Osteogenic Supplements Research Articles

Borosilicate bioactive glasses with added Mg/Sr enhances human adipose-derived stem cells osteogenic commitment and angiogenic properties

Bioactive glasses are one of the most promising materials for applications in bone tissue engineering. In this study, the focus was on borosilicate bioactive glasses with composition 47.12 SiO2 - 6.73 B2O3 - 21.77-x-y CaO - 22.65 Na2O - 1.72 P2O5 - x MgO - y SrO (mol%). These compositions are based on silicate S53P4 bioactive glass, from where 12.5% of SiO2 is replaced with B2O3, and additionally, part of CaO is substituted for MgO and/or SrO. The impact of ion release, both as extract and in direct contact, on human adipose-derived stem cells’ (hADSCs) viability, proliferation, ECM maturation, osteogenic commitment and endothelial marker expression was assessed. Osteogenic media supplements were utilized with the extracts, and in part of the direct cell/material culturing conditions. While it has been reported in other studies that boron release can induce cytotoxicity, the glasses in this study supported cells viability and proliferation. Moreover, borosilicate’s, especially with further Mg/Sr substitutions, upregulated several osteogenic markers (such as RUNX2a, OSTERIX, DLX5, OSTEOPONTIN), as well as angiogenic factors (e.g., vWF and PECAM-1). Furthermore, the studied glasses supported collagen-I production even in the absence of osteogenic supplements, when hADSCs were cultured in contact with the glasses, suggesting that while the bioactive glass degradation products are beneficial for osteogenesis, the glasses surface physico-chemical properties play a significant role on hADSCs differentiation. This study brings critical information on the impact of bioactive glass compositional modification to control glass dissolution and the subsequent influence on stem cells proliferation and differentiation. Furthermore, the role of the material surface chemistry on promoting cell differentiation is reported.Graphical

Driving Osteocytogenesis from Mesenchymal Stem Cells in Osteon-like Biomimetic Nanofibrous Scaffolds.

The treatment of critical-sized bone defects caused by tumor removal, skeletal injuries, or infections continues to pose a major clinical challenge. A popular potential alternative solution to autologous bone grafts is a tissue-engineered approach that utilizes the combination of mesenchymal stromal/stem cells (MSCs) with synthetic biomaterial scaffolds. This approach aims to support new bone formation by mimicking many of the biochemical and biophysical cues present within native bone. Regrettably, osteocyte cells, crucial for bone maturation and homeostasis, are rarely produced within MSC-seeded scaffolds, thereby restricting the development of fully mature cortical bone from these synthetic implants. In this work, we have constructed a multimodal scaffold by combining electrospun poly(lactic-co-glycolic acid) (PLGA) fibrous scaffolds with poly(ethylene glycol) (PEG)-based hydrogels that mimic the functional unit of cortical bone, osteon (osteon-mimetic) scaffolds. These scaffolds were decorated with a novel bone morphogenic protein-6 (BMP6) peptide (BMP6p) after our findings revealed that the BMP6p drives higher levels of Smad signaling than the full-length protein counterpart, soluble or when bound to the PEG hydrogel backbone. We show that our osteon-mimetic scaffolds, in presenting concentric layers of BMP6p-PEG hydrogel overlaid on MSC-seeded PLGA nanofibers, promoted the rapid formation of osteocyte-like cells with a phenotypic dendritic morphology, producing early osteocyte markers, including E11/gp38 (E11). Maturation of these osteocyte-like cells was further confirmed by the observation of significant dentin matrix protein 1 (DMP1) throughout our bilayered scaffolds after 3 weeks, even when cultured in a medium without dexamethasone (DEX) or any other osteogenic supplements. These results demonstrate that these osteon-mimetic scaffolds, in presenting biochemical and topographical cues reminiscent of the forming osteon, can drive the formation of osteocyte-like cells in vitro from hBMSCs without the need for any osteogenic factor media supplementation.

Managing Oxidative Stress Using Vitamin C to Improve Biocompatibility of Polycaprolactone for Bone Regeneration In Vitro.

