A 3D bioreactor model to study osteocyte differentiation and mechanobiology under perfusion and compressive mechanical loading.

Osteocytes perceive and process mechanical stimuli in the lacuno-canalicular network in bone. As a result, they secrete signaling molecules that mediate bone formation and resorption. To date, few three-dimensional (3D) models exist to study the response of mature osteocytes to biophysical stimuli that mimic fluid shear stress and substrate strain in a mineralized, biomimetic bone-like environment. Here we established a biomimetic 3D bone model by utilizing a state-of-art perfusion bioreactor platform where immortomouse/Dmp1-GFP-derived osteoblastic IDG-SW3 cells were differentiated into mature osteocytes. We evaluated proliferation and differentiation properties of the cells on 3D microporous scaffolds of decellularized bone (dBone), poly(L-lactide-co-trimethylene carbonate) lactide (LTMC), and beta-tricalcium phosphate (ß-TCP) under physiological fluid flow conditions over 21 days. Osteocyte viability and proliferation were similar on the scaffolds with equal distribution of IDG-SW3 cells on dBone and LTMC scaffolds. After seven days, the differentiation marker alkaline phosphatase (Alpl), dentin matrix acidic phosphoprotein 1 (Dmp1), and sclerostin (Sost) were significantly upregulated in IDG-SW3 cells (p = 0.05) on LTMC scaffolds under fluid flow conditions at 1.7 ml/min, indicating rapid and efficient maturation into osteocytes. Osteocytes responded by inducing the mechanoresponsive genes FBJ osteosarcoma oncogene (Fos) and prostaglandin-endoperoxide synthase 2 (Ptgs2) under perfusion and dynamic compressive loading at 1 Hz with 5% strain. Together, we successfully created a 3D biomimetic platform as a robust tool to evaluate osteocyte differentiation and mechanobiology in vitro while recapitulating in vivo mechanical cues such as fluid flow within the lacuno-canalicular network. STATEMENT OF SIGNIFICANCE: This study highlights the importance of creating a three-dimensional (3D) in vitro model to study osteocyte differentiation and mechanobiology, as cellular functions are limited in two-dimensional (2D) models lacking in vivo tissue organization. By using a perfusion bioreactor platform, physiological conditions of fluid flow and compressive loading were mimicked to which osteocytes are exposed in vivo. Microporous poly(L-lactide-co-trimethylene carbonate) lactide (LTMC) scaffolds in 3D are identified as a valuable tool to create a favorable environment for osteocyte differentiation and to enable mechanical stimulation of osteocytes by perfusion and compressive loading. The LTMC platform imitates the mechanical bone environment of osteocytes, allowing the analysis of the interaction with other cell types in bone under in vivo biophysical stimuli.

Correlation between Ca Release and Osteoconduction by 3D-Printed Hydroxyapatite-Based Templates.

The application of hydroxyapatite (HA)-based templates is quite often seen in bone tissue engineering since that HA is an osteoconductive bioceramic material, which mimics the inorganic component of mineralized tissues. However, the reported osteoconductivity varies in vitro and in vivo, and the levels of calcium (Ca) release most favorable to osteoconduction have yet to be determined. In this study, HA-based templates were fabricated by melt-extrusion 3D-printing and characterized in order to determine a possible correlation between Ca release and osteoconduction. The HA-based templates were blended with poly(lactide-co-trimethylene carbonate) (PLATMC) at three different HA ratios: 10, 30, and 50%. The printability and physical properties of the HA templates were compared with those of pristine PLATMC. In vitro, osteoconductivity was assessed using seeded human bone marrow-derived mesenchymal stem cells. A mild rate of Ca release was observed for HA10 templates, which exhibited higher mineralized extracellular matrix (ECM) secretion than PLATMC at 14 and 21 days. In contrast, the high rate of Ca release exhibited by HA30 and HA50 templates was associated with reduced osteoconduction and impeded mineralized ECM secretion in vitro. Similar results were observed in vivo. In the calvarial defect model in rabbit, PLATMC and HA10 templates exhibited the highest amount of new bone formation, with obvious contact osteogenesis on their surfaces. In contrast, HA30 and HA50 exhibited distant osteogenesis and reduced amounts of new bone ingrowth. It is concluded that HA-based templates are osteoconductive only at low rates of Ca release.

Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model.

BACKGROUND: There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. METHODS: EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. RESULTS: Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. CONCLUSIONS: This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

The use of mesenchymal stromal cell secretome to enhance guided bone regeneration in comparison with leukocyte and platelet-rich fibrin.

OBJECTIVES: Secretomes of mesenchymal stromal cells (MSC) represent a novel strategy for growth-factor delivery for tissue regeneration. The objective of this study was to compare the efficacy of adjunctive use of conditioned media of bone-marrow MSC (MSC-CM) with collagen barrier membranes vs. adjunctive use of conditioned media of leukocyte- and platelet-rich fibrin (PRF-CM), a current growth-factor therapy, for guided bone regeneration (GBR). METHODS: MSC-CM and PRF-CM prepared from healthy human donors were subjected to proteomic analysis using mass spectrometry and multiplex immunoassay. Collagen membranes functionalized with MSC-CM or PRF-CM were applied on critical-size rat calvaria defects and new bone formation was assessed via three-dimensional (3D) micro-CT analysis of total defect volume (2 and 4 weeks) and 2D histomorphometric analysis of central defect regions (4 weeks). RESULTS: While both MSC-CM and PRF-CM revealed several bone-related proteins, differentially expressed proteins, especially extracellular matrix components, were increased in MSC-CM. In rat calvaria defects, micro-CT revealed greater total bone coverage in the MSC-CM group after 2 and 4 weeks. Histologically, both groups showed a combination of regular new bone and 'hybrid' new bone, which was formed within the membrane compartment and characterized by incorporation of mineralized collagen fibers. Histomorphometry in central defect sections revealed greater hybrid bone area in the MSC-CM group, while the total new bone area was similar between groups. CONCLUSION: Based on the in vitro and in vivo investigations herein, functionalization of membranes with MSC-CM represents a promising strategy to enhance GBR.

Assessment of Health-Related Quality of Life in Children with Totally Corrected Tetralogy of Fallot in Sulaymaniyah, Iraq.

PURPOSE: This study examined the health-related quality of life among children who have undergone surgery for tetralogy of Fallot (TOF) compared to healthy children. DESIGN: Cross-sectional study. METHODS: The study was carried out at Children's Heart Hospital and Primary Healthcare Centers in Sulaymaniyah, Iraq, between July 1, 2022, and November 1, 2022. The study involved 400 participants, comprised of 200 parents of healthy children and 200 parents with children with repaired TOF. Data collection involved utilizing a 5-point Likert Scale questionnaire administered through direct face-to-face interviews with the parents of the children. FINDINGS: Children with surgically repaired TOF had markedly lower mean scores (P ≤ 0.001) in the overall dimension of QoL (Mean rank = 137) when compared to healthy children (Mean rank = 263). Furthermore, it was observed that children with repaired TOF had significantly lower mean scores (P < 0.001) across all subdimensions of QoL compared to their healthy counterparts. In the subject of children with repaired TOF, the impact of maternal education (illiterate and lower education) on overall QoL was found to be highly significant (P < 0.001). In addition, children with repaired TOF from lower socioeconomic status (SES) families had worse quality of life than those from medium and high SES families, with a p-value <0.001. CONCLUSIONS: Repaired TOF children exhibited notable deficiencies across all dimensions of QoL compared to healthy children. Moreover, SES emerged as a significant determinant influencing the QoL outcomes of repaired TOF children. CLINICAL EVIDENCE: Despite undergoing corrective surgery for TOF, the QoL in children with repaired TOF continues to be lower than that of their healthy counterparts. Notably, the economic and educational status of the family significantly impacts the way these children perceive and experience their QoL. This finding underscores the critical significance of factoring in socioeconomic elements when addressing the well-being of this particular group of children.

The neuroprotective potential of mesenchymal stem cells from bone marrow and human exfoliated deciduous teeth in a murine model of demyelination.

