Bone Tissue Engineering Constructs Research Articles

3D Printing of a Self-Healing, Bioactive, and Dual-Cross-Linked Polysaccharide-Based Composite Hydrogel as a Scaffold for Bone Tissue Engineering.

Although 3D printing is becoming a dominant technique for scaffold preparation in bone tissue engineering (TE), developing hydrogel-based ink compositions with bioactive and self-healing properties remains a challenge. This research focuses on developing a bone scaffold based on a composite hydrogel, which maintains its self-healing properties after incorporating bioactive glass and is 3D-printable. The plain hydrogel ink was synthesized using natural polymers of 1 wt % N-carboxyethyl chitosan, 2 wt % hyaluronic acid aldehyde, 0.3 wt % adipic acid hydrazide, and alginate (ALG) (2, 5, and 10 wt %). Bioactive glass (BG) (0 and 5 w/v %) particles were incorporated into the plain matrix to obtain an osteogenic composite hydrogel. The material was characterized via rheology, field emission scanning electron microscopy/energy-dispersive X-ray spectroscopy (FESEM/EDS), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), swelling, degradation, bioactivity, and in vitro cellular assessments. Rheological evaluations confirmed that the specimen with 0 w/v % BG and 5 wt % ALG exhibited the highest G', G″, and viscosity values. All specimens exhibited self-healing, provided by two reversible dynamic bonds, namely, imine and acylhydrazone. Bioactivity evaluation by SBF immersion revealed the formation of HA particles on the composite hydrogels. MTT cytotoxicity assay on MG63 indicated that the composite sample containing 5 w/v % BG and 10 wt % ALG had the highest cell viability (95 ± 1.02%) by culture day 3. The developed approach presents a promising hydrogel ink formulation with a high potential for extrusion-based 3D printing of bone TE constructs.

Abstract A029: Integrated modeling of renal cancer bone metastasis to illuminate tumor progression and therapy response

Abstract Bone metastatic clear cell renal cell carcinoma (BM ccRCC) is a persistent clinical challenge and a source of significant morbidity and lethality for up to 40% of metastatic patients. Despite the recent approval of life-prolonging agents (antiangiogenic agents, immunotherapy) patients with BM ccRCC will eventually progress, due to emerging resistance. Bidirectional communication between tumor cells and bone stroma has emerged as a key determinant of disease progression and recurrence. However, a major challenge in addressing the black box of therapy response in bone is the lack of efficient and informative systems that allow us to analyze the heterotypic stromal-epithelial interaction, further complicated by the anatomical inaccessibility of bone. For these reasons, the evolution of the metastatic niche and its implications in therapy response remain elusive. In order to improve clinical results, it is critical to establish biologically informed pre-clinical models to provide a mechanistic understanding of tumor progression in bone and treatment outcomes. To this purpose, we have established clinically relevant models of ccRCC bone metastasis that span in vivo and ex vivo multiphoton microscopy (iMPM and eMPM), tissue engineered bone window systems, and computational oncology. iMPM displays both sensitivity and time-resolution to identify dynamic interactions between ccRCC cells and bone 3D adaptive niches, which support therapy response and resistance. However, iMPM in bone is limited due to its cortical thickness, increased light scattering, and complex topology. As a novel alternative, we created a tissue-engineered bone construct (TEBC) that, after direct implantation of cancer cells, is combined with an adjacent skin window, allowing for non- destructive intravital examination of tumor growth. To flank these in vivo dynamic analyses, we established a pipeline for ex vivo extraction of topological information related to the molecular and cellular niches involved in tumor progression and response to treatment (eMPM). This method allows reconstruction of whole lesions, including zones more distant from the bone interface, paired with an extensive panel of molecular markers. To further investigate the effects of a broad multi-parameter space on tumor regression or persistence upon treatment, in silico, we developed an in vivo-inspired agent-based model (ABM) of ccRCC in bone. A computational approach combined to ad hoc biological experimentation can refine the experimental design, test clinically relevant hypotheses (including impact of treatments on disease progression) and predict scenarios that guide biological testing towards more successful outcomes. Consequently, by integrating these approaches, we are currently studying the progression of the bone metastatic niche and its response to therapy, with the ultimate goal of overcoming therapy failure. Citation Format: Sergio Barrios, Luca Marsilio, Stefan Maksimovic, Matthew Campbell, Stefano Casarin, Eleonora Dondossola. Integrated modeling of renal cancer bone metastasis to illuminate tumor progression and therapy response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A029.