Increased oxidative stress in bone cells is known to negatively alter favorable bone regeneration. This study aimed to develop a porous polycaprolactone (PCL) membrane incorporated with 25 wt % Vitamin C (PCL-Vit C) and compared it to the PCL membrane to control oxidative stress and enhance biomineralization in vitro. Both membranes were characterized using SEM-EDS, FTIR spectroscopy, and surface hydrophilicity. Vitamin C release was quantified colorimetrically. Assessments of the viability and attachment of human fetal osteoblast (hFOB 1.19) cells were carried out using XTT assay, SEM, and confocal microscopy, respectively. ROS generation and wound healing percentage were measured using flow cytometry and ImageJ software, respectively. Mineralization study using Alizarin Red in the presence or absence of osteogenic media was carried out to measure the calcium content. Alkaline phosphatase assay and gene expression of osteogenic markers (alkaline phosphatase (ALP), collagen Type I (Col1), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN)) were analyzed by real-time PCR. SEM images revealed smooth, fine, bead-free fibers in both membranes. The FTIR spectrum of pure vitamin C was replaced with peaks at 3436.05 and 2322.83 cm-1 in the PCL-Vit C membrane. Vitamin C release was detected at 15 min and 1 h. The PCL-Vit C membrane was hydrophilic, generated lower ROS, and showed significantly higher viability than the PCL membrane. Although both PCL and PCL-Vit C membranes showed similar cellular and cytoskeletal morphology, more cell clusters were evident in the PCL-Vit C membrane. Lower ROS level in the PCL-Vit C membrane displayed improved cell functionality as evidenced by enhanced cellular differentiation with more intense alizarin staining and higher calcium content, supported by upregulation of osteogenic markers ALP, Col1, and OPN even in the absence of osteogenic supplements. The presence of Vitamin C in the PCL-Vit C membrane may have mitigated oxidative stress in hFOB 1.19 cells, resulting in enhanced biomineralization facilitating bone regeneration.

Clinical potential of plasma-functionalized graphene oxide ultrathin sheets for bone and blood vessel regeneration: Insights from cellular and animal models

Graphene and graphene oxide (GO), due to their unique chemical and physical properties, possess biochemical characteristics that can trigger intercellular signals promoting tissue regeneration. Clinical applications of thin GO-derived sheets have inspired the development of various tissue regeneration and repair approaches. In this study, we demonstrate that ultrathin sheets of plasma-functionalized and reduced GO, with the oxygen content ranging from 3.2 % to 22 % and the nitrogen content from 0 % to 8.3 %, retain their essential mechanical and molecular integrity, and exhibit robust potential for regenerating bone tissue and blood vessels across multiple cellular and animal models. Initially, we observed the growth of blood vessels and bone tissue in vitro using these functionalized GO sheets on human adipose-derived mesenchymal stem cells and umbilical vein endothelial cells. Remarkably, our study indicates a 2.5-fold increase in mineralization and two-fold increase in tubule formation even in media lacking osteogenic and angiogenic supplements. Subsequently, we observed the initiation, conduction, and formation of bone and blood vessels in a rat tibial osteotomy model, evident from a marked 4-fold increase in the volume of low radio-opacity bone tissue and a significant elevation in connectivity density, all without the use of stem cells or growth factors. Finally, we validated these findings in a mouse critical-size calvarial defect model (33 % higher healing rate) and a rat skin lesion model (up to 2.5-fold increase in the number of blood vessels, and 35 % increase in blood vessels diameter). This study elucidates the pro-osteogenic and pro-angiogenic properties of both pristine and plasma-treated GO ultrathin films. These properties suggest their significant potential for clinical applications, and as valuable biomaterials for investigating fundamental aspects of bone and blood vessel regeneration.

Sustained adenosine release: Revealing its impact on osteogenic signalling pathways of human mesenchymal stromal cells

Non-healing fractures, a global health concern arising from trauma, osteoporosis, and tumours, can lead to severe disabilities. Adenosine, integral to cellular energy metabolism, gains prominence in bone regeneration via adenosine A2B receptor activation. This study introduces a controlled-release system for localized adenosine delivery, fostering human mesenchymal stromal cell (hMSC) differentiation into functional bone cells. The study investigates how the ratio of lactic acid to glycolic acid in microparticles can influence adenosine release and explores the downstream effects on gene expression and metabolic profiles of osteogenic differentiation in hMSCs cultured in growth and osteoinductive media. Insights into adenosine-modulated signalling pathways during MSC differentiation, with osteogenic factors, provide a comprehensive understanding of the pathways involved. Analysing gene expression and metabolic profiles unravels adenosine's regulatory mechanisms in MSC differentiation. Sustained adenosine release from microparticles induces mineralization, synergizing with osteogenic media supplements, showcasing the potential of adenosine for treating critical bone defects and metabolic disorders. This study highlights the efficacy of a polymeric microparticle-based delivery system, offering novel strategies for bone repair. Unveiling adenosine's roles and associated signalling pathways advances our comprehension of molecular mechanisms steering bone regeneration, propelling innovative biomaterial, combined with metabolites, approaches for clinical use.

