Mineralized Matrix Formation Research Articles

Decellularized Periosteum-Derived Hydrogels Promote the Proliferation, Migration and Osteogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells.

Human umbilical cord mesenchymal stem cells (hUCMSCs) are promising for bone tissue engineering, which have a non-invasive harvesting process, high cell yield, favorable proliferation capacity, and low immunogenicity. However, the osteogenic efficacy of hUCMSCs is relatively lower than that of bone marrow mesenchymal stem cells (BMSCs). Hydrogels from decellularized extracellular matrix (dECM) preserve the biological compositions and functions of natural ECM, which can provide tissue-specific cues to regulate phenotypic expression and cell fate. It is unknown, however, whether hydrogels from periosteum can serve as pro-osteogenic carriers of hUCMSCs. Herein, a decellularized periosteum-derived hydrogel (dPH) was fabricated to reveal the effects of periosteum-specific cues on the bioactivities of hUCMSCs. A widely used non-bone/periosteum-derived ECM hydrogel product, Matrigel, was used as the control group. After decellularization, the absence of nuclei in the histological analysis indicated a successful removal of cellular components, which was also confirmed by DNA content quantification. The storage modulus of dPH increased (from 164.49 ± 29.92 Pa to 855.20 ± 20.67 Pa) with increasing concentration (from 0.5% to 1%). With a highly porous, fibrous microstructure, dPH had a more hydrophilic surface than Matrigel, of which the water contact angle reduced 62.62 ± 0.04%. Furthermore, dPH prominently promoted the initial cellular spreading with a significantly higher cell surface area (1.47-fold), cell spreading length (1.45-fold) and proliferation (approximately 1.05–1.13-fold) of hUCMSCs than those of Matrigel. Additionally, dPH was conducive to cell migration, whereas no cells migrated to Matrigel in the Transwell model. Compared with those of the Matrigel group, the osteogenesis-related genes expression levels (runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN)) and mineralized matrix formation (9.74-fold) of the hUCMSCs significantly increased in the dPH group. Our study indicated that dPH could provide a pro-osteogenic microenvironment for hUCMSCs, thereby revealing a promising application potential to repair bone defects.

Mapping Bone Marrow Cell Response from Senile Female Rats on Ca-P-Doped Titanium Coating.

Chemical and topographical surface modifications on dental implants aim to increase the bone surface contact area of the implant and improve osseointegration. This study analyzed the cellular response of undifferentiated mesenchymal stem cells (MSC), derived from senile rats’ femoral bone marrow, when cultured on a bioactive coating (by plasma electrolytic oxidation, PEO, with Ca2+ and P5+ ions), a sandblasting followed by acid-etching (SLA) surface, and a machined surface (MSU). A total of 102 Ti-6Al-4V discs were divided into three groups (n = 34). The surface chemistry was analyzed by energy dispersive spectroscopy (EDS). Cell viability assay, gene expression of osteoblastic markers, and mineralized matrix formation were investigated. The cell growth and viability results were higher for PEO vs. MSU surface (p = 0.001). An increase in cell proliferation from 3 to 7 days (p < 0.05) and from 7 to 10 days (p < 0.05) was noted for PEO and SLA surfaces. Gene expression for OSX, ALP, BSP, and OPN showed a statistical significance (p = 0.001) among groups. In addition, the PEO surface showed a higher mineralized matrix bone formation (p = 0.003). In conclusion, MSC from senile female rats cultured on SLA and PEO surfaces showed similar cellular responses and should be considered for future clinical investigations.

Polydopamine Codoped BaTiO3-Functionalized Polyvinylidene Fluoride Coating as a Piezo-Biomaterial Platform for an Enhanced Cellular Response and Bioactivity.