Hepatocyte growth factor and insulin-like growth factor-1 were used to repair bone-cartilage defects in bone marrow mesenchymal stem cells in a rabbit model of postmenopausal

In postmenopausal osteoporosis (PMOP), an imbalance exists in the differentiation of bone marrow mesenchymal stem cells (BMSCs), with a decrease in osteogenic differentiation and an increase in adipogenic differentiation. This imbalance leads to bone marrow adiposity, bone loss, bone fragility, and a substantial rise in fracture risk. After a patient experiences an osteochondral defect due to trauma, it struggles to heal naturally, presenting a clinical challenge for treatment. Our study delved into the abnormal differentiation of BMSCs in PMOP by conducting transcriptome sequencing on BMSCs from a PMOP model (PMOP-BMSCs) and a healthy control model (Normal-BMSCs). We identified insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) genesas significantly low-expressed protein-coding genes during the osteogenic cartilage differentiation process of PMOP-BMSCs. Due to the downregulation of its expression, it leads to the deletion of the proteins it encodes IGF-1 and HGF. In order to verify the sequencing results, the feasibility of co-culture the above two growth factors with PMOP-BMSCs to repair osteochondral defects was discussed. The findings indicated that the inclusion of elements enhanced the DNA replication activity and extracellular matrix mineralization of PMOP-BMSCs. It also promoted the construction of tissue-engineered bone in vitro and the up- regulation of Runx2, BMP4, OCN, ACAN, collagen type Ⅰ, II, and Sox9 osteochondral differentiation markers. In the rabbit model of knee osteochondral injury with PMOP, the group treated with both growth factors and PMOP-BMSCs showed superior outcomes in repairing cartilage and subchondral bone defects compared to the other groups. We suggest that the addition of HGF and IGF-1 increases the expression of osteoblast and cartilage-related genes and proteins, promoting the proliferation and differentiation of osteochondrous bone in PMOP-BMSCs. These findings could offer a novel cell therapy strategy for treating postmenopausal osteoporosis, utilizing growth factors.

Biomimetic Mineralized 3D-Printed Polycaprolactone Scaffold Induced by Self-Adaptive Nanotopology to Accelerate Bone Regeneration.

Three-dimensional (3D)-printed biodegradable polymer scaffolds are at the forefront of personalized constructs for bone tissue engineering. However, it remains challenging to create a biological microenvironment for bone growth. Herein, we developed a novel yet feasible approach to facilitate biomimetic mineralization via self-adaptive nanotopography, which overcomes difficulties in the surface biofunctionalization of 3D-printed polycaprolactone (PCL) scaffolds. The building blocks of self-adaptive nanotopography were PCL lamellae that formed on the 3D-printed PCL scaffold via surface-directed epitaxial crystallization and acted as a linker to nucleate and generate hydroxyapatite crystals. Accordingly, a uniform and robust mineralized layer was immobilized throughout the scaffolds, which strongly bound to the strands and had no effect on the mechanical properties of the scaffolds. In vitro cell culture experiments revealed that the resulting scaffold was biocompatible and enhanced the proliferation and osteogenic differentiation of mouse embryolous osteoblast cells. Furthermore, we demonstrated that the resulting scaffold showed a strong capability to accelerate in vivo bone regeneration using a rabbit bone defect model. This study provides valuable opportunities to enhance the application of 3D-printed scaffolds in bone repair, paving the way for translation to other orthopedic implants.

Direct inkjet writing of polylactic acid/β-tricalcium phosphate composites for bone tissue regeneration: A proof-of-concept study.

There is an ever-evolving need of customized, anatomic-specific grafting materials for bone regeneration. More specifically, biocompatible and osteoconductive materials, that may be configured dynamically to fit and fill defects, through the application of an external stimulus. The objective of this study was to establish a basis for the development of direct inkjet writing (DIW)-based shape memory polymer-ceramic composites for bone tissue regeneration applications and to establish material behavior under thermomechanical loading. Polymer-ceramic (polylactic acid [PLA]/β-tricalcium phosphate [β-TCP]) colloidal gels were prepared of different w/w ratios (90/10, 80/20, 70/30, 60/40, and 50/50) through polymer dissolution in acetone (15% w/v). Cytocompatibility was analyzed through Presto Blue assays. Rheological properties of the colloidal gels were measured to determine shear-thinning capabilities. Gels were then extruded through a custom-built DIW printer. Space filling constructs of the gels were printed and subjected to thermomechanical characterization to measure shape fixity (Rf) and shape recovery (Rr) ratios through five successive shape memory cycles. The polymer-ceramic composite gels exhibited shear-thinning capabilities for extrusion through a nozzle for DIW. A significant increase in cellular viability was observed with the addition of β-TCP particles within the polymer matrix relative to pure PLA. Shape memory effect in the printed constructs was repeatable up to 4 cycles followed by permanent deformation. While further research on scaffold macro-/micro-geometries, and engineered porosities are warranted, this proof-of-concept study suggested suitability of this polymer-ceramic material and the DIW 3D printing workflow for the production of customized, patient specific constructs for bone tissue engineering.