Strontium and Zinc Co-Doped Mesoporous Bioactive Glass Nanoparticles for Potential Use in Bone Tissue Engineering Applications.

Mesoporous bioactive glass nanoparticles (MBGNs) have attracted significant attention as multifunctional nanocarriers for various applications in both hard and soft tissue engineering. In this study, multifunctional strontium (Sr)- and zinc (Zn)-containing MBGNs were successfully synthesized via the microemulsion-assisted sol-gel method combined with a cationic surfactant (cetyltrimethylammonium bromide, CTAB). Sr-MBGNs, Zn-MBGNs, and Sr-Zn-MBGNs exhibited spherical shapes in the nanoscale range of 100 ± 20 nm with a mesoporous structure. Sr and Zn were co-substituted in MBGNs (60SiO2-40CaO) to induce osteogenic potential and antibacterial properties without altering their size, morphology, negative surface charge, amorphous nature, mesoporous structure, and pore size. The synthesized MBGNs facilitated bioactivity by promoting the formation of an apatite-like layer on the surface of the particles after immersion in Simulated Body Fluid (SBF). The effect of the particles on the metabolic activity of human mesenchymal stem cells was concentration-dependent. The hMSCs exposed to Sr-MBGNs, Zn-MBGNs, and Sr-Zn-MBGNs at 200 μg/mL enhanced calcium deposition and osteogenic differentiation without osteogenic supplements. Moreover, the cellular uptake and internalization of Sr-MBGNs, Zn-MBGNs, and Sr-Zn-MBGNs in hMSCs were observed. These novel particles, which exhibited multiple functionalities, including promoting bone regeneration, delivering therapeutic ions intracellularly, and inhibiting the growth of Staphylococcus aureus and Escherichia coli, are potential nanocarriers for bone regeneration applications.

Using osteogenic medium in the in vitro evaluation of bone biomaterials: Artefacts due to a synergistic effect

In vitro tests using bone cells to evaluate the osteogenic potential of biomaterials usually employ the osteogenic medium (OM). The lack of correlation frequently reported between in vitro and in vivo studies in bone biomaterials, makes necessary the evaluation of the impact of osteogenic supplements on these results. This study analysed the proteomic profiles of human osteoblasts (HOb) cultured in the media with and without osteogenic agents (ascorbic acid and β-glycerol phosphate). The cells were incubated for 1 and 7 days, on their own or in contact with Ti. The comparative Perseus analysis identified 2544 proteins whose expression was affected by osteogenic agents. We observed that the OM strongly alters protein expression profiles with a complex impact on multiple pathways associated with adhesion, immunity, oxidative stress, coagulation, angiogenesis and osteogenesis. OM-triggered changes in the HOb intracellular energy production mechanisms, with key roles in osteoblast maturation. HOb cultured with and without Ti showed enrichment in the skeletal system development function due to the OM. However, differentially expressed proteins with key regenerative functions were associated with a synergistic effect of OM and Ti. This synergy, caused by the Ti–OM interaction, could complicate the interpretation of in vitro results, highlighting the need to analyse this phenomenon in biomaterial testing.

Osteogenic differentiation of human amniotic mesenchymal stem cells by phycocyanin and phycoerythrin pigments isolated from Spirulina platensis and Gracilaria gracilis algae

Bone regeneration is a multistep and regular physiological process that occurs normally in fracture repair and bone defects. However, some factors such as aging, particular diseases and some drugs prevent or slowdown bone natural healing. Cell therapy using stem cells and differentiation activating factors is an effective treatment method for bone regeneration triggering in unusual conditions. Therefore, in the present study the effect of phycocyanin and phycoerythrin pigments which isolated from Spirulina platensis and Gracilaria gracilis algae was investigate on osteogenic differentiation potency of human Amniotic Mesenchymal Stem Cells (hAMSCs). For this purpose, hAMSCs were exposed to 300, 500, and 700 µg/ml concentrations of phycocyanin and phycoerythrin pigments and then the cells viability was measured with MTT assay in 48 and 72 h after treatment. The osteo-differentiation level of cells was studied by measuring ALP activity using calorimetric method and Alizarin red staining for calcium deposition in 7 and 21 days after treatment. Also, total RNA of cells was extracted in different time periods and then cDNA synthesized with specific primers, and relative expression of Runx2, β-catenin and Osteocalcin genes were investigated using SYBR Green RT-qPCR technique. Osteogenic differentiation of hAMSCs that treated with pigments was confirmed by mineral deposits staining and increased level of ALP activity. Furthermore, these pigments elevated significantly the expression of osteogenic marker genes compared to control samples and caused hAMSCs to differentiate into osteoblast cells. According to these results, phycocyanin and phycoerythrin may suggest as suitable osteogenic supplements with low toxicity, low cost and high efficiency, although the molecular mechanism of its efficacy is not available yet.