For a number of clinical applications, Ti6Al4V implants with bioactive coatings are used. However, the deposition of a functional polymeric coating with desired physical properties, biocompatibility, and long-term stability remains largely unexplored. Among widely investigated synthetic biomaterials, polyvinylidene fluoride (PVDF) with β-polymorph and barium titanate (BaTiO3, BT) are considered as good examples of piezo-biopolymers and bioceramics, respectively. In this work, an adherent PVDF-based nanocomposite coating is deposited onto a Ti6Al4V substrate to explore the impact of its functional characteristics (piezoactivity) on cellular behavior and bioactivity (apatite growth and mineralized matrix formation). The precursor solution was prepared by physically grafting PVDF with polydopamine (pDOPA), forming mPVDF. Subsequently, mPVDF was reinforced with BaTiO3 nanoparticles in dimethylformamide/acetone solution, and the resulting nanocomposite (mPVDF-BT) was then spray-coated onto a roughened Ti6Al4V substrate using an airbrush at 140 °C under a pressure of 2 bar. The reproducibility of this simple yet effective processing approach to deposit chemically stable and adherent coatings was established. Remarkably, the modification with pDOPA and reinforcement with BaTiO3 nanoparticles resulted in an enhanced β-fraction of PVDF up to 96%. This nanocomposite encouraged cellular viability of preosteoblasts (∼158% at day 5) and characteristic spreading, in vitro. Our findings indicate that the mPVDF-BT coating facilitated faster nucleation and growth of the biomineralized apatite layer with ∼70% coverage within 3 days of incubation in the simulated body fluid. In addition, the coupling among surface polar energy (5.5 mN/m), fractional polarity (∼117%), roughness (8.7 μm), and fibrous morphology also endorsed better cellular behavior. Taken together, this coating deposition strategy will pave the pathway toward designing cell-instructive surface-modified Ti6Al4V biomaterials with tailored biomineralization and bioactivity properties for musculoskeletal reconstruction and regeneration applications.

Dentin Matrix Protein 1 on Titanium Surface Facilitates Osteogenic Differentiation of Stem Cells.

Dentin matrix protein 1 (DMP1) contains a large number of acidic domains, multiple phosphorylation sites, a functional arginine-glycine-aspartate (RGD) motif, and a DNA binding domain, and has been shown to play essential regulatory function in dentin and bone mineralization. DMP1 could also orchestrate bone matrix formation, but the ability of DMP1 on Ti to human mesenchymal stem cell (hMSC) conversion to osteoblasts has not been studied. There is importance to test if the DMP1 coated Ti surface would promote cell migration and attachment to the metal surface and promote the differentiation of the attached stem cells to an osteogenic lineage. This study aimed to study the human mesenchymal stem cells (hMSCs) attachment and proliferation on DMP1 coated titanium (Ti) disks compared to non-coated disks, and to assess possible osteoblastic differentiation of attached hMSCs. Sixty-eight Ti disks were divided into two groups. Group 1 disks were coated with dentin matrix protein 1 and group 2 disks served as control. Assessment with light microscopy was used to verify hMSC attachment and proliferation. Cell viability was confirmed through fluorescence microscopy and mitochondrial dehydrogenase activity. Real-time polymerase chain reaction analysis was done to study the gene expression. The proliferation assay showed significantly greater cell proliferation with DMP1 coated disks compared to the control group (p-value < 0.001). Cell vitality analysis showed a greater density of live cells on DMP1 coated disks compared to the control group. Alkaline phosphatase staining revealed higher enzyme activity on DMP1 coated disks and showed itself to be significantly higher than the control group (p-value < 0.001). von Kossa staining revealed higher positive areas for mineralized deposits on DMP1 coated disks than the control group (p-value < 0.05). Gene expression analysis confirmed upregulation of runt-related transcription factor 2, osteoprotegerin, osteocalcin, osteopontin, and alkaline phosphatase on DMP1 coated disks (p-value < 0.001). The dentin matrix protein promoted the adhesion, proliferation, facilitation differentiation of hMSC, and mineralized matrix formation.

Microenvironment Influences on Human Umbilical Cord Mesenchymal Stem Cell-Based Bone Regeneration.