Abstract 2830: Intravital multiphoton microscopy of bone metastatic renal cell carcinoma

Abstract Clear cell renal carcinoma (ccRCC) represents 80% of renal neoplasms, with bone as a major site for distant spread (35-40% of patients). These secondary lesions cause a variety of skeletal-related complications, including pain, spinal cord compression, hypercalcemia, mobility issues and fractures, posing a significant negative impact on patient quality of life and survival. Furthermore, bone is well-known as a site of therapy resistance, which confers to patients treated with systemic therapies significantly worse time-to-treatment failure and progression-free and overall survival compared to those without bone metastases. Because of the complexity of the bone microenvironment and the paucity of suitable models, preclinical investigation of bone metastasis and therapy response is challenging. Preclinical models of bone metastasis that integrate tissue engineering and advanced imaging techniques can facilitate the study of the mechanism of tumor progression and therapy response. We recently developed a window model amenable to intravital multiphoton microscopy (MPM) to longitudinally monitor human ccRCC lesions in tissue engineered bone constructs (TEBCs) in immunodeficient mice. TEBCs were generated by functionalizing polymeric polycaprolactone scaffolds with bone morphogenetic protein 7. Human ccRCC lesions, after implantation into the bone cavity, were longitudinally monitored for growth and niche development, using multi-parameter recording of collagen/bone matrix (SHG), bone surface (THG), blood vessels/stromal phagocytes (fluorescent dextran), and tumor cells (GFP). However, this model lacked key components of the immune system (including B and T-cells), which are of major interest in the study of tumor progression and (immune) therapy response. Therefore, we developed a tissue-engineered preclinical model that incorporate species-specific interactions between cancer and bone stromal cells, and account for an intact immune system. To this purpose, we optimized TEBC formation in C57BL/6 fluorescent reporter mice (Sp7-mCherry, osteoblasts; Trap-td-Tomato, osteoclasts; GFP, immune cells, αSMA-RFP fibroblasts; and Flk1-GFP, blood vessels), coupled to detection by intravital or ex vivo multiphoton microscopy analysis, to monitor the evolution and fate of the TEBC. Interestingly, the bone marrow in the core of the TEBC was replaced by a desmoplastic tissue with features like the foreign body response, including foreign body giant cells, which was not present in immunodeficient mice. By applying dual color GFP/αSMA-RFP mice, which expressed red fluorescence in activated fibroblasts, we confirmed the fibrotic nature of this core. To avoid its formation, we generated scaffold-free ossicle, by direct implantation of BMP7 and VEGF in the subcutaneous tissue. To conclude, we established a protocol for generating ectopic ossicles in immunocompetent mice. Citation Format: Stefan Maksimovic, Nina Constanza Boscolo, Sergio Barrios, Antonios Mikos, Matthew T. Campbell, Eleonora Dondossola. Intravital multiphoton microscopy of bone metastatic renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2830.

Microgels-Encapsulated Magnesium/Emodin-based metal organic framework nanorods for diabetic bone regeneration

Diabetic bone defect repair is one of the major challenges in clinic, because the pathological microenvironments such as hyperglycemia, oxidative stress, and inflammation exist in the bone defect area of diabetes mellitus. Although the current tissue-engineered bone has achieved favorable bone regeneration and functional reconstruction, it is still unsatisfactory for diabetic bone defect repair only by correcting a single pathological factor. In this study, we develop a multifunctional nano-releasing system of GelMA/HAMA microgels-encapsulated Mg2+/Emodin (MgEm) nanorods based on MOF design principle (denoted as MEGH) for reversing the diabetic pathological microenvironment. For the slow-release system of MEGH microgels, Emodin exert the hypoglycemic, antioxidant, and anti-inflammatory function, while Mg2+ ions could promote angiogenesis for bone regeneration. Subsequently, we combine decalcified bone matrix with the bone regenerative units based on BMSCs-loaded MEGH microgels (BMSCs@MEGH-D) for the construction of tissue-engineered bone. Finally, the pre-constructed BMSCs@MEGH-D scaffolds successfully promote the vascularized bone regeneration in diabetic rabbit skull models due to the effective correction of diabetic pathological microenvironment. The underlying mechanism indicates a synergetic efficacy of regulate glucose, inflammation, oxygen-related bioprocesses, as well as osteogenesis-related pathways. Our findings provide a promising treatment for reversing the diabetic pathological microenvironment to accelerate bone defect repair.

