transverse flow [TF]) for 7 days of the PC followed by 7 days of the OI. The flow configuration during the PC not only changed growth kinetics but also influenced cell distribution and potency of osteogenic differentiation and mineralization during the subsequent OI. While shear stress resulted from the TF stimulated cell proliferation during PC, the convective removal of de novo extracellular matrix (ECM) proteins and growth factors (GFs) reduced cell proliferation on OI. In contrast, the effective retention of de novo ECM proteins and GFs in the PC constructs under the PF maintained cell proliferation under the OI but resulted in localized cell aggregations, which influenced their osteogenic differentiation. The results revealed the contrasting roles of the convective flow as a mechanical stimulus, the redistribution of the cells and macromolecules in 3D constructs, and their divergent impacts on cellular events, leading to bone construct formation. The results suggest that the modulation of the flow configuration in the perfusion bioreactor is an effective strategy that regulates the construct properties and maximizes the functional outcome.

Bone is a highly vascularized tissue, and its development, maturation, remodeling, and regeneration are dependent on a tight regulation of blood vessel supply. This condition also has to be taken into consideration in the context of the development of artificial tissue substitutes. In classic tissue engineering, bone-forming cells such as primary osteoblasts or mesenchymal stem cells are introduced into suitable scaffolds and implanted in order to treat critical-size bone defects. However, such tissue substitutes are initially avascular. Because of the occurrence of hypoxic conditions, especially in larger tissue substitutes, this leads to the death of the implanted cells. Therefore, it is necessary to devise vascularization strategies aiming at fast and efficient vascularization of implanted artificial tissues. In this review article, we present and discuss the current vascularization strategies in bone tissue engineering. These are based on the use of angiogenic growth factors, the co-implantation of blood vessel forming cells, the ex vivo microfabrication of blood vessels by means of bioprinting, and surgical methods for creating surgically transferable composite tissues.

Cellular strategies play an important role in bone tissue engineering and regenerative medicine (BTE/RM). Variability in cell culture procedures (e.g., cell types, cell isolation and expansion, cell seeding methods, and preculture conditions before in vivo implantation) may influence experimental outcome. Meanwhile, outcomes from initial clinical trials are far behind those of animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the BTE/RM constructs as some complex clinical implementations require bone regeneration in too large a quantity. Coculture strategies, in which angiogenic cells are introduced into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, preclinical studies have demonstrated that cell-based tissue-engineered constructs generally induce more bone formation compared with acellular constructs. Further, cocultures have been shown to enhance vascularization and bone formation compared with monocultures. However, translational efficacy from animal studies to clinical use requires improvement, and the role implanted cells play in clinical bone regeneration needs to be further elucidated. In view of this, the present review provides an overview of the critical procedures during in vitro and in vivo phases for cell-based strategies (both monoculture and coculture) in BTE/RM to achieve more standardized culture conditions for future studies, and hence enhance bone formation.

PurposeAutologous bone grafting (BG) remains the standard reconstruction strategy for large craniofacial defects. Calcium phosphate (CaP) biomaterials, such as biphasic calcium phosphate (BCP), do not yield consistent results when used alone and must then be combined with cells through bone tissue engineering (BTE). In this context, total bone marrow (TBM) and bone marrow-derived mesenchymal stem cells (MSC) are the primary sources of cellular material used with biomaterials. However, several other BTE strategies exist, including the use of growth factors, various scaffolds, and MSC isolated from different tissues. Thus, clinicians might be unsure as to which method offers patients the most benefit. For this reason, the aim of this study was to compare eight clinically relevant BTE methods in an “all-in-one” study.MethodsWe used a transgenic rat strain expressing green fluorescent protein (GFP), from which BG, TBM, and MSC were harvested. Progenitor cells were then mixed with CaP materials and implanted subcutaneously into nude mice. After eight weeks, bone formation was evaluated by histology and scanning electron microscopy, and GFP-expressing cells were tracked with photon fluorescence microscopy.Results/ConclusionsBone formation was observed in only four groups. These included CaP materials mixed with BG or TBM, in which abundant de novo bone was formed, and BCP mixed with committed cells grown in two- and three-dimensions, which yielded limited bone formation. Fluorescence microscopy revealed that only the TBM and BG groups were positive for GFP expressing-cells, suggesting that these donor cells were still present in the host and contributed to the formation of bone. Since the TBM-based procedure does not require bone harvest or cell culture techniques, but provides abundant de novo bone formation, we recommend consideration of this strategy for clinical applications.

Use of allogeneic mesenchymal stem cells (allo-MSCs) in bone tissue engineering strategies can overcome the limitations associated with autologous MSCs, but unfortunately, the immunogenicity of allo-MSCs leads to a high rate of rejection, unless immunosuppressive agents are used. B and T lymphocyte attenuator (BTLA) is a newly discovered immunoglobulin superfamily inhibitory receptor, and Herpesvirus-entry mediator (HVEM), a member of the tumor necrosis factor receptor family, is the only ligand of BTLA. Both BTLA and HVEM are widely expressed in B and T lymphocytes and other immune cells and play significant roles in the negative regulation of an immunoreaction. Therefore, we hypothesized that MSCs could be modified to maintain their bone differentiation ability through negative regulation of the immune response, and to test this hypothesis, we generated HVEM-expressing MSCs and tested their potential for osteogenic differentiation and bone repair in a simulated immune activation condition in vitro and in a mice femoral defect model. We found that osteogenic differentiation of allo-MSCs was decreased significantly in the activated immune microenvironment and that HVEM expression by allo-MSCs inhibited the immune response, resulting in improved osteogenic differentiation in vitro and new bone formation by allo-MSCs in a mouse femoral defect model. Our results also preliminarily suggested that the mechanism by which HVEM-expressing allo-MSCs overcome inflammation and enhance osteogenesis may be related to inhibition of interleukin-17. Overall, the data obtained in the present study provide support for the further development of HVEM-modified allo-MSCs as potentially ideal seed cells for bone tissue engineering applications.

Bone defect repair remains a major challenge in modern medicine. Although bone marrow mesenchymal stem cells (BMSCs) possess multilineage differentiation potential, traditional BMSC constructs are often limited in clinical applications due to insufficient osteogenic differentiation efficiency and inadequate vascularization. This study developed an innovative bone tissue engineering strategy by combining BMSCs with gelatin/polycaprolactone (GT/PCL) nanofiber membranes to form cell sheets, which were then modified with endothelial cells (ECs) on the surface. The sheets were subsequently rolled into three-dimensional scaffolds to systematically evaluate their osteogenic potential and underlying mechanisms. Results showed that electrospun GT/PCL nanofiber membranes exhibited uniform fiber structure (diameter 200-500nm), successfully mimicking the microstructure of natural extracellular matrix. In vitro experiments demonstrated that after 14days of culture, EC modification significantly enhanced the osteogenic differentiation of BMSCs compared to unmodified controls, with approximately 3-fold increase in ALP expression (p < 0.05) and 2.5-fold increase in angiogenic factor VEGF expression (p < 0.01). Subcutaneous implantation in nude mice revealed superior bone formation capability of EC-modified constructs at both 4 and 8weeks: micro-CT analysis showed bone density reaching 350mg/cm3, bone surface area approaching 400mm2, and bone volume fraction of approximately 20%, significantly higher than control groups (p < 0.0001). Immunohistochemical evaluation further confirmed more mature trabecular bone structure and richer vascular networks in EC-modified groups. Mechanistic studies revealed that EC modification promoted bone regeneration through three key pathways: optimization of local vascular microenvironment for improved nutrient supply, activation of intercellular synergistic signaling pathways, and reconstruction of physiological bone tissue microenvironment. This study not only validates the application value of this composite strategy in bone tissue engineering but also provides important theoretical basis for developing novel bone regeneration solutions.

Dental pulp stem cells (DPSCs) can undergo several types of differentiation, including osteogenic differentiation. One osteogenesis-inducing factor that has been previously described is in vitro low-power laser irradiation of cells. Laser irradiation promotes the acceleration of bone matrix mineralization of the cell strain. However, no consensus exists regarding the dose and treatment time. We used DPSC strains from cleft lip and palate patients because new bone tissue engineering strategies have used DPSCs in preclinical and clinical trials for the rehabilitation of alveolar bone clefts. Optimizing bone tissue engineering techniques for cleft and lip palate patients by applying low-power laser therapy (LPLT) to DPSCs obtained from these patients can help improve current strategies to quickly close large alveolar clefts. The aim of this study was to investigate the effects of LPLT at different energy densities in DPSC strains obtained from cleft lip and palate patients during in vitro osteogenic differentiation. Ten DPSC strains were obtained from cleft lip and palate patients and then used in the following study groups: group 1: control, the strains underwent osteogenic differentiation for 21 days; and groups 2, 3, and 4: the strains were irradiated each day with a low-power red laser (660 nm) (5, 10, and 20 J) during 21 days of osteogenic differentiation. Using Bonferroni's test, a statistically significant difference in the mean values was found between the irradiated groups (2, 3, and 4) and the control group (p < 0.001). However, no significant difference in osteogenic potential was found among the irradiated groups. Our findings showed that the osteogenic potential of DPSCs increases with red laser irradiation at 5, 10, and 20 J, and this treatment could be considered a new approach for preconditioning these cells to be used in bone tissue engineering.