Development, Establishment, and Validation of a Model for the Mineralization of Periodontium Remodelling Cells: Cementoblasts.

Chronic kidney disease (CKD) patients undergoing dialysis are at high risk of bone fractures. CKD-induced mineral and bone disorder is extended to periodontal disease due to changes in the ionic composition of saliva in CKD patients, dysregulating mineralization, hindering regeneration and thereby promoting the progression of dental complications. Despite the importance of cementum for overall oral health, the mechanisms that regulate its development and regeneration are not well comprehended, and a lack of sufficient in vitro experimental models has hindered research progress. In this study, the impact of experimental conditions on the calcification of cementoblasts was systematically investigated, aimed at establishing a standardized and validated model for the calcification of cementoblasts. The effects of phosphate, calcium, ascorbic acid, β-glycerolphosphate, dexamethasone, and fetal calf serum on the calcification process of cementoblasts were analyzed over a wide range of concentrations and time points by investigating calcium content, cell viability, gene expression and kinase activity. Cementoblasts calcified in a concentration- and time-dependent manner with higher concentrations of supplements cause a higher degree of calcification but decreased cell viability. Phosphate and calcium have a significantly stronger effect on cementoblast calcification processes compared to osteogenic supplements: ascorbic acid, β-glycerolphosphate, and dexamethasone induce calcification over a wide range of osteogenic signalling pathways, with osteopontin being a central target of gene regulation. Conversely, treatment with ascorbic acid, β-glycerolphosphate, and dexamethasone leads to activating only selected pathways, especially promoting bone sialoprotein expression. The developed and validated cementoblast calcification protocol, incubating up to 60% confluent cementoblasts with 1.9 mmol L-1 of phosphate supplementation for a reasonable, multi-pathway calcification induction and 10 mmol L-1 β-glycerolphosphate, 75 µmol L-1 ascorbic acid and 10 nmol L-1 dexamethasone for a reasonable osteogenic differentiation-based calcification induction, provides standard in vitro experimental models for better understanding cementoblast function and regeneration.

Micellized protein transduction domain-bone morphogenetic protein-2 accelerates bone healing in a rat tibial distraction osteogenesis model

The clinical application of growth factors such as recombinant human bone morphogenetic protein-2 (rh-BMP-2), for functional bone regeneration remains challenging due to limited in vivo efficacy and adverse effects of previous modalities. To overcome the instability and short half-life of rh-BMP-2 in vivo, we developed a novel osteogenic supplement by fusing a protein transduction domain (PTD) with BMP-2, effectively creating a prodrug of BMP-2. In this study, we first created an improved PTD-BMP-2 formulation using lipid nanoparticle (LNP) micellization, resulting in downsizing from micrometer to nanometer scale and achieving a more even distribution. The micellized PTD-BMP-2 (mPTD-BMP-2) demonstrated improved distribution and aggregation profiles. As a prodrug of BMP-2, mPTD-BMP-2 successfully activated Smad1/5/8 and induced mineralization with osteogenic gene induction in vitro. In vivo pharmacokinetic analysis revealed that mPTD-BMP-2 had a much more stable pharmacokinetic profile than rh-BMP-2, with a 7.5-fold longer half-life. The in vivo BMP-responsive element (BRE) reporter system was also successfully activated by mPTD-BMP-2. In the in vivo rat tibia distraction osteogenesis (DO) model, micro-computed tomography (micro-CT) scan findings indicated that mPTD-BMP-2 significantly increased bone volume, bone surface, axis moment of inertia (MOI), and polar MOI. Furthermore, it increased the expression of osteogenesis-related genes, and induced bone maturation histologically. Based on these findings, mPTD-BMP-2 could be a promising candidate for the next-generation osteogenesis drug to promote new bone formation in DO surgery. Statement of SignificanceThis study introduces micellized bone morphogenetic protein-2 (mPTD-BMP-2), a next-generation osteogenic supplement that combines protein transduction domain (PTD) and nano-sized micelle formulation technique to improve transduction efficiency and stability. The use of PTD represents a novel approach, and our results demonstrate the superiority of mPTD-BMP-2 over rh-BMP-2 in terms of in vivo pharmacokinetic profile and osteogenic potential, particularly in a rat tibial model of distraction osteogenesis. These findings have significant scientific impact and potential clinical applications in the treatment of bone defects that require distraction osteogenesis. By advancing the field of osteogenic supplements, our study has the potential to contribute to the development of more effective treatments for musculoskeletal disorders.