Mechanoregulation analysis of bone formation in tissue engineered constructs requires a volumetric method using time-lapsed micro-computed tomography

Bone can adapt its microstructure to mechanical loads through mechanoregulation of the (re)modeling process. This process has been investigated in vivo using time-lapsed micro-computed tomography (micro-CT) and micro-finite element (FE) analysis using surface-based methods, which are highly influenced by surface curvature. Consequently, when trying to investigate mechanoregulation in tissue engineered bone constructs, their concave surfaces make the detection of mechanoregulation impossible when using surface-based methods. In this study, we aimed at developing and applying a volumetric method to non-invasively quantify mechanoregulation of bone formation in tissue engineered bone constructs using micro-CT images and FE analysis. We first investigated hydroxyapatite scaffolds seeded with human mesenchymal stem cells that were incubated over 8 weeks with one mechanically loaded and one control group. Higher mechanoregulation of bone formation was measured in loaded samples with an area under the curve for the receiver operating curve (AUCformation) of 0.633–0.637 compared to non-loaded controls (AUCformation: 0.592–0.604) during culture in osteogenic medium (p < 0.05). Furthermore, we applied the method to an in vivo mouse study investigating the effect of loading frequencies on bone adaptation. The volumetric method detected differences in mechanoregulation of bone formation between loading conditions (p < 0.05). Mechanoregulation in bone formation was more pronounced (AUCformation: 0.609–0.642) compared to the surface-based method (AUCformation: 0.565–0.569, p < 0.05). Our results show that mechanoregulation of formation in bone tissue engineered constructs takes place and its extent can be quantified with a volumetric mechanoregulation method using time-lapsed micro-CT and FE analysis. Statement of significanceMany efforts have been directed towards optimizing bone scaffolds for tissue growth. However, the impact of the scaffolds mechanical environment on bone growth is still poorly understood, requiring accurate assessment of its mechanoregulation. Existing surface-based methods were unable to detect mechanoregulation in tissue engineered constructs, due to predominantly concave surfaces in scaffolds. We present a volumetric approach to enable the precise and non-invasive quantification and analysis of mechanoregulation in bone tissue engineered constructs by leveraging time-lapsed micro-CT imaging, image registration, and finite element analysis. The implications of this research extend to diverse experimental setups, encompassing culture conditions, and material optimization, and investigations into bone diseases, enabling a significant stride towards comprehensive advancements in bone tissue engineering and regenerative medicine.

Hypoxia as a stimulus for tissue formation: The concept of organogenesis in microsurgically vascularized tissue engineering constructs

Axial vascularization of tissue constructs is essential to maintain an adequate blood supply for a stable regeneration of a clinically relevant tissue size. The versatility of the arterio-venous loop (AVL) has been previously shown in various small and large animal models as well as in clinical reports for bone regeneration. We have previously demonstrated the capability of the AVL to induce axial vascularization and to support the nourishment of tissue constructs in small animal models after applying high doses of ionizing radiation comparable to those applied for adjuvant radiotherapy after head and neck cancer. We hypothesize that this robust ability to induce regeneration after irradiation could be related to a state of hypoxia inside the constructs that triggers the HIF1 (hypoxia induced factor 1) - SDF1 (stromal derived factor 1) axis leading to chemotaxis of progenitor cells and induction of tissue regeneration and vascularization. We analyzed the expression of HIF1 and SDF1 via immunofluorescence in axially vascularized bone tissue engineering constructs in Lewis rats 2 and 5 weeks after local irradiation with 9Gy or 15Gy. We also analyzed the expression of various genes for osteogenic differentiation (collagen 1, RUNX, alkaline phosphatase and osteonectin) via real time PCR analysis. The expression of HIF1 and SDF1 was enhanced two weeks after irradiation with 15Gy in comparison to non-irradiated constructs. The expression of osteogenic markers was enhanced at the 5-weeks time point with significant results regarding collagen, alkaline phosphatase and osteonectin. These results indicate that the hypoxia within the AVL constructs together with an enhanced SDF1 expression probably play a role in promoting tissue differentiation. The process of tissue generation triggered by hypoxia in the vicinity of a definite vascular axis with enhanced tissue differentiation over time resembles hereby the well-known concept of organogenesis in fetal life.