The microenvironment, or niche, regulates stem cell fate and improves differentiation efficiency. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are ideal cell source for bone tissue engineering. However, the role of the microenvironments in hUC-MSC-based bone regeneration is not yet fully understood. This study is aimed at investigating the effects of the in vitro culture microenvironment (hUC-MSCs, nano-hydroxyapatite/collagen/poly (L-lactide) (nHAC/PLA), osteogenic media (OMD), and recombinant human bone morphogenetic protein-7 (rhBMP-7)) and the in vivo transplanted microenvironment (ectopic and orthotopic) on bone regeneration ability of hUC-MSCs. The isolated hUC-MSCs showed self-renewal potential and MSCs' characteristics. In the in vitro two-dimensional culture microenvironment, OMD or OMD with rhBMP-7 significantly enhanced hUC-MSCs' osteocalcin immunofluorescence staining, alkaline phosphatase, and Alizarin red staining; OMD with rhBMP-7 exhibited the highest ALP secretion and mineralized matrix formation. In the in vitro three-dimensional culture microenvironment, nHAC/PLA supported hUC-MSCs' adhesion, proliferation, and differentiation; the microenvironment containing OMD or OMD and rhBMP-7 shortened cell proliferation progression and made osteogenic differentiation progression advance; rhBMP-7 significantly attenuated the inhibiting effect of OMD on hUC-MSCs' proliferation and significantly enhanced the promoting effect of OMD on gene expression and protein secretion of osteogenic differentiation markers, calcium and phosphorous concentration, and mineralized matrix formation. The in vitro three-dimensional culture microenvironment containing OMD and rhBMP-7 induced hUC-MSCs to form the most new bones in ectopic or orthotopic microenvironment as proved by microcomputed tomography and hematoxylin and eosin staining, but bone formation in orthotopic microenvironment was significantly higher than that in ectopic microenvironment. The results indicated that the combination of in vitro hUC-MSCs+nHAC/PLA+OMD+rhBMP-7 microenvironment and in vivo orthotopic microenvironment provided a more optimized niche for bone regeneration of hUC-MSCs. This study elucidates that hUC-MSCs and their local microenvironment, or niche, play an important role in hUC-MSC-based bone regeneration. The endogenously produced BMP may serve an important regulatory role in the process.

The influence of chitosan, cellulose and alginate chemical nature on mineral matrix formation

Cell carriers such as chitosan, alginate and cellulose play a crucial role in cell behavior, however the influence of their chemical nature on osteogenesis is little studied. Here, rat bone marrow cells were grown on the surface of those polymers. The cells remained viable and produced a mineral matrix, which was deposited on the surface of all the polymers. The amount of inorganic matrix significantly varied according to the polymer, and the morphology of the inorganic matrix was influenced by the nature of the substrates, generating perfect hexagonal crystals at the surface of chitosan, alginate and cellulose.

Decellularized Cartilage Extracellular Matrix Incorporated Silk Fibroin Hybrid Scaffolds for Endochondral Ossification Mediated Bone Regeneration

Tissue engineering strategies promote bone regeneration for large bone defects by stimulating the osteogenesis route via intramembranous ossification in engineered grafts, which upon implantation are frequently constrained by insufficient integration and functional anastomosis of vasculature from the host tissue. In this study, we developed a hybrid biomaterial incorporating decellularized cartilage extracellular matrix (CD-ECM) as a template and silk fibroin (SF) as a carrier to assess the bone regeneration capacity of bone marrow-derived mesenchymal stem cells (hBMSC’s) via the endochondral ossification (ECO) route. hBMSC’s were primed two weeks for chondrogenesis, followed by six weeks for hypertrophy onto hybrid CD-ECM/SF or SF alone scaffolds and evaluated for the mineralized matrix formation in vitro. Calcium deposition biochemically determined increased significantly from 4-8 weeks in both SF and CD-ECM/SF constructs, and retention of sGAG’s were observed only in CD-ECM/SF constructs. SEM/EDX revealed calcium and phosphate crystal localization by hBMSC’s under all conditions. Compressive modulus reached a maximum of 40 KPa after eight weeks of hypertrophic induction. μCT scanning at eight weeks indicated a cloud of denser minerals in groups after hypertrophic induction in CD-ECM/SF constructs than SF constructs. Gene expression by RT-qPCR revealed that hBMSC’s expressed hypertrophic markers VEGF, COL10, RUNX2, but the absence of early hypertrophic marker ChM1 and later hypertrophic marker TSBS1 and the presence of osteogenic markers ALPL, IBSP, OSX under all conditions. Our data indicate a new method to prime hBMSC’S into the late hypertrophic stage in vitro in mechanically stable constructs for ECO-mediated bone tissue regeneration.

Photobiomodulation combined with adipose-derived stem cells encapsulated in methacrylated gelatin hydrogels enhances in vivo bone regeneration.

Reconstruction of bone defects is still a significant challenge. The aim of this study was to evaluate the effect of application of photobiomodulation (PBM) to enhance in vivo bone regeneration and osteogenic differentiation potential of adipose-derived stem cells (ADSCs) encapsulated in methacrylated gelatin (GEL-MA) hydrogels. Thirty-six Sprague-Dawley rats were randomly separated into 3 experimental groups (n = 12 each). The groups were control/blank defect (I), GEL-MA hydrogel (II), and ADSC-loaded GEL-MA (GEL-MA+ADSC) hydrogel (III). Biparietal critical sized bone defects (6 mm in size) are created in each animal. Half of the animals from each group (n = 6 each) were randomly selected for PBM application using polychromatic light in the near infrared region, 600-1200 nm. PBM was administered from 10 cm distance cranially in 48 h interval. The calvaria were harvested at the 20th week, and macroscopic, microtomographic, and histologic evaluation were performed for further analysis. Microtomographic evaluation demonstrated the highest result for mineralized matrix formation (MMF) in group III. PBM receiving samples of group III showed mean MMF of 79.93±3.41%, whereas the non-PBM receiving samples revealed mean MMF of 60.62±6.34 % (p=0.002). In terms of histologic evaluation of bone defect repair, the higher scores were obtained in the groups II and III when compared to the control group (2.0 for both PBM receiving and non-receiving specimens; p<0.001). ADSC-loaded microwave-induced GEL-MA hydrogels and periodic application of photobiomodulation with polychromatic light appear to have beneficial effect on bone regeneration and can stimulate ADSCs for osteogenic differentiation.

Regeneration of pulp-dentine complex-like tissue in a rat experimental model under an inflammatory microenvironment using high phosphorous-containing bioactive glasses.

To investigate the effects of a bioactive glass with a high proportion of phosphorus (BG-hP) on the repair and regeneration of dental pulps in rats under an inflammatory microenvironment. Human dental pulp cells (hDPCs) stimulated with 1μgmL-1 lipopolysaccharide (LPS) were co-cultured with 0.1mgmL-1 BG-hP. Cell proliferation was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assays. The expression of inflammation-related genes and odontogenic differentiation-related genes was determined by real-time PCR. Alizarin red staining was used to detect the formation of mineralized nodules. Coronal pulp tissues of rat molars were stimulated with 10mgmL-1 LPS and then treated with BG-hP. The expression of inflammation-related genes in pulp tissue was determined by real-time PCR. Haematoxylin-eosin staining and Masson staining were performed to observe the inflammatory response and mineralized matrix formation, after subcutaneous implantation in nude mice, at 3days and 4weeks, respectively. Analysis of variance was performed to measure statistical significance (P<0.05). BG-hP significantly reduced expression of interleukin-6 (IL-6) and IL-8 and significantly upregulated the expression of IL-10, IL-4 and transforming growth factor-β1 of the LPS-stimulated hDPCs (P<0.05). BG-hP significantly inhibited the initial cell number (P<0.05), but the hDPCs stimulated by LPS and co-cultured with BG-hP maintained the same proliferation rate as the untreated hDPCs. BG-hP significantly promoted the expression of dentine matrix protein-1 and dentine sialophosphoprotein and the mineralization capacity of the LPS-stimulated hDPCs (P<0.05). Furthermore, BG-hP significantly downregulated the expression of Il-6 and reduced the inflammatory response of the LPS-stimulated pulp tissue 3days after subcutaneous implantation (P<0.05). Four weeks after subcutaneous implantation, BG-hP induced the formation of a continuous layer of dentine-like structure with dentinal tubules and polarizing odontoblast-like cells aligned along it in the LPS-stimulated pulp tissue. The present preliminarily results demonstrated that the bioactive glass with a high proportion of phosphorus inhibited the inflammatory response and promoted the formation of a pulp-dentine complex in a rat experimental model. This study provides a foundation for the construction of materials with the dual functions of exerting anti-inflammatory effects and promoting tissue regeneration to meet the needs of dental pulp repair and regeneration.