The molecular weight of hyaluronic acid influences metabolic activity and osteogenic differentiation of periodontal ligament cells

ObjectiveWhile HA is present naturally in periodontal tissues, its molecular weight can vary widely in vivo. The objective of this study was to directly compare the biological reactions of periodontal ligament cells to four distinct molecular weights of hyaluronic acid (HA).Materials and methodsImmortalized human periodontal ligament cells (PDL-hTERT) were cultured for 21 days in culture medium alone (control) or enriched with osteogenic supplements (OS group). Other 4 experimental groups were cultured in OS medium with the addition of HA with different molecular weights (HMW, MMW, LMW, and ULMW). The cell morphology was examined daily. WST1 assays were performed to evaluate metabolic activity. Von Kossa staining and calcium deposition assay were used to analyze osteogenic differentiation and mineralization.ResultsCell morphology remained unaltered in all groups. Cells stimulated with OS alone or with the addition of hyaluronan showed all the typical microscopic appearance of osteogenic differentiation. Metabolic activity increased in all groups over time. Hyaluronan stimulated greater metabolic activity than the control group, with LMW HA and MMW HA showing the most significant increase. All groups showed mineral deposits and calcium deposition after 21 days of stimulation.ConclusionOur results suggest that hyaluronan can promote metabolic activity and mineralization of PDL-hTERT cells, with LMW HA being the most effective.Clinical relevanceThese results shed light on how the various molecular weight fractions of HA promote tissue regeneration and repair, as well as help to identify an optimal molecular weight range for this application in periodontal tissues.

Thiol-Functionalized, Antioxidant, and Osteogenic Mesoporous Silica Nanoparticles for Osteoporosis.

Osteoporosis is a chronic bone disorder characterized by decreased bone mass, leading to brittle bones and fractures. Oxidative stress has been identified as the most profound trigger for the initiation and progression of osteoporosis. Current treatment strategies do not induce new bone formation and fail to address a high level of reactive oxygen species (ROS). Mesoporous silica nanoparticles (MSNs) have been explored in bone tissue regeneration owing to their inherent osteogenic property, but they lack antioxidant and cell adhesion properties, required in such applications. We have developed thiolated, bioactive mesoporous silica nanoparticles (MSN-SH) to address this challenge. MSNs were fabricated using the Stöber method, and 11% of the surface was functionalized post-synthesis with thiol groups using MPTMS to obtain MSN-SH. The particle size measured by the dynamic light scattering technique was found to be around 300 nm. The surface morphology was investigated using HR-TEM, and their physical and chemical properties were characterized using various spectroscopic techniques. They exhibited more than 90% antioxidant activity, neutralized ROS formed in cells, and provided protection against ROS-induced cell damage. The cell viability assay in murine osteoblast precursor cells (MC3T3) showed that MSN-SH is cell-proliferative in nature with 140% cell viability. Osteogenic potential was evaluated by measuring the ALP activities, calcium deposition, and gene expression levels of osteogenic markers, such as RUNX2, ALP, OCN, and OPN, and results revealed that MSN-SH increases calcium deposition and induces osteogenesis through upregulation of osteogenic genes and markers without the involvement of any osteogenic supplements. Besides promoting osteogenesis, MSN-SH was found to inhibit osteoclastogenesis. The nanomaterial was found to be regenerative in nature, and it stimulated migration of osteoblast cells and caused a complete wound closure within 48 h. We were able to achieve a multifunctional nanomaterial by simply modifying the surface. MSNs have been explored for bone tissue engineering/osteoporosis as a composite system incorporating metals, like gold and cerium, or as a nanocarrier loaded with growth factors or active drugs. This study offers a simple and economical method to enhance the existing properties of MSNs and impart new activities by a single-step surface modification. It can be concluded that MSN-SH holds promise as a complementary and alternate treatment for osteoporosis along with the standardized therapy.