Tissue-engineered bone construct promotes early osseointegration of implants with low primary stability in oversized osteotomy

ObjectivesTo evaluate the histological parameters and bone mechanical properties around implants with low primary stability (PS) in grafted bone substitutes within an oversized osteotomy.Materials and methodsAn oversized osteotomy penetrating the double cortical bone layers was made on both femora of 24 New Zealand white rabbits. Bilaterally in the femur of all animals, 48 implants were installed, subdivided into four groups, corresponding to four prepared tissue-engineering bone complexes (TEBCs), which were placed between the implant surface and native bone wall: A: tricalcium phosphate β (TCP-β); B: autologous adipose derived-stem cells with TCP-β (ASCs/TCP-β); C: ASCs transfected with the enhanced-GFP gene with TCP-β (EGFP-ASCs/TCP-β); D: ASCs transfected with the BMP-2 gene with TCP-β (BMP2-ASCs/TCP-β). Trichrome fluorescent labeling was conducted. Animals were sacrificed after eight weeks. The trichromatic fluorescent labeling (%TFL), area of new bone (%NB), residual material (%RM), bone-implant contact (%BIC), and the removal torque force (RTF, N/cm) were assessed.ResultsASCs were successfully isolated from adipose tissue, and the primary ASCs were induced into osteogenic, chondrogenic, and adipogenic differentiation. The BMP-2 overexpression of ASCs sustained for ten days and greatly enhanced the expression of osteopontin (OPN). At eight weeks post-implantation, increased %NB and RTF were found in all groups. The most significant value of %TFL, %BIC and lowest %RM was detected in group D.ConclusionThe low PS implants osseointegrate with considerable new bone in grafted TEBCs within an oversized osteotomy. Applying BMP-2 overexpressing ASCs-based TEBC promoted earlier osseointegration and more solid bone mechanical properties on low PS implants. Bone graft offers a wedging effect for the implant with low PS at placement and promotes osteogenesis on their surface in the healing period.

TRIAZINE-TRIONE BASED COMPOSITE MATERIALS FOR POTENTIAL BONE TISSUE ENGINEERING

Several synthetic polymers have been widely investigated for their use in bone tissue engineering applications, but the ideal material is yet to be engineered. Triazine-trione (TATO) based materials and their derivatives are novel in the field of biomedical engineering but have started to draw interest. Different designs of the TATO monomers and introduction of different chemical linkages and end-groups widens the scope of the materials due to a range of mechanical properties.The aim of our work is to investigate novel TATO based materials, with or without hydroxyapatite filler, for their potential in bone tissue engineering constructs. Initially the biocompatibility of the materials was tested, indirectly and directly, according to ISO standards. Following this the osteoconductive properties were investigated with primary osteoblasts and an osteoblastic cell line. Bone marrow derived mesenchymal stem cells were used to evaluate the osteogenic differentiation and consequently the materials potential in bone tissue engineering applications.

Investigation of the biological activity of hydroxyapatite on vascular endothelial cells

ObjectiveThis study aims to explore the impact of nano-hydroxyapatite (na-HA) and micron-hydroxyapatite (mi-HA) on human umbilical vein endothelial cells (HUVEC) using in vitro experiments, assessing their influence on cellular biological activity. These findings offer crucial experimental data for informing the development of more vascularized tissue-engineered bone constructs. MethodsWe employed the Cell Counting Kit-8 (CCK-8) assay to assess the impact of various concentrations of both HA extracts on HUVEC metabolic activity post 48, 72, and 96 h of treatment. Transwell experiments were conducted to evaluate the influence of HA extract on HUVEC migratory capabilities. The cell proliferation activity was assessed using the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, elucidating the impact of varying concentrations of both HA extracts on cell proliferation. Lumen formation experiments were conducted to assess the capacity of HA-treated HUVECs to form lumen-like structures. The Enzyme-Linked Immunosorbent Assay (ELISA) was employed to measure the impact of HA extract on vascular endothelial growth factor (VEGF) secretion by HUVECs. Western blotting (WB) was utilized to analyze alterations in the expression levels of PI3K/Akt signaling pathway-related proteins following HA extract treatment of cells. ResultsAt extract concentrations of 100 g/L and 12.5 g/L, both the mi-HA and na-HA groups demonstrated suppression of cell metabolic activity, migration, and proliferation. Conversely, at 25 g/L, increased cell metabolic activity and proliferative activity were observed. Lumen formation experiments demonstrated that both HA extracts at 100 g/L concentration facilitated lumen formation, with the na-HA group at 25 g/L concentration displaying a more pronounced impact on lumen formation. The ELISA results indicated a notable reduction in VEGF secretion within the mi-HA group at a concentration of 100 g/L. WB experiments revealed that within the na-HA group, treatment of HUVECs with 25 g/L and 12.5 g/L extract concentrations led to upregulation of PI3K and Akt protein expression, while at 100 g/L concentration, Akt protein expression decreased. In the mi-HA group, intracellular expression of both PI3K and Akt proteins exhibited reduction. ConclusionHydroxyapatite extract at both high and low concentrations impacts the biological activity of vascular endothelial cells, with the potential mechanism of action involving the PI3K/Akt signaling pathway.