INTRODUCTION: Multiple sclerosis (MS) is characterized by chronic inflammation, demyelination, and axonal degeneration within the central nervous system (CNS), for which there is no current treatment available with the ability to promote neuroprotection or remyelination. Some aspects of the progressive form of MS are displayed in the murine cuprizone model, where demyelination is induced by the innate immune system without major involvement of the adaptive immune system. Mesenchymal stem cells (MSCs) are multipotent cells with immunomodulatory and neuroprotective potential. In this study, we aimed to assess the neuroprotective potential of MSCs from bone marrow (BM-MSCs) and stem cells from human exfoliated deciduous teeth (SHED) in the cuprizone model. METHODS: Human BM-MSCs and SHED were isolated and characterized. Nine-week-old female C57BL/6 mice were randomized to receive either human BM-MSCs, human SHED or saline intraperitoneally. Treatments were administered on day -1, 14 and 21. Outcomes included levels of local demyelination and inflammation, and were assessed with immunohistochemistry and histology. RESULTS: BM-MSCs were associated with increased myelin content and reduced microglial activation whereas mice treated with SHED showed reduced microglial and astroglial activation. There were no differences between treatment groups in numbers of mature oligodendrocytes or axonal injury. MSCs were identified in the demyelinated corpus callosum in 40% of the cuprizone mice in both the BM-MSC and SHED group. CONCLUSION: Our results suggest a neuroprotective effect of MSCs in a toxic MS model, with demyelination mediated by the innate immune system.

Proteomic Analysis of Mesenchymal Stromal Cells Secretome in Comparison to Leukocyte- and Platelet-Rich Fibrin.

Secretomes of mesenchymal stromal cells (MSCs) are emerging as a novel growth factor (GF)-based strategy for periodontal and bone regeneration. The objective of this study was to compare the secretome of human bone marrow MSC (BMSC) to that of leukocyte- and platelet-rich fibrin (L-PRF), an established GF-based therapy, in the context of wound healing and regeneration. Conditioned media from human BMSCs (BMSC-CM) and L-PRF (LPRF-CM) were subjected to quantitative proteomic analysis using liquid chromatography with tandem mass spectrometry. Global profiles, gene ontology (GO) categories, differentially expressed proteins (DEPs), and gene set enrichment (GSEA) were identified using bioinformatic methods. Concentrations of selected proteins were determined using a multiplex immunoassay. Among the proteins identified in BMSC-CM (2157 proteins) and LPRF-CM (1420 proteins), 1283 proteins were common. GO analysis revealed similarities between the groups in terms of biological processes (cellular organization, protein metabolism) and molecular functions (cellular/protein-binding). Notably, more DEPs were identified in BMSC-CM (n = 550) compared to LPRF-CM (n = 118); these included several key GF, cytokines, and extracellular matrix (ECM) proteins involved in wound healing. GSEA revealed enrichment of ECM (especially bone ECM)-related processes in BMSC-CM and immune-related processes in LPRF-CM. Similar trends for intergroup differences in protein detection were observed in the multiplex analysis. Thus, the secretome of BMSC is enriched for proteins/processes relevant for periodontal and bone regeneration. The in vivo efficacy of this therapy should be evaluated in future studies.

Unique osteogenic profile of bone marrow stem cells stimulated in perfusion bioreactor is Rho-ROCK-mediated contractility dependent.

The fate determination of bone marrow mesenchymal stem/stromal cells (BMSC) is tightly regulated by mechanical cues, including fluid shear stress. Knowledge of mechanobiology in 2D culture has allowed researchers in bone tissue engineering to develop 3D dynamic culture systems with the potential for clinical translation in which the fate and growth of BMSC are mechanically controlled. However, due to the complexity of 3D dynamic cell culture compared to the 2D counterpart, the mechanisms of cell regulation in the dynamic environment remain relatively undescribed. In the present study, we analyzed the cytoskeletal modulation and osteogenic profiles of BMSC under fluid stimuli in a 3D culture condition using a perfusion bioreactor. BMSC subjected to fluid shear stress (mean 1.56 mPa) showed increased actomyosin contractility, accompanied by the upregulation of mechanoreceptors, focal adhesions, and Rho GTPase-mediated signaling molecules. Osteogenic gene expression profiling revealed that fluid shear stress promoted the expression of osteogenic markers differently from chemically induced osteogenesis. Osteogenic marker mRNA expression, type 1 collagen formation, ALP activity, and mineralization were promoted in the dynamic condition, even in the absence of chemical supplementation. The inhibition of cell contractility under flow by Rhosin chloride, Y27632, MLCK inhibitor peptide-18, or Blebbistatin revealed that actomyosin contractility was required for maintaining the proliferative status and mechanically induced osteogenic differentiation in the dynamic culture. The study highlights the cytoskeletal response and unique osteogenic profile of BMSC in this type of dynamic cell culture, stepping toward the clinical translation of mechanically stimulated BMCS for bone regeneration.