Multifunctional Zn and Ag co-doped bioactive glass nanoparticles for bone therapeutic and regeneration

Bone cancer has traditionally been treated using surgery, radiotherapy, and/or chemotherapy. The nonspecific distribution of chemotherapy and implantable infections are significant risk factors for the failure of the bone to heal. Multifunctional zinc and silver co-doped bioactive glass nanoparticles (yAg–xZn-BGNPs) with a diameter of 150 ± 30 nm were successfully synthesized using modified sol–gel and two-step post-functionalization processes, tailored to provide antibacterial and anticancer activity whilst maintaining osteogenesis ability. Co-doped BGNPs with Zn and Ag did not significantly alter physicochemical properties, including size, morphology, glass network, and amorphous nature. Apatite-like layer was observed on the surface of yAg–xZn-BGNPs and resorbed in the simulated body fluid solution, which could increase their bioactivity. Human fetal osteoblast cell line (hFOB 1.19) treated with particles showed calcified tissue formation and alkaline phosphatase activity in the absence of osteogenic supplements in vitro, especially with 0.5Ag–1Zn-BGNPs. Moreover, these particles preferentially disrupted the metabolic activity of bone cancer cells (MG-63) and had an antibacterial effect against B. subtilis, E. coli, and S. aureus via the disc diffusion method. This novel 0.5Ag–1Zn-BGNP and 1Ag–1Zn-BGNPs, with wide-ranging ability to stimulate bone regeneration, to inhibit bone cancer cell proliferation, and to prevent bacterial growth properties, may provide a feasible strategy for bone cancer treatment. The 0.5Ag–1Zn-BGNPs and 1Ag–1Zn-BGNPs can be applied for the preparation of scaffolds or filler composites using in bone tissue engineering.

Proliferative and Osteogenic Supportive Effect of VEGF-Loaded Collagen-Chitosan Hydrogel System in Bone Marrow Derived Mesenchymal Stem Cells.

The use of hydrogel (HG) in regenerative medicine is an emerging field and thus several approaches have been proposed recently to find an appropriate hydrogel system. In this sense, this study developed a novel HG system using collagen, chitosan, and VEGF composites for culturing mesenchymal stem cells (MSCs), and investigated their ability for osteogenic differentiation and mineral deposition. Our results showed that the HG loaded with 100 ng/mL VEGF (HG-100) significantly supported the proliferation of undifferentiated MSCs, the fibrillary filament structure (HE stain), mineralization (alizarin red S and von Kossa stain), alkaline phosphatase, and the osteogenesis of differentiated MSCs compared to other hydrogels (loaded with 25 and 50 ng/mL VEGF) and control (without hydrogel). HG-100 showed a higher VEGF releasing rate from day 3 to day 7 than other HGs, which substantially supports the proliferative and osteogenic properties of HG-100. However, the HGs did not increase the cell growth in differentiated MSCs on days 14 and 21 due to the confluence state (reach stationary phase) and cell loading ability, regardless of the VEGF content. Similarly, the HGs alone did not stimulate the osteogenesis of MSCs; however, they increased the osteogenic ability of MSCs in presence of osteogenic supplements. Accordingly, a fabricated HG with VEGF could be used as an appropriate system to culture stem cells for bone and dental regeneration.

Osteogenic potential of solid and porous phosphate glass microspheres as pulp capping materials

This study investigated the morphology (Scanning Electron Microscope), chemistry (Elemental Analysis (EDX), Raman Spectroscopy and X-ray diffraction), thermal characteristics, cytocompatibility, and osteogenic potential of solid and porous phosphate-based glass microspheres for potential use as pulp-capping materials. Cytocompatibility was investigated using human dental pulp stem cells (hDPSCs) through both direct and indirect (extracts) contact tests. Cells grown in normal cell culture medium and 10% dimethyl sulfoxide served as positive and negative controls, respectively. Unlike solid glass microspheres (GMs), porous GMs showed higher CaO mol% and crystalline structure. Both types of GMs supported the attachment, spreading, and viability of hDPSCs similar to the positive control. Cells grown in GMs’ extracts showed even higher viability, but less mineralization than those grown in direct contact with GMs. The presence of osteogenic supplements however did not have a significant effect on mineralisation. Accordingly, both solid and porous GMs show useful applicability as pulp-capping materials.

Composite Graded Melt Electrowritten Scaffolds for Regeneration of the Periodontal Ligament-to-Bone Interface.

Periodontitis is a ubiquitous chronic inflammatory, bacteria-triggered oral disease affecting the adult population. If left untreated, periodontitis can lead to severe tissue destruction, eventually resulting in tooth loss. Despite previous efforts in clinically managing the disease, therapeutic strategies are still lacking. Herein, melt electrowriting (MEW) is utilized to develop a compositionally and structurally tailored graded scaffold for regeneration of the periodontal ligament-to-bone interface. The composite scaffolds, consisting of fibers of polycaprolactone (PCL) and fibers of PCL-containing magnesium phosphate (MgP) were fabricated using MEW. To maximize the bond between bone (MgP) and ligament (PCL) regions, we evaluated two different fiber architectures in the interface area. These were a crosshatch pattern at a 0/90° angle and a random pattern. MgP fibrous scaffolds were able to promote in vitro bone formation even in culture media devoid of osteogenic supplements. Mechanical properties after MgP incorporation resulted in an increase of the elastic modulus and yield stress of the scaffolds, and fiber orientation in the interfacial zone affected the interfacial toughness. Composite graded MEW scaffolds enhanced bone fill when they were implanted in an in vivo periodontal fenestration defect model in rats. The presence of an interfacial zone allows coordinated regeneration of multitissues, as indicated by higher expression of bone, ligament, and cementoblastic markers compared to empty defects. Collectively, MEW-fabricated scaffolds having compositionally and structurally tailored zones exhibit a good mimicry of the periodontal complex, with excellent regenerative capacity and great potential as a defect-specific treatment strategy.

In Vitro Evaluation of Human Demineralised Teeth Matrix on Osteogenic Differentiation of Gingival Mesenchymal Stem Cells

The use of tooth-derived material as a scaffold has gained attention recently due to its ease of availability and bioactive properties. Hence, the objective of this study was to determine in vitro interaction of human gingival mesenchymal stem cells (hGMSCs) with human demineralised teeth matrix (hDTM) on osteogenic potential with or without osteogenic inducers. The hGMSCs were established and characterised on their morphology, proliferation, population doubling time (PDT), viability, colony-forming ability, expression of cell surface markers and adipogenic differentiation. Further, the effect of hDTM on the biocompatibility and osteogenic differentiation ability of hGMSCs was evaluated. The hGMSCs displayed a fibroblast-like appearance and exhibited a greater proliferative activity. The cells showed > 91% viability, and PDT varied between 39.34 hours and 62.59 hours. Further, hGMSCs indicated their propensity to form clusters/ colonies, and expressed the markers, such as CD29, CD44, CD73 and CD90, but were negative for CD34 and CD45. When treated with adipogenic induction medium, hGMSCs were able to exhibit the formation of neutral lipid vacuoles. The hGMSCs cultured with hDTM did not show any cytotoxic changes including morphology and viability. Mineralisation of calcium nodules was observed in hGMSCs when cultured in osteogenic induction (OI) medium as an indication of osteogenesis. hGMSCs when cultured with hDTM confirmed the presence of a mineralised matrix. Further, when the cells were cultured with hDTM along with OI, they showed slightly enhanced differentiation into osteocytes. In conclusion, hGMSCs were shown to be biocompatible with hDTM, and demonstrated their enhanced osteogenic potential in the presence of hDTM and osteogenic supplements.

Amnion and chorion matrix maintain hMSC osteogenic response and enhance immunomodulatory and angiogenic potential in a mineralized collagen scaffold.