Effect of Uniaxial Compression Frequency on Osteogenic Cell Responses in Dynamic 3D Cultures.

The application of mechanical stimulation on bone tissue engineering constructs aims to mimic the native dynamic nature of bone. Although many attempts have been made to evaluate the effect of applied mechanical stimuli on osteogenic differentiation, the conditions that govern this process have not yet been fully explored. In this study, pre-osteoblastic cells were seeded on PLLA/PCL/PHBV (90/5/5 wt.%) polymeric blend scaffolds. The constructs were subjected every day to cyclic uniaxial compression for 40 min at a displacement of 400 μm, using three frequency values, 0.5, 1, and 1.5 Hz, for up to 21 days, and their osteogenic response was compared to that of static cultures. Finite element simulation was performed to validate the scaffold design and the loading direction, and to assure that cells inside the scaffolds would be subjected to significant levels of strain during stimulation. None of the applied loading conditions negatively affected the cell viability. The alkaline phosphatase activity data indicated significantly higher values at all dynamic conditions compared to the static ones at day 7, with the highest response being observed at 0.5 Hz. Collagen and calcium production were significantly increased compared to static controls. These results indicate that all of the examined frequencies substantially promoted the osteogenic capacity.

Biofabricating the vascular tree in engineered bone tissue.

The development of tissue engineering strategies for treatment of large bone defects has become increasingly relevant, given the growing demand for bone substitutes. Native bone is composed of a dense vascular network necessary for the regulation of bone development, regeneration and homeostasis. A major obstacle in fabricating living, clinically relevant-sized bone mimics (1-10 cm3) is the limited supply of nutrients, including oxygen to the core of the construct. Therefore, strategies to support vascularization are pivotal for the development of tissue engineered bone constructs. Creating a functional bone construct integrated with a vascular network, capable of delivering the necessary nutrients for optimal tissue development is imperative for translation into the clinics. The vascular system is composed of a complex network that runs throughout the body in a tree-like hierarchical branching fashion. A significant challenge for tissue engineering approaches lies in mimicking the intricate, multi-scale structures consisting of larger vessels (macro-vessels) which interconnect with multiple sprouting vessels (microvessels) in a closed network. The advent of biofabrication has enabled complex, out of plane channels to be generated and has laid the groundwork for the creation of multi-scale vasculature in recent years. This review highlights the key state-of-the-art achievements for the development of vascular networks of varying scales in the field of biofabrication with a particular focus for its application in developing a functional tissue engineered bone construct. STATEMENT OF SIGNIFICANCE: There is a growing need for bone substitutes to overcome the limited supply of patient-derived bone. Bone tissue engineering aims to overcome this by combining stem cells with scaffolds to restore missing bone. The current bottleneck in upscaling is the lack of an integrated vascular network, required for the delivery of nutrients to cells. 3D bioprinting techniques has enabled the creation of complex hollow structures of varying dimensions that resemble native blood vessels. The convergence of multiple materials, cell types and fabrication approaches, opens the possibility of developing clinically-relevant sized vascularized bone constructs. This review provides an up-to-date insight of the technologies currently available for the generation of complex vascular networks, with a focus on their application in bone tissue engineering.

Periodontal ligament stem cell-based bioactive constructs for bone tissue engineering.

Objectives: Stem cell-based tissue engineering approaches are promising for bone repair and regeneration. Periodontal ligament stem cells (PDLSCs) are a promising cell source for tissue engineering, especially for maxillofacial bone and periodontal regeneration. Many studies have shown potent results via PDLSCs in bone regeneration. In this review, we describe recent cutting-edge researches on PDLSC-based bone regeneration and periodontal tissue regeneration. Data and sources: An extensive search of the literature for papers related to PDLSCs-based bioactive constructs for bone tissue engineering was made on the databases of PubMed, Medline and Google Scholar. The papers were selected by three independent calibrated reviewers. Results: Multiple types of materials and scaffolds have been combined with PDLSCs, involving xeno genic bone graft, calcium phosphate materials and polymers. These PDLSC-based constructs exhibit the potential for bone and periodontal tissue regeneration. In addition, various osteo inductive agents and strategies have been applied with PDLSCs, including drugs, biologics, gene therapy, physical stimulation, scaffold modification, cell sheets and co-culture. Conclusoin: This review article demonstrates the great potential of PDLSCs-based bioactive constructs as a promising approach for bone and periodontal tissue regeneration.

Lnc Tmem235 promotes repair of early steroid-induced osteonecrosis of the femoral head by inhibiting hypoxia-induced apoptosis of BMSCs

Bone marrow mesenchymal stem cells (BMSCs) have been used in the treatment of early steroid-induced osteonecrosis of the femoral head (SONFH). However, the hypoxic microenvironment in the osteonecrotic area leads to hypoxia-induced apoptosis of transplanted BMSCs, which limits their efficacy. Therefore, approaches that inhibit hypoxia-induced apoptosis of BMSCs are promising for augmenting the efficacy of BMSC transplantation. Our present study found that under hypoxia, the expression of the long noncoding RNA (Lnc) transmembrane protein 235 (Tmem235) was downregulated, the expression of Bcl-2-associated X protein was upregulated, the expression of B-cell lymphoma-2 protein was downregulated, and the apoptotic rate of BMSCs was over 70%. However, overexpression of Lnc Tmem235 reversed hypoxia-induced apoptosis of BMSCs and promoted their survival. These results demonstrated that Lnc Tmem235 effectively inhibited hypoxia-induced apoptosis of BMSCs. Mechanistically, we found that Lnc Tmem235 exhibited competitive binding to miR-34a-3p compared with BIRC5 mRNA, which is an inhibitor of apoptosis; this competitive binding relieved the silencing effect of miR-34a-3p on BIRC5 mRNA to ultimately inhibit hypoxia-induced apoptosis of BMSCs by promoting the expression of BIRC5. Furthermore, we cocultured BMSCs overexpressing Lnc Tmem235 with xenogeneic antigen-extracted cancellous bone to construct tissue-engineered bone to repair a model of early SONFH in vivo. The results showed that overexpression of Lnc Tmem235 effectively reduced apoptosis of BMSCs in the hypoxic microenvironment of osteonecrosis and improved the effect of BMSC transplantation. Taken together, our findings show that Lnc Tmem235 inhibited hypoxia-induced apoptosis of BMSCs by regulating the miR-34a-3p/BIRC5 axis, thus improving the transplantation efficacy of BMSCs for treating early SONFH.

Osteogenic Potential of Sheep Mesenchymal Stem Cells Preconditioned with BMP-2 and FGF-2 and Seeded on an nHAP-Coated PCL/HAP/β-TCP Scaffold.

Mesenchymal stem cells (MSCs) attract interest in regenerative medicine for their potential application in bone regeneration. However, direct transplantation of cells into damaged tissue is not efficient enough to regenerate large bone defects. This problem could be solved with a biocompatible scaffold. Consequently, bone tissue engineering constructs based on biomaterial scaffolds, MSCs, and osteogenic cytokines are promising tools for bone regeneration. The aim of this study was to evaluate the effect of FGF-2 and BMP-2 on the osteogenic potential of ovine bone marrow-derived MSCs seeded onto an nHAP-coated PCL/HAP/β-TCP scaffold in vitro and its in vivo biocompatibility in a sheep model. In vitro analysis revealed that cells preconditioned with FGF-2 and BMP-2 showed a better capacity to adhere and proliferate on the scaffold than untreated cells. BM-MSCs cultured in an osteogenic medium supplemented with FGF-2 and BMP-2 had the highest osteogenic differentiation potential, as assessed based on Alizarin Red S staining and ALP activity. qRT-PCR analysis showed increased expression of osteogenic marker genes in FGF-2- and BMP-2-treated BM-MSCs. Our pilot in vivo research showed that the implantation of an nHAP-coated PCL/HAP/β-TCP scaffold with BM-MSCs preconditioned with FGF-2 and BMP-2 did not have an adverse effect in the sheep mandibular region and induced bone regeneration. The biocompatibility of the implanted scaffold-BM-MSC construct with sheep tissues was confirmed by the expression of early (collagen type I) and late (osteocalcin) osteogenic proteins and a lack of an elevated level of proinflammatory cytokines. These findings suggest that FGF-2 and BMP-2 enhance the osteogenic differentiation potential of MSCs grown on a scaffold, and that such a tissue engineering construct may be used to regenerate large bone defects.

Fluid shear stress promotes osteogenesis of bone mesenchymal stem cells at early matrix maturity phase through Lamin A/ METTL3 signal axis

During the construction of tissue-engineered bone in vitro, bionic mechanical stimulation has been considered to be critical to cellular response and bone tissue formation. Bone mesenchymal stem cells (BMSCs) at different osteogenesis phases in the bone marrow cavity are subjected to fluid shear stress (FSS). However, the responses of BMSCs at different phases of osteogenic differentiation to FSS remain unknown. Therefore, FSS was applied to BMSCs at different osteogenesis phases in vitro by parallel plate flow chamber, and the osteogenic ability and relaed underlying mechanisms were explored. Our study showed that BMSCs underwent rapid proliferation, early matrix maturity and late mineralization phases at 1, 7 and 14 days of osteogenic induction, respectively. FSS significantly promoted the osteogenic differentiation of BMSCs at early matrix maturation phase. Applying FSS on BMSCs at early matrix maturity phase resulted in significant remodeling of F-actin, forming of stress fibers, and the transduction of mechanical signal by the cytoskeleton-Lamin A pathway. Through the interaction between Lamin A and Methyltransferase-like 3 (METTL3), the levels of METTL3 protein and the m6A methylation genes were increased, promoting the osteoblastic differentiation of BMSCs. The results provided a new insight into the construction of bone tissue in vitro and the treatment strategy for bone defects. Data AvailabilityThe data used to support the findings of this study are available from the corresponding author upon request.