Functionalizing Collagen Membranes with MSC-Conditioned Media Promotes Guided Bone Regeneration in Rat Calvarial Defects.

Functionalizing biomaterials with conditioned media (CM) from mesenchymal stromal cells (MSC) is a promising strategy for enhancing the outcomes of guided bone regeneration (GBR). This study aimed to evaluate the bone regenerative potential of collagen membranes (MEM) functionalized with CM from human bone marrow MSC (MEM-CM) in critical size rat calvarial defects. MEM-CM prepared via soaking (CM-SOAK) or soaking followed by lyophilization (CM-LYO) were applied to critical size rat calvarial defects. Control treatments included native MEM, MEM with rat MSC (CEL) and no treatment. New bone formation was analyzed via micro-CT (2 and 4 weeks) and histology (4 weeks). Greater radiographic new bone formation occurred at 2 weeks in the CM-LYO group vs. all other groups. After 4 weeks, only the CM-LYO group was superior to the untreated control group, whereas the CM-SOAK, CEL and native MEM groups were similar. Histologically, the regenerated tissues showed a combination of regular new bone and hybrid new bone, which formed within the membrane compartment and was characterized by the incorporation of mineralized MEM fibers. Areas of new bone formation and MEM mineralization were greatest in the CM-LYO group. Proteomic analysis of lyophilized CM revealed the enrichment of several proteins and biological processes related to bone formation. In summary, lyophilized MEM-CM enhanced new bone formation in rat calvarial defects, thus representing a novel 'off-the-shelf' strategy for GBR.

Responses of Rat Mesenchymal Stromal Cells to Nanocellulose with Different Functional Groups.

Cellulose nanofibrils (CNFs) are multiscale hydrophilic biocompatible polysaccharide materials derived from wood and plants. TEMPO-mediated oxidation of CNFs (TO-CNF) turns some of the primary hydroxyl groups to carboxylate and aldehyde groups. Unlike carboxylic functional groups, there is little or no information about the biological role of the aldehyde groups on the surface of wood-based CNFs. In this work, we replaced the aldehyde groups in the TO-CNF samples with carboxyl groups by another oxidation treatment (TO-O-CNF) or with primary alcohols with terminal hydroxyl groups by a reduction reaction (TO-R-CNF). Rat mesenchymal stem/stromal cells (MSCs) derived from bone marrow were seeded on polystyrene tissue culture plates (TCP) coated with CNFs with and without aldehyde groups. TCP and TCP coated with bacterial nanocellulose (BNC) were used as control groups. Protein adsorption measurements demonstrated that more proteins were adsorbed from cell culture media on all CNF surfaces compared to BNC. Live/dead and lactate dehydrogenase assays confirmed that all nanocellulose biomaterials supported excellent cell viability. Interestingly, TO-R-CNF samples, which have no aldehyde groups, showed better cell spreading than BNC and comparable results to TCP. Unlike TO-O-CNF surfaces, which have no aldehyde groups either, TO-R-CNF stimulated cells, in osteogenic medium, to have higher alkaline phosphatase activity and to form more biomineralization than TCP and TO-CNF groups. These findings indicate that the presence of aldehyde groups (280 ± 14 µmol/g) on the surface of TEMPO-oxidized CNFs might have little or no effect on attachment, proliferation, and osteogenic differentiation of MSCs.

Composition of Crude Palm Oil Extracted Using Hot Compressed Water Extraction.