Physicochemical properties of anodized-hydrothermally treated titanium with a nanotopographic surface structure promote osteogenic differentiation in dental pulp stem cells

Purpose Implants made of anodized-hydrothermally treated commercially pure titanium with a nanotopographic surface structure (SA-treated c.p.Ti) may advantageously promote contact osteogenesis during the early stages of healing. We hypothesized that utilizing SA-treated c.p.Ti with dental pulp stem cells (DPSCs) might improve osteoconduction during the process of osseointegration. This in vitro study investigated the effect of initial adhesion of DPSCs to SA-treated c.p.Ti compared with conventional c.p.Ti and anodic oxide (AO) c.p.Ti.Methods DPSCs were obtained from the mandibular incisors of Sprague-Dawley rats and cultured without osteogenic induction medium on c.p.Ti, AO c.p.Ti, and SA-treated c.p.Ti disks for up to 14 days. The morphology, proliferation, and differentiation of DPSCs were assessed by scanning electron microscopy, an MTT assay, and Alizarin Red S staining, respectively. A real-time quantitative polymerase chain reaction was used to quantify the mRNA expression of osteocalcin, osteopontin, and bone sialoprotein.Results On all disks, the DPSCs appeared flattened with the formation of extensions over time. The filopodium-like extensions were closely bound to the SA-treated c.p.Ti surface. The proliferation of DPSCs was not significantly different among the c.p.Ti treatments. However, DPSCs on SA-treated c.p.Ti showed the greatest mRNA levels of osteopontin, osteocalcin, and bone sialoprotein, as well as increased Alizarin Red S staining.Conclusions The results of the present in vitro study demonstrate that the surface properties of SA-treated c.p.Ti disks enhance osteogenic differentiation of DPSCs and may facilitate mineralized matrix formation on SA-treated c.p.Ti implant surfaces, which can enhance early bone regeneration.

Porous Tantalum VS. Titanium Implants: Enhanced Mineralized Matrix Formation after Stem Cells Proliferation and Differentiation.

Titanium dental implants are used routinely, with surgical procedure, to replace missing teeth. Even though they lead to satisfactory results, novel developments with implant materials can still improve implant treatment outcomes. The aim of this study was to investigate the efficiency of porous tantalum (Ta) dental implants for osseointegration, in comparison to classical titanium (Ti). Mesenchymal stem cells from the dental pulp (DPSC) were incubated on Ta, smooth titanium (STi), and rough titanium (RTi) to assess their adhesion, proliferation, osteodifferentiation, and mineralized matrix production. Cell proliferation was measured at 4 h, 24 h, 48 h with MTT test. Early osteogenic differentiation was followed after 4, 8, 12 days by alkaline phosphatase (ALP) quantification. Cells organization and matrix microstructure were studied with scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). Collagen production and matrix mineralization were evaluated by immunostaining and histological staining. MTT test showed significantly higher proliferation of DPSC on Ta at 24 h and 48 h. However, APL quantification after 8 and 12 days was significantly lower for Ta, revealing a delayed differentiation, where cells were proliferating the more. After 3 weeks, collagen immunostaining showed an efficient production of collagen on all samples. However, Red Alizarin staining clearly revealed a higher calcification on Ta. The overall results tend to demonstrate that DPSC differentiation is delayed on Ta surface, due to a longer proliferation period until cells cover the 3D porous Ta structure. However, after 3 weeks, a more abundant mineralized matrix is produced on and inside Ta implants. Cell populations on porous Ta proliferate greater and faster, leading to the production of more calcium phosphate deposits than cells on roughened and smooth titanium surfaces, revealing a potential enhanced capacity for osseointegration.

Collagen maturity and mineralization in mesenchymal stem cells cultured on the hydroxyapatite-based bone scaffold analyzed by ATR-FTIR spectroscopic imaging