The SIRT1 activator SRT2104 promotes BMP9-induced osteogenic and angiogenic differentiation in mesenchymal stem cells

Bone defects resulting from trauma, bone tumors, infections and skeletal abnormalities are a common osteoporotic condition with respect to clinical treatment. Of the known bone morphogenetic proteins (BMPs), BMP9 has the strongest osteogenic differentiation potential, which could be beneficial in the construction of tissue-engineered bone. Silent mating type information regulator 2 homolog-1 (SIRT1) is a highly conserved nicotinamide adenine dinucleotide-dependent deacetylase that deacetylates and modulates histone or non-histone substrates. However, the role of SIRT1 in BMP9-induced osteogenic differentiation of stem cells has not been studied. Furthermore, it is unclear whether SIRT1 interacts with the BMP/Smad and BMP/MAPK pathways in stem cells. We found that SIRT1 expression decreased gradually in a time-dependent manner during BMP9-induced osteogenic differentiation of MSCs. Interactions between SIRT1 and Smad7 promoted degradation of Smad7 and increased Smad1/5/8 phosphorylation. SRT2104, an activator of SIRT, enhanced the expression of osteogenic- and angiogenic-related proteins in BMP9-induced MSCs. In addition, we found that activation of the BMP/MAPK pathway led to osteogenic and angiogenic differentiation of MSCs. Our study demonstrated that SIRT1 expression decreased during BMP9-induced differentiation. The SIRT1 activator SRT2104 promoted BMP9-induced osteogenic and angiogenic differentiation of MSCs through the BMP/Smad and BMP/MAPK signaling pathways.

Promotion of angiogenesis in vitro by Astragalus polysaccharide via activation of TLR4 signaling pathway.

During the implantation of functional tissue-engineered constructs for treating bone defects, a functional vascular network is critical for the survival of the construct. One strategy to achieve rapid angiogenesis for this application is the co-culture of outgrowth endothelial cells (OECs) and primary human osteoblasts (POBs) within a scaffold prior to implantation. In the present study, we aim to investigate whether Astragalus polysaccharide (APS) promotes angiogenesis or vascularization via the TLR4 signaling pathway in a co-culture of OECs and POBs. The co-cultures were treated with various concentrations of APS for 24 h and, subsequently, another 7 days, followed by CD31 staining and analysis of micro-vessel-formation areas using software. Additionally, APS (0.4mg/ml for 24 h) was added to monocultures of OECs or POBs for evaluating proliferation, apoptosis, angiogenesis, osteogenesis, TLR4 signaling pathway, and inflammatory cytokine release. We found that APS promoted angiogenesis in the co-culture at the optimal concentration of 0.4mg/ml. TLR4 activation by APS up-regulated the expression level of TLR4/MyD88 and enhanced angiogenesis and osteogenesis in monocultures of OECs and POBs. The levels of E-selectin adhesion molecules, three cytokines (IL-6, TNF-α, and IFN-γ), and VEGF and PDGF-BB, which can induce angiogenesis, increased significantly (p < .05) following APS treatment. Therefore, APS appears to promote angiogenesis and ossification in the co-culture system via the TLR4 signaling pathway. PRACTICAL APPLICATIONS: This study demonstrates that APS may promote angiogenesis and osteocyte proliferation in OEC and POB co-culture systems through the MyD88-dependent TLR4 signaling pathway. APS might represent a potential therapeutic strategy in tissue-engineered bone implantation for the treatment of large bone defects; additionally, it has the advantage of safety, as it exhibits low or no side effects. In the future, it is expected to be used in vitro for the construction of tissue-engineered bone and in vivo after implantation in patients with bone defects for promoting rapid vascularization and ossification of tissue-engineered bone and early fusion with the recipient's bone. In addition, as a food additive, Astragalus membranaceus can be used as a tonic material in patients recovering from a fracture for promoting blood-vessel formation at the fracture site and fracture recovery. Combining traditional Chinese medicine with tissue engineering can provide further strategies for promoting the development of regenerative medicine.