Extraction process are one of the critical units in palm oil milling process which dictates its efficiency. In this study, hot compressed water extraction (HCWE) is utilized for the crude palm oil (CPO) extraction. With regards to CPO quality, the extracted CPO using HCWE was analysed based on overall composition, triacylglycerol (TAG) composition and fatty acid profile composition. This is to evaluate the possible product degradation during the process. From the results, the CPO extracted using HCWE process contain low FFA of 0.15±0.01% and low DAG of 2.145% which reflect to higher quality of CPO. This CPO also show the equal mixture of saturated and unsaturated fatty acid composition at 50.63% and 49.93% respectively, within the CPO composition range in the literature. No significant changing of the fatty acid composition is observed between CPO extracted using HCWE and commercial CPO indicated that no possible undesired reaction during the extraction process. HCWE is a promising method for screw press system replacement, but the economic analysis is essential to evaluate its prospective.

The effect of resolvin D1 on bone regeneration in a rat calvarial defect model.

Resolvin D1 (RvD1) is a pro-resolving lipid mediator of inflammation, endogenously synthesized from omega-3 docosahexaenoic acid. The purpose of this study was to investigate the effect of RvD1 on bone regeneration using a rat calvarial defect model. Collagen 3D nanopore scaffold (COL) and Pluronic F127 hydrogel (F127) incorporated with RvD1 (RvD1-COL-F127 group) or COL and F127 (COL-F127 group) were implanted in symmetrical calvarial defects. After implantation, RvD1 was administrated subcutaneously every 7 days for 4 weeks. The rats were sacrificed at weeks 1 and 8 post-implantation. Tissue samples were analyzed by real-time reverse transcriptase-polymerase chain reaction and histology at week 1. Radiographical and histological analyses were done at week 8. At week 1, calvarial defects treated with RvD1 exhibited decreased numbers of inflammatory cells and tartrate-resistant acid phosphatase (TRAP) positive cells, greater numbers of newly formed blood vessels, upregulated gene expression of vascular endothelial growth factor and alkaline phosphatase, and downregulated gene expression of receptor activator of nuclear factor-κB ligand, interleukin-1ß and tumor necrosis factor-α. At week 8, the radiographical results showed that osteoid area fraction of the RvD1-COL-F127 group was higher than that of the COL-F127 group, and histological examination exhibited enhanced osteoid formation and newly formed blood vessels in the RvD1-COL-F127 group. In conclusion, this study showed that RvD1 enhanced bone formation and vascularization in rat calvarial defects.

Impact of Resolvin D1 on the inflammatory phenotype of periodontal ligament cell response to hypoxia.

OBJECTIVE: Periodontal ligament cells (PDLCs) are critical for wound healing and regenerative capacity of periodontal diseases. Within an inflammatory periodontal pocket, a hypoxic environment can aggravate periodontal inflammation, where PDLCs response to the inflammation would change. Resolvin D1 (RvD1) is an endogenous lipid mediator, which can impact intracellular inflammatory pathways of periodontal/oral cells and periodontal regeneration. It is not clear how hypoxia and RvD1 impact the inflammatory responses of pro-inflammatory PDLCs phenotype. Therefore, this study aimed to test hypoxia could induce changes in pro-inflammatory phenotype of PDLCs and RvD1 could reverse it. METHODS: Human PDLCs were cultured from periodontal tissues from eight healthy individuals and were characterized by immunofluorescence staining of vimentin and cytokeratin. Cell viability was examined by Methyl-thiazolyl-tetrazolium (MTT) assay. To examine the effects of hypoxia and RvD1 on the inflammatory responses of pro-inflammatory PDLCs phenotype, protein levels and gene expressions of inflammatory cytokines and signal transduction molecules were measured by enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and real-time quantitative reverse transcription PCR (real-time qRT-PCR). Alizarin red S staining and real-time qRT-PCR were employed to study the effects of hypoxia and RvD1 on the osteogenic differentiation of pro-inflammatory PDLCs phenotype. RESULTS: It was found that hypoxia increases the expression of inflammatory factors at the gene level (p < .05). RvD1 reduced the expression of IL-1ß (p < .05) in PDLCs under hypoxia both at the protein and RNA levels. There were increases in the expression of p38 mitogen-activated protein kinase (p38 MAPK, p < .01) and protein kinase B (Akt, p < .05) in response to RvD1. Also, a significantly higher density of calcified nodules was observed after treatment with RvD1 for 21 days under hypoxia. CONCLUSION: Our results indicate that hypoxia up-regulated the inflammatory level of PDLCs. RvD1 can reduce under-hypoxia-induced pro-inflammatory cytokines in the inflammatory phenotype of PDLCs. Moreover, RvD1 promotes the calcium nodules in PDLCs, possibly by affecting the p38 MAPK signaling pathway through Akt and HIF-1α.

Efficacy of treating segmental bone defects through endochondral ossification: 3D printed designs and bone metabolic activities.

Three-dimensional printing (3D printing) is a promising technique for producing scaffolds for bone tissue engineering applications. Porous scaffolds can be printed directly, and the design, shape and porosity can be controlled. 3D synthetic biodegradable polymeric scaffolds intended for in situ bone regeneration must meet stringent criteria, primarily appropriate mechanical properties, good 3D design, adequate biocompatibility and the ability to enhance bone formation. In this study, healing of critical-sized (5 â€‹mm) femur defects of rats was enhanced by implanting two different designs of 3D printed poly(l-lactide-co-ε-caprolactone) (poly(LA-co-CL)) scaffolds seeded with rat bone marrow mesenchymal stem cells (rBMSC), which had been pre-differentiated in vitro into cartilage-forming chondrocytes. Depending on the design, the scaffolds had an interconnected porous structure of 300-500 â€‹µm and porosity of 50-65%. According to a computational simulation, the internal force distribution was consistent with scaffold designs and comparable between the two designs. Moreover, the defects treated with 3D-printed scaffolds seeded with chondrocyte-like cells exhibited significantly increased bone formation up to 15 weeks compared with empty defects. In all experimental animals, bone metabolic activity was monitored by positron emission tomography 1, 3, 5, 7, 11 and 14 weeks after surgery. This demonstrated a time-dependent relationship between scaffold design and metabolic activity. This confirmed that successful regeneration was highly reproducible. The in vitro and in vivo data indicated that the experimental setups had promising outcomes and could facilitate new bone formation through endochondral ossification.

Extracellular Vesicles Derived from Primed Mesenchymal Stromal Cells Loaded on Biphasic Calcium Phosphate Biomaterial Exhibit Enhanced Macrophage Polarization.

Mesenchymal stromal cells (MSC) loaded on biphasic calcium phosphate biomaterial (MSC + BCP) have been used as an advanced therapy medicinal product to treat complex maxillofacial bone defects in patients. Further, MSC-derived extracellular vesicles (EVs) are established vehicles of paracrine factors, supporting inter-cellular communication between MSC and other interacting cell types, such as monocytes/macrophages. However, the information about the immunomodulatory potential of EVs derived from MSC and biomaterial constructs (MSC + BCP:EV) and inflammatory primed constructs (MSCp + BCP:EV) are scarce. Hence, we isolated and characterized EVs from these different systems, and compared their cytokine contents with plastic-adherent MSC-derived EVs (MSC:EV). When EVs from all three MSC systems were added to the primary blood-derived macrophages in vitro, significantly higher numbers of M0 (naive) macrophages shifted to M2-like (anti-inflammatory) by MSCp + BCP:EV treatment. Further, this treatment led to enhanced switching of M1 polarized macrophages to M2 polarized, and conversely, M2 to M1, as evaluated by determining the M1/M2 ratios after treatment. The enhanced macrophage modulation by MSCp + BCP:EV was attributed to their higher immunomodulatory (TNFα, IL1ß, IL5), angiogenic (VEGF), and chemokine-rich (RANTES, MCP1, MIP1ß) cytokine cargo. In conclusion, we successfully isolated and characterized EVs from MSC + BCP constructs and demonstrated that, depending upon the tissue microenvironment, these EVs contribute towards modulating the macrophage-mediated inflammation and healing responses. The study offers new insights into the use of biomaterial-induced EVs for MSC secretome delivery, as a step towards future 'cell-free' bone regenerative therapies.

Systemic and local innate immune responses to surgical co-transplantation of mesenchymal stromal cells and biphasic calcium phosphate for bone regeneration.