Modern bone tissue engineering is based on the use of implants in the form of biomaterials, which are used as scaffolds for osteoprogenitor or stem cells. The task of the scaffolds is to temporarily sustain the function, proliferation and differentiation of bone tissue to enable its regeneration. The aim of this work is to use the macro ATR-FTIR spectroscopic imaging for analysis of the ceramic-based biomaterial (chitosan/β-1,3-glucan/hydroxyapatite). Specifically, during long-term culture of mesenchymal cells derived from adipose tissue (ADSCs) and bone marrow (BMDSCs) on the surface of scaffold. Infrared spectroscopy allows the acquisition of information on both the organic and inorganic parts of the tested composite. This innovative spectroscopic approach proved to be very suitable for studying the formation of new bone tissue and ECM components, sample staining and demineralization are not required and consequently the approach is rapid and cost-effective. The novelty of this study focuses on the innovatory use of ATR-FTIR imaging to evaluate the molecular structure and maturity of collagen as well as mineral matrix formation and crystallization in the context of bone regenerative medicine. Our research has shown that the biomaterial investigated on this work facilitates the formation of valid bone ECM of the stem cells types studied, as a result of the synthesis of type I collagen and mineral content deposition. Nevertheless, ADSC cells have been proven to produce a greater amount of collagen with a lower content of helical secondary structures, at the same time showing a higher mineralization intensity compared to BMDSC cells. Considering the above results, it could be stated that the developed scaffold is a promising material for biomedical applications, including modification of bone implants to increase their biocompatibility.

Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells

Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFβ, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.

Calcium aluminate cement-based blends for application to fill in bone defects

The need to treat bone defects is increasing dramatically as the population grows old. Calcium aluminate cement, CAC, has been applied to fill in bone defects. CAC has been used in orthopedics, as it can avoid some problems of commercial products, polymethylmethacrylate, PMMA, regarding its too low consistency and strong odor and exothermic reactions which can damage the surrounding tissue. Besides that, CAC can be molded in situ to provide intimate fitting to the defect rims. The injectability of cements is an important aspect when minimally invasive surgical techniques are used and for applications with limited accessibility and narrow cavities. At present work, CAC was initially analyzed via zeta potential and viscosity when mixed with different dispersant additives. Then, CAC blends containing different materials, alumina, zirconia, zinc oxide, hydroxyapatite, tricalcium phosphate, chitosan, collagen, or zinc oxide-bismuth oxide, were evaluated regarding their injectability and by means of in vitro mineralization and ex vivo uniaxial cold crushing strength tests. The best injectability conditions, 76 wt% solids, hypodermic needle, were used for the ex vivo tests. In the latter, the compositions selected by in vitro tests were injected to repair 3-mm defects on mouse femurs cut into pieces of 10 mm, whereas collagen and PMMA were inserted with a spatula. After setting the materials for 24 h at 37 °C in body fluid solution, the pieces were submitted to uniaxial cold crushing strength tests. The blends alumina, collagen, and chitosan resulted in greater reddish-stained areas indicating enhanced mineralized matrix formation for these groups, which also presented higher values of calcium deposits indicating a successful in vitro bone-like nodule formation. The filling of defects with CACH and its blends increased the resistance of bones when compared to bones with the added defect, with lower effect verified in the presence of chitosan and collagen.

Combined Use of Recombinant Human BMP-7 and Osteogenic Media May Have No Ideal Synergistic Effect on Leporine Bone Regeneration of Human Umbilical Cord Mesenchymal Stem Cells Seeded on Nanohydroxyapatite/Collagen/Poly (l-Lactide).

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a promising alternative source of mesenchymal stem cells (MSCs) that are enormously attractive for clinical use. This study was designed to investigate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) and/or osteogenic media (OMD) on bone regeneration of hUC-MSCs seeded on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA) in a rabbit model. The characteristics of stem cells were analyzed by plastic adherence, cell phenotype, and multilineage differentiation potential. Cell proliferation was examined using cell counting kit-8 assay. Osteogenic differentiation was evaluated by quantitative Ca2+ concentration, PO43- concentration, alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralized matrix formation. Bone regeneration was investigated in jaw bone defect repair in rabbit by microcomputed tomography, fluorescent labeling, and hematoxylin and eosin staining. Except for initial stress response, OMD and OMD + rhBMP-7 inhibited the proliferation of hUC-MSCs seeded on nHAC/PLA; rhBMP-7 inhibited cell proliferation in the nonlogarithmic phase and attenuated the inhibitory effect of OMD on cell proliferation. The inhibitory effects of OMD, rhBMP-7, and OMD + rhBMP-7 on cell proliferation were ranked as OMD > OMD + rhBMP-7 > rhBMP-7. OMD, rhBMP-7, and OMD + rhBMP-7 promoted Ca2+ concentration, PO43- concentration, ALP activity, OCN secretion, and mineralized matrix formation of hUC-MSCs seeded on nHAC/PLA. The promoting effects of OMD, rhBMP-7, and OMD+rhBMP-7 on Ca2+ concentration, PO43- concentration, ALP activity, OCN secretion, and mineralized matrix formation were ranked as rhBMP-7 > OMD > OMD + rhBMP-7, OMD > OMD + rhBMP-7 > rhBMP-7, OMD > rhBMP-7 > OMD + rhBMP-7, rhBMP-7 > OMD + rhBMP-7 > OMD, and OMD > rhBMP-7 > OMD + rhBMP-7, respectively. In rabbit jaw bone defect repair, OMD, rhBMP-7, and OMD + rhBMP-7 enhanced bone regeneration of hUC-MSCs seeded on nHAC/PLA, but the largest bone mineral apposition rate and bone formation were presented in cultures with rhBMP-7. These findings suggested that the combined use of rhBMP-7 and OMD may have no ideal synergistic effect on bone regeneration of hUC-MSCs seeded on nHAC/PLA in rabbit jaw bone defect.

Effect of sodium ascorbyl phosphate on osteoblast viability and differentiation.

Sodium ascorbyl phosphate (SAP) is a hydrophilic and stable L-ascorbic acid derivative, being converted by the cell phosphatases into free ascorbic acid (AA), which allows its sustained release in the medium. AA participates in the maintenance and healing of the periodontium. It presents a regulatory role of the osteoblastic activity, stimulating the deposition of collagen extracellular matrix followed by the induction of genes associated with the osteoblastic phenotype. It also acts in the elimination of reactive oxygen species, abundantly produced by defense cells in periodontal disease. The aim of this study was to evaluate the effect of SAP on osteoblast viability and differentiation. Mouse preosteoblastic cells of the MC3T3-E1 strain were used. Cell viability was assessed by the trypan blue dye exclusion assay and the expression of genes related to osteoblast differentiation by quantitative PCR. Collagen I secretion was evaluated by ELISA, and mineralized matrix formation was assayed by Alizarin red S staining. The results showed that SAP at concentrations from 50 to 500µmol/L does not influence preosteoblast cell viability, but stimulates their differentiation, observed by the induction of RUNX2, COL1A1, and BGLAP2; by the higher secreted levels of collagen I; and also by the increase in the mineralization of the extracellular matrix in cells exposed to this agent at 200 or 400µmol/L, compared with those not exposed. By its stability and capacity to induce preosteoblastic cell differentiation, our results indicate that the incorporation of SAP into local release devices, membranes/scaffolds or biomaterials, could favor bone tissue formation and therefore periodontal healing.

A simvastatin-releasing scaffold with periodontal ligament stem cell sheets for periodontal regeneration.

Simvastatin (SIM) has been documented to induce the osteogenic differentiation of periodontal ligament stem cells (PDLSCs). To establish an efficient release system for periodontal regeneration, a polycaprolactone (PCL) membrane scaffold containing SIM was electrospun and evaluated. The obtained PCL-SIM membrane scaffold showed sustained release up to 28 days, without deleterious effect on proliferation of PDLSCs on the scaffolds. PDLSCs were seeded onto scaffolds and their osteogenic differentiation was evaluated. After 21 days, expressions of collagen type I, alkaline phosphatase and bone sialoprotein genes were significantly upregulated and mineralized matrix formation was increased on the PCL-SIM scaffolds compared with the PCL scaffolds. In a heterotopic periodontal regeneration model, a cell sheet-scaffold construct was assembled by placement of multilayers of PDLSC sheets on PCL or PCL-SIM scaffolds, and these were then placed between dentin and ceramic bovine bone for subcutaneous implantation in athymic mice. After 8 weeks, the PCL-SIM membrane showed formation of significantly more ectopic cementum-like mineral on the dentin surface. These findings demonstrated that the PCL-SIM membrane scaffold promotes cementum-like tissue formation by sustained drug release, suggesting the feasibility of its therapeutic use with PDLSC sheets to improve periodontal regeneration.

Prostate Cancer Phenotype Influences Bone Mineralization at Metastasis: A Study Using an In Vitro Prostate Cancer Metastasis Testbed.

ABSTRACTIn this study, two types of prostate cancer cell lines, highly metastatic PC‐3 and low metastatic MDA PCa 2b (PCa) were cultured on bone mimetic scaffolds to recapitulate metastasis to bone. A unique in vitro 3D tumor model that uses a sequential culture (SC) of human mesenchymal stem cells followed by seeding with cancer cells after bone formation was initiated to study the phenotype‐specific interaction between prostate cancer cells and bone microenvironment. The PCa cells were observed to be less prolific and less metastatic, and to form multicellular tumoroids in the bone microenvironment, whereas PC‐3 cells were more prolific and were highly metastatic, and did not form multicellular tumoroids in the bone microenvironment. The metastatic process exhibited by these two prostate cancer cell lines showed a significant and different effect on bone mineralization and extracellular matrix formation. Excessive bone formation in the presence of PC‐3 and significant osteolysis in the presence of PCa were observed, which was also indicated by osteocalcin and MMP‐9 expression as measured by ELISA and qRT‐PCR. The field emission scanning electron microscopy images revealed that the structure of mineralized collagen in the presence of PC‐3 is different than the one observed in healthy bone. All experimental results indicated that both osteolytic and osteoblastic bone lesions can be recapitulated in our tumor testbed model and that different cancer phenotypes have a very different influence on bone at metastasis. The 3D in vitro model presented in this study provides an improved, reproducible, and controllable system that is a useful tool to elucidate osteotropism of prostate cancer cells. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Notch Signaling Inhibition by LY411575 Attenuates Osteoblast Differentiation and Decreased Ectopic Bone Formation Capacity of Human Skeletal (Mesenchymal) Stem Cells.

Background Chemical biology approaches using small molecule inhibitors targeting specific signaling pathways are useful tools to dissect the molecular mechanisms governing stem cell differentiation and for their possible use in therapeutic interventions. Methods Stem cell signaling small molecule library functional screen was performed employing human bone marrow skeletal (mesenchymal) stem cells (hBMSCs). Alkaline phosphatase (ALP) activity and formation of mineralized matrix visualized by Alizarin red staining were employed as markers for osteoblastic differentiation. Global gene expression profiling was conducted using the Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. Pathway analyses were conducted using the Ingenuity Pathway Analysis (IPA) tool. In vivo ectopic bone formation was performed using hBMSC mixed with hydroxyapatite–tricalcium phosphate granules that were implanted subcutaneously in 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius red staining were performed to identify bone formation in vivo. Results Among the tested molecules, LY411575, a potent γ-secretase and Notch signaling inhibitor, exhibited significant inhibitory effects on osteoblastic differentiation of hBMSCs manifested by reduced ALP activity, mineralized matrix formation, and decreased osteoblast-specific gene expression as well as in vivo ectopic bone formation. Global gene expression profiling of LY411575-treated cells revealed changes in multiple signaling pathways, including focal adhesion, insulin, TGFβ, IL6, and Notch signaling, and decreased the expression of genes associated with functional categories of tissue development. Among the affected signaling networks were TGFβ1, SPP1, and ERK regulatory networks. Conclusions We identified γ-secretase inhibitor (LY411575) as a potent regulator of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with ectopic bone formation.

Effect of selenium incorporation on the structure and in vitro bioactivity of 45S5 bioglass

In vitro bioactivity, biodegradation, and biocompatibility behavior of a new bioactive glass systems were investigated with the incorporation of selenium oxide, for the replacement of sodium oxide in the traditional 45S5 bioglass composition. The apatite-forming ability of melt-derived bioactive glasses was evaluated by immersion studies in simulated body fluid while monitoring the concentration of silicon, calcium, phosphorus, sodium, and selenium in the medium. The weight loss of bioactive glasses and pH change in the tris-(hydroxymethyl)-amino methane buffer solution was determined to observe the biodegradation behavior of glass samples. The glasses were characterized by a Fourier transform infrared spectroscopy, scanning electron microscopy, inductively coupled plasma, and Vickers hardness measurements. The biocompatibility evaluation of the glasses was determined through in vitro osteogenesis assays by cell viability, alkaline phosphatase activity, and mineralized matrix formation. The incorporation of selenium enhanced the hydroxyapatite formation on the bioactive glass surface and microhardness of glasses. The hardness of glasses was found to decrease with immersion duration. The results indicate that selenium incorporated bioactive glasses can be used as bioactive material in bone tissue engineering applications.