Craniomaxillofacial (CMF) bone injuries present a major surgical challenge and cannot heal naturally due to their large size and complex topography. We are developing a mineralized collagen scaffold that mimics extracellular matrix (ECM) features of bone. These scaffolds induce in vitro human mesenchymal stem cell (hMSC) osteogenic differentiation and in vivo bone formation without the need for exogenous osteogenic supplements. Here, we seek to enhance pro-regenerative potential via inclusion of placental-derived products in the scaffold architecture. The amnion and chorion membranes are distinct components of the placenta that each have displayed anti-inflammatory, immunomodulatory, and osteogenic properties. While potentially a powerful modification to our mineralized collagen scaffolds, the route of inclusion (matrix-immobilized or soluble) is not well understood. Here we compare the effect of introducing amnion and chorion membrane matrix versus soluble extracts derived from these membranes into the collagen scaffolds on scaffold biophysical features and resultant hMSC osteogenic activity. While inclusion of amnion and chorion matrix into the scaffold microarchitecture during fabrication does not influence their porosity, it does influence compression properties. Incorporating soluble extracts from the amnion membrane into the scaffold post-fabrication induces the highest levels of hMSC metabolic activity and equivalent mineral deposition and elution of the osteoclast inhibitor osteoprotegerin (OPG) compared to the conventional mineralized collagen scaffolds. Mineralized collagen-amnion composite scaffolds elicited enhanced early stage osteogenic gene expression (BGLAP, BMP2), increased immunomodulatory gene expression (CCL2, HGF, and MCSF) and increased angiogenic gene expression (ANGPT1, VEGFA) in hMSCs. Mineralized collagen-chorion composite scaffolds promoted immunomodulatory gene expression in hMSCs (CCL2, HGF, and IL6) while unaffecting osteogenic gene expression. Together, these findings suggest that mineralized collagen scaffolds modified using matrix derived from amnion and chorion membranes represent a promising environment conducive to craniomaxillofacial bone repair.

A Tissue Engineering Acoustophoretic (TEA) Set-up for the Enhanced Osteogenic Differentiation of Murine Mesenchymal Stromal Cells (mMSCs).

Quickly developing precision medicine and patient-oriented treatment strategies urgently require novel technological solutions. The randomly cell-populated scaffolds usually used for tissue engineering often fail to mimic the highly anisotropic characteristics of native tissue. In this work, an ultrasound standing-wave-based tissue engineering acoustophoretic (TEA) set-up was developed to organize murine mesenchymal stromal cells (mMSCs) in an in situ polymerizing 3-D fibrin hydrogel. The resultant constructs, consisting of 17 cell layers spaced at 300 µm, were obtained by continuous wave ultrasound applied at a 2.5 MHz frequency. The patterned mMSCs preserved the structured behavior within 10 days of culturing in osteogenic conditions. Cell viability was moderately increased 1 day after the patterning; it subdued and evened out, with the cells randomly encapsulated in hydrogels, within 21 days of culturing. Cells in the structured hydrogels exhibited enhanced expression of certain osteogenic markers, i.e., Runt-related transcription factor 2 (RUNX2), osterix (Osx) transcription factor, collagen-1 alpha1 (COL1A1), osteopontin (OPN), osteocalcin (OCN), and osteonectin (ON), as well as of certain cell-cycle-progression-associated genes, i.e., Cyclin D1, cysteine-rich angiogenic inducer 61 (CYR61), and anillin (ANLN), when cultured with osteogenic supplements and, for ANLN, also in the expansion media. Additionally, OPN expression was also augmented on day 5 in the patterned gels cultured without the osteoinductive media, suggesting the pro-osteogenic influence of the patterned cell organization. The TEA set-up proposes a novel method for non-invasively organizing cells in a 3-D environment, potentially enhancing the regenerative properties of the designed anisotropic constructs for bone healing.

Highly elastic and bioactive bone biomimetic scaffolds based on platelet lysate and biomineralized cellulose nanocrystals

Bone is a vascularized organic-inorganic composite tissue that shows a heavily-mineralized extracellular matrix (ECM) on the nanoscale. Herein, the nucleation of calcium phosphates during the biomineralization process was mimicked using negatively-charged cellulose nanocrystals (CNCs). These mineralized-CNCs were combined with platelet lysate to produce nanocomposite scaffolds through cryogelation to mimic bone ECM protein-mineral composite nature and take advantage of the bioactivity steaming from platelet-derived biomolecules. The nanocomposite scaffolds showed high microporosity (94–95%), high elasticity (recover from 75% strain cycles), injectability, and modulated platelet-derived growth factors sequestration and release. Furthermore, they increased alkaline phosphatase activity (up to 10-fold) and up-regulated the expression of bone-related markers (up to 2-fold), without osteogenic supplementation, demonstrating their osteoinductive properties. Also, the scaffolds promoted the chemotaxis of endothelial cells and enhanced the expression of endothelial markers, showing proangiogenic potential. These results suggest that the mineralized nanocomposite scaffolds can enhance bone regeneration by simultaneously promoting osteogenesis and angiogenesis.