Bone regeneration from mesenchymal stromal cells (MSC) is attributed to comprehensive immune modulation mediated by the MSC. However, the temporal and spatial regulation of these immune responses has not yet been described. The aim of the present study was to assess the local and systemic innate immune responses to implantation of biphasic calcium phosphate biomaterial (BCP) alone, or with bone marrow derived MSC (BCP+MSC), in critical-sized calvarial bone defects of Lewis rats. Four weeks after implantation, flow cytometry analysis of innate immune cells revealed increased numbers of circulating classical monocyte-macrophages (MM) and decreased non-classical MM in the BCP+MSC group. At week 8, this differential systemic MM response was associated with an increased presence of local tissue anti-inflammatory macrophages expressing CD68 and CD163 markers (M2-like). In the BCP group without MSC, NK cells increased at weeks 1 and 4, and neutrophils increased in circulation at weeks 2 and 8. At week 8, the increase in number of neutrophils in circulation was associated with decreased local tissue neutrophils, in the BCP+MSC group. Gene expression analysis of tissue biopsies from defects implanted with BCP+MSC, in comparison to BCP alone, revealed upregulated expression of early osteogenesis genes along with macrophage differentiation-related genes at weeks 1 and 8 and neutrophil chemotaxis-related genes at week 1. This study is the first to demonstrate that surgical implantation of BCP or BCP+MSC grafts differentially regulate both systemic and local tissue innate immune responses which enhance bone formation. The results provide new insights into immune mechanisms underlying MSC-mediated bone regeneration. STATEMENT OF SIGNIFICANCE: The suitability of biphasic calcium phosphate and mesenchymal stromal cell construct (BCP+MSC) transplantation is evident from their progress in clinical trials for treating challenging maxillofacial bone defects. But less is known about the overall immune response generated by this surgical process and how it later impacts the bone formation. To this end, it is crucial to understand for both clinicians and researchers, the systemic immune response to transplanting MSC in patients for ensuring both the safety and efficacy of cell therapies. In this study, we used rat calvarial bone defect model and showed that both systemic and local innate immunes responses (monocyte-macrophages and neutrophils) are favorably directed towards enhanced bone formation in BCP+MSC implanted defects, as compared to BCP alone.

Influence of Bone Substitutes on Mesenchymal Stromal Cells in an Inflammatory Microenvironment.

Bone regeneration is driven by mesenchymal stromal cells (MSCs) via their interactions with immune cells, such as macrophages (MPs). Bone substitutes, e.g., bi-calcium phosphates (BCPs), are commonly used to treat bone defects. However, little research has focused on MSC responses to BCPs in the context of inflammation. The objective of this study was to investigate whether BCPs influence MSC responses and MSC-MP interactions, at the gene and protein levels, in an inflammatory microenvironment. In setup A, human bone marrow MSCs combined with two different BCP granules (BCP 60/40 or BCP 20/80) were cultured with or without cytokine stimulation (IL1ß + TNFα) to mimic acute inflammation. In setup B, U937 cell-line-derived MPs were introduced via transwell cocultures to setup A. Monolayer MSCs with and without cytokine stimulation served as controls. After 72 h, the expressions of genes related to osteogenesis, healing, inflammation and remodeling were assessed in the MSCs via quantitative polymerase chain reactions. Additionally, MSC-secreted cytokines related to healing, inflammation and chemotaxis were assessed via multiplex immunoassays. Overall, the results indicate that, under both inflammatory and non-inflammatory conditions, the BCP granules significantly regulated the MSC gene expressions towards a pro-healing genotype but had relatively little effect on the MSC secretory profiles. In the presence of the MPs (coculture), the BCPs positively regulated both the gene expression and cytokine secretion of the MSCs. Overall, similar trends in MSC responses were observed with BCP 60/40 and BCP 20/80. In summary, within the limits of in vitro models, these findings suggest that the presence of BCP granules at a surgical site may not necessarily have a detrimental effect on MSC-mediated wound healing, even in the event of inflammation.

Conditioned Medium from Bone Marrow Mesenchymal Stem Cells Restored Oxidative Stress-Related Impaired Osteogenic Differentiation.

Oxidative stress from high levels of intracellular reactive oxygen species (ROS) has been linked to various bone diseases. Previous studies indicate that mesenchymal stem cells (MSC) secrete bioactive factors (conditioned medium (MSC-CM)) that have antioxidant effects. However, the antioxidant role of MSC-CM on osteogenesis has not been fully studied. We aimed to identify antioxidant proteins in MSC-CM using mass spectrometry-based proteomics and to explore their effects on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSC) exposed to oxidative stress induced by hydrogen peroxide (H2O2). Our analysis revealed that MSC-CM is comprised of antioxidant proteins that are involved in several biological processes, including negative regulation of apoptosis and positive regulation of cell proliferation. Then, hBMSC exposed to H2O2 were treated with MSC-CM, and the effects on their osteogenic differentiation were evaluated. MSC-CM restored H2O2-induced damage to hBMSC by increasing the antioxidant enzyme-SOD production and the mRNA expression level of the anti-apoptotic BCL-2. A decrease in ROS production and cellular apoptosis was also shown. MSC-CM also modulated mRNA expression levels of osteogenesis-related genes, runt-related transcription factor 2, collagen type I, bone morphogenic protein 2, and osteopontin. Furthermore, collagen type I protein secretion, alkaline phosphatase activity, and in vitro mineralization were increased. These results indicate that MSC-CM contains several proteins with antioxidant and anti-apoptotic properties that restored the impaired hBMSC osteogenic differentiation associated with oxidative stress.

Bone regeneration in rat calvarial defects using dissociated or spheroid mesenchymal stromal cells in scaffold-hydrogel constructs.

BACKGROUND: Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSC). 3D printing offers the possibility to produce customized scaffolds for complex bone defects. The aim of this study was to compare the potential of human BMSC cultured as 2D monolayers or 3D spheroids encapsulated in constructs of 3D-printed poly-L-lactide-co-trimethylene carbonate scaffolds and modified human platelet lysate hydrogels (PLATMC-HPLG) for bone regeneration. METHODS: PLATMC-HPLG constructs with 2D or 3D BMSC were assessed for osteogenic differentiation based on gene expression and in vitro mineralization. Subsequently, PLATMC-HPLG constructs with 2D or 3D BMSC were implanted in rat calvarial defects for 12 weeks; cell-free constructs served as controls. Bone regeneration was assessed via in vivo computed tomography (CT), ex vivo micro-CT and histology. RESULTS: Osteogenic gene expression was significantly enhanced in 3D versus 2D BMSC prior to, but not after, encapsulation in PLATMC-HPLG constructs. A trend for greater in vitro mineralization was observed in constructs with 3D versus 2D BMSC (p > 0.05). In vivo CT revealed comparable bone formation after 4, 8 and 12 weeks in all groups. After 12 weeks, micro-CT revealed substantial regeneration in 2D BMSC (62.47 ± 19.46%), 3D BMSC (51.01 ± 24.43%) and cell-free PLATMC-HPLG constructs (43.20 ± 30.09%) (p > 0.05). A similar trend was observed in the histological analysis. CONCLUSION: Despite a trend for superior in vitro mineralization, constructs with 3D and 2D BMSC performed similarly in vivo. Regardless of monolayer or spheroid cell culture, PLATMC-HPLG constructs represent promising scaffolds for bone tissue engineering applications.

Induction of osteogenic differentiation of bone marrow stromal cells on 3D polyester-based scaffolds solely by subphysiological fluidic stimulation in a laminar flow bioreactor.

The fatal determination of bone marrow mesenchymal stem/stromal cells (BMSC) is closely associated with mechano-environmental factors in addition to biochemical clues. The aim of this study was to induce osteogenesis in the absence of chemical stimuli using a custom-designed laminar flow bioreactor. BMSC were seeded onto synthetic microporous scaffolds and subjected to the subphysiological level of fluid flow for up to 21 days. During the perfusion, cell proliferation was significantly inhibited. There were also morphological changes, with F-actin polymerisation and upregulation of ROCK1. Notably, in BMSC subjected to flow, mRNA expression of osteogenic markers was significantly upregulated and RUNX2 was localised in the nuclei. Further, under perfusion, there was greater deposition of collagen type 1 and calcium onto the scaffolds. The results confirm that an appropriate level of fluid stimuli preconditions BMSC towards the osteoblastic lineage on 3D scaffolds in the absence of chemical stimulation, which highlights the utility of flow bioreactors in bone tissue engineering.