Extracellular Matrix Mineralization Research Articles

Scaffold Pore Geometry Guides Gene Regulation and Bone-like Tissue Formation in Dynamic Cultures

Cells sense and respond to scaffold pore geometry and mechanical stimuli. Many fabrication methods used in bone tissue engineering render structures with poorly controlled pore geometries. Given that cell-scaffold interactions are complex, drawing a conclusion on how cells sense and respond to uncontrolled scaffold features under mechanical loading is difficult. In this study, monodisperse templated scaffolds (MTSC) were fabricated and used as well-defined porous scaffolds to study the effect of dynamic culture conditions on bone-like tissue formation. Human bone marrow-derived stromal cells were cultured on MTSC or conventional salt-leached scaffolds (SLSC) for up to 7 weeks, either under static or dynamic conditions (wall shear stress [WSS] using spinner flask bioreactors). The influence of controlled spherical pore geometry of MTSC subjected to static or dynamic conditions on osteoblast cell differentiation, bone-like tissue formation, structure, and distribution was investigated. WSS generated within the two idealized geometrical scaffold features was assessed. Distinct response to fluid flow in osteoblast cell differentiation were shown to be dependent on scaffold pore geometry. As revealed by collagen staining and microcomputed tomography images, dynamic conditions promoted a more regular extracellular matrix (ECM) formation and mineral distribution in both scaffold types compared with static conditions. The results showed that regulation of bone-related genes and the amount and the structure of mineralized ECM were dependent on scaffold pore geometry and the mechanical cues provided by the two different culture conditions. Under dynamic conditions, SLSC favored osteoblast cell differentiation and ECM formation, whereas MTSC enhanced ECM mineralization. The spherical pore shape in MTSC supported a more trabecular bone-like structure under dynamic conditions compared with MTSC statically cultured or to SLSC under either static or dynamic conditions. These results suggest that cell activity and bone-like tissue formation is driven not only by the pore geometry but also by the mechanical environment. This should be taken into account in the future design of complex scaffolds, which should favor cell differentiation while guiding the formation, structure, and distribution of the engineered bone tissue. This could help to mimic the anatomical complexity of the bone tissue structure and to adapt to each bone defect needs. Impact statement Aging of the human population leads to an increasing need for medical implants with high success rate. We provide evidence that cell activity and the amount and structure of bone-like tissue formation is dependent on the scaffold pore geometry and on the mechanical environment. Fabrication of complex scaffolds comprising concave and planar pore geometries might represent a promising direction toward the tunability and mimicry the structural complexity of the bone tissue. Moreover, the use of fabrication methods that allow a systematic fabrication of reproducible and geometrically controlled structures would simplify scaffold design optimization.

Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition.

ABSTRACTCollagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation.This article has an associated First Person interview with the first author of the paper.

Osteogenic capability of strontium and icariin-loaded TiO2 nanotube coatings in vitro and in osteoporotic rats.

Titanium (Ti) and Ti alloys are widely used biomaterials, but they lack osteogenic capability for rapid bone integration. To improve osseointegration of Ti implants, TiO2 nanotubes were prepared using the anodizing oxidation technique, and strontium (Sr) combined with icariin (ICA) was loaded on TiO2 nanotube coatings. Cell adhesion and proliferation of MC3T3-E1 cells, alkaline phosphatase (ALP) activity, mineralization of extracellular matrix, and bone formation around titanium implants in ovariectomized rats, were examined separately. The results showed that compared with pure Ti, TiO2 and Sr-loaded TiO2 coatings, the coatings loaded with both Sr and ICA showed better effect on cell adhesion and proliferation, higher ALP activity and more red-stained mineralized nodules. Furthermore, more bone was formed around implants loaded with both Sr and ICA in osteoporotic rats. Therefore, coating with Sr and ICA is valuable for clinical application to strengthen the osseointegration of titanium implants, especially in osteoporotic patients.

Lactoferrin/Calcium Phosphate-Modified Porous Ti by Biomimetic Mineralization: Effective Infection Prevention and Excellent Osteoinduction

The surface modification of titanium (Ti) can enhance the osseointegration and antibacterial properties of implants. In this study, we modified porous Ti discs with calcium phosphate (CaP) and different concentrations of Lactoferrin (LF) by biomimetic mineralization and examined their antibacterial effects and osteogenic bioactivity. Firstly, scanning electron microscopy (SEM), the fluorescent tracing method, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and the releasing kinetics of LF were utilized to characterize the modified Ti surface. Then, the antibacterial properties against S. sanguis and S. aureus were investigated. Finally, in vitro cytological examination was performed, including evaluations of cell adhesion, cell differentiation, extracellular matrix mineralization, and cytotoxicity. The results showed that the porous Ti discs were successfully modified with CaP and LF, and that the LF-M group (200 μg/mL LF in simulated body fluid) could mildly release LF under control. Further, the LF-M group could effectively inhibit the adhesion and proliferation of S. sanguis and S. aureus and enhance the osteogenic differentiation in vitro with a good biocompatibility. Consequently, LF-M-modified Ti may have potential applications in the field of dental implants to promote osseointegration and prevent the occurrence of peri-implantitis.

Cell Surface Glycoprotein CD24 Marks Bone Marrow-Derived Human Mesenchymal Stem/Stromal Cells with Reduced Proliferative and Differentiation Capacity In Vitro.

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) are fundamental to bone regenerative therapies, tissue engineering, and postmenopausal osteoporosis. Donor variation among patients, cell heterogeneity, and unpredictable capacity for differentiation reduce effectiveness of BMSCs for regenerative cell therapies. The cell surface glycoprotein CD24 exhibits the most prominent differential expression during osteogenic versus adipogenic differentiation of human BMSCs. Therefore, CD24 may represent a selective biomarker for subpopulations of BMSCs with increased osteoblastic potential. In undifferentiated human BMSCs, CD24 cell surface expression is variable among donors (range: 2%-10%) and increased by two to fourfold upon osteogenic differentiation. Strikingly, FACS sorted CD24pos cells exhibit delayed mineralization and reduced capacity for adipocyte differentiation. RNAseq analysis of CD24pos and CD24neg BMSCs identified a limited number of genes with increased expression in CD24pos cells that are associated with cell adhesion, motility, and extracellular matrix. Downregulated genes are associated with cell cycle regulation, and biological assays revealed that CD24pos cells have reduced proliferation. Hence, expression of the cell surface glycoprotein CD24 identifies a subpopulation of human BMSCs with reduced capacity for proliferation and extracellular matrix mineralization. Functional specialization among BMSCs populations may support their regenerative potential and therapeutic success by accommodating cell activities that promote skeletal tissue formation, homeostasis, and repair.

A comparative study on silicon nitride, titanium and polyether ether ketone on mouse pre-osteoblast cells.

The current study provides more insights about the surface bioactivity of the silicon nitride (Si3N4) as a potential candidate for bone regeneration in craniofacial and orthopaedic applications compared with conventional implantation materials. Current skeletal reconstructive materials such as titanium and polyether ether ketone (PEEK) are limited by poor long-term stability, biocompatibility and prolonged healing. Si3N4 is an FDA-approved material for an intervertebral spacer in spinal fusion applications. It is biocompatible and has antimicrobial properties. Here, we hypothesize that Si3N4 was found to be an osteoconductive material and conducts the growth, differentiation of MC3T3-E1 cells for extracellular matrix deposition, mineralization and eventual bone regeneration for craniofacial and orthopaedic applications. MC3T3-E1 cells were used to study the osteoblastic differentiation and mineralization on sterile samples of Si3N4, titanium alloy and PEEK. The samples were then analysed for extracellular matrix deposition and mineralization by FTIR, Raman spectroscopy, SEM, EDX, Alizarin Red, qRT-PCR and ELISA. The in vitro study indicates the formation of collagen fibres and mineral deposition on all three sample surfaces. There was more profound and faster ECM deposition and mineralization on Si3N4 surface as compared to titanium and PEEK. The FTIR and Raman spectroscopy show formation of collagen and mineral deposition at 30 days for Si3N4 and titanium and not PEEK. The peaks shown by Raman for Si3N4 resemble closely to natural bone. Results also indicate the upregulation of osteogenic transcription factors such as RUNX2, SP7, collagen type I and osteocalcin. The authors concluded that Si3N4 rapidly conducts mineralized tissue formation via extracellular matrix deposition and biomarker expression in mouse calvarial pre-osteoblast cells. Thus, this study confirms that the bioactive Si3N4 could be a potential material for craniofacial and orthopaedic applications leading to rapid bone regeneration that resemble the natural bone structure.

Cyanogenic glycoside amygdalin influences functions of human osteoblasts in vitro

Amygdalin has been promoted as an alternative cancer cure. However, it is still unclear how this cyanogenic glycoside affects non-cancer cells including bone cells. This study first investigated the impact of amygdalin on viability, morphology and expression of important genes in human osteoblasts in vitro. Primary human osteoblast cultures were exposed to amygdalin at concentrations 0; 0.1; 1 and 10 mg/mL in growth medium for 72 h. Cell viability, osteoblasts morphology and expression of 10 genes associated with osteoblast-specific pathways, oxidative stress and cell death were determined. Osteoblasts viability was significantly decreased (–27.26%) and their size was reduced (–23.20%) at the highest concentration of amygdalin (10 mg/mL). This concentration of amygdalin down-regulated the expression of COL1A1 and ALPL genes, whereas the expression of BGLAP, TNFSF11 and WNT5A genes was increased. The osteoblast cultivation with 0.1 mg/mL amygdalin caused down-regulation of COL1A1 gene. No changes in expression were determined for RUNX2, BAX, CASP1, SOD1 and GPX1 genes among all tested concentrations of amygdalin. In conclusion, amygdalin in a high concentration negatively affected mineralization of extracellular matrix, increased bone resorption and decreased osteoblast viability. These changes were accompanied by modified expression profiles of responsible genes.

Vitamin K2 promotes the osteogenic differentiation of periodontal ligament stem cells via the Wnt/β-catenin signaling pathway

ObjectiveVitamin K2 (MK-4, menaquinone 4) plays an important role in osteoprotection. The present study aimed to examine the effect of MK-4 on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro and probed the potential signaling pathway. DesignPDLSCs were isolated from extracted premolars by tissue block culture method and were identified by flow cytometry. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to determine the effect of MK-4 on the proliferation of PDLSCs. Alkaline phosphatase (ALP) activity was analyzed quantitatively, and extracellular matrix mineralization was examined by Alizarin Red S staining. The mRNA and protein expression levels of ALP, Runx Family Transcription Factor 2 (Runx2), osteocalcin (OCN), and Sp7 Transcription Factor (SP7; Osterix) were measured by qRT-PCR and Western blot. In addition, after adding the inhibitor XAV-939, Western blot was used to assess the correlation with the Wnt/β-catenin signaling pathway. The above results were obtained by observing at least three fields randomly, and each experiment was repeated at least three times. ResultsThis study found that 10−5 M MK-4 significantly promoted the osteogenic differentiation of PDLSCs. Gene and protein expression levels of ALP, Runx2, OCN, and Osterix were all upregulated compared with control. Remarkably, after blocking the Wnt/β-catenin signaling pathway with XAV-939, the effect of MK-4 was apparently reversed. ConclusionThese results demonstrate that MK-4 can promote the osteogenic differentiation of PDLSCs, which is likely related to the activation of the Wnt/β-catenin signaling pathway.

Pathways involved in pony body size development

BackgroundThe mechanism of body growth in mammals is poorly understood. Here, we investigated the regulatory networks involved in body growth through transcriptomic analysis of pituitary and epiphyseal tissues of smaller sized Debao ponies and Mongolian horses at the juvenile and adult stages.ResultsWe found that growth hormone receptor (GHR) was expressed at low levels in long bones, although growth hormone (GH) was highly expressed in Debao ponies compared with Mongolian horses. Moreover, significant downregulated of the GHR pathway components m-RAS and ATF3 was found in juvenile ponies, which slowed the proliferation of bone osteocytes. However, WNT2 and PLCβ2 were obviously upregulated in juvenile Debao ponies, which led to premature mineralization of the bone extracellular matrix. Furthermore, we found that the WNT/Ca2+ pathway may be responsible for regulating body growth. GHR was demonstrated by q-PCR and Western blot analyses to be expressed at low levels in long bones of Debao ponies. Treatment with WNT antagonistI decreased the expression of WNT pathway components (P < 0.05) in vitro. Transduction of ATDC5 cells with a GHR-RNAi lentiviral vector decreased the expression of the GHR pathway components (P < 0.05). Additionally, the expression of the IGF-1 gene in the liver was lower in Debao ponies than in Mongolian horses at the juvenile and adult stages. Detection of plasma hormone concentrations showed that Debao ponies expressed higher levels of IGF-1 as juveniles and higher levels of GH as adults than Mongolian horses, indicating that the hormone regulation in Debao ponies differs from that in Mongolian horses.ConclusionOur work provides insights into the genetic regulation of short stature growth in mammals and can provide useful information for the development of therapeutic strategies for small size.

Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

In in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

Effects of normobaric cyclic hypoxia exposure on mesenchymal stem-cell differentiation-pilot study on bone parameters in elderly.

BACKGROUNDMesenchymal stem cells (MSC) of bone marrow are the progenitor of osteoblasts and adipocytes. MSC tend to differentiate into adipocytes, instead of osteoblasts, with aging. This favors the loss of bone mass and development of osteoporosis. Hypoxia induces hypoxia inducible factor 1α gene encoding transcription factor, which regulates the expression of genes related to energy metabolism and angiogenesis. That allows a better adaptation to low O2 conditions. Sustained hypoxia has negative effects on bone metabolism, favoring bone resorption. Yet, surprisingly, cyclic hypoxia (CH), short times of hypoxia followed by long times in normoxia, can modulate MSC differentiation and improve bone health in aging.AIMTo evaluate the CH effect on MSC differentiation, and whether it improves bone mineral density in elderly.METHODSMSC cultures were induced to differentiate into osteoblasts or adipocytes, in CH (3% O2 for 1, 2 or 4 h, 4 d a week). Extracellular-matrix mineralization and lipid-droplet formation were studied in MSC induced to differentiate into osteoblast or adipocytes, respectively. In addition, gene expression of marker genes, for osteogenesis or adipogenesis, have been quantified by quantitative real time polymerase chain reaction. The in vivo studies with elderly (> 75 years old; n = 10) were carried out in a hypoxia chamber, simulating an altitude of 2500 m above sea level, or in normoxia, for 18 wk (36 CH sessions of 16 min each). Percentages of fat mass and bone mineral density from whole body, trunk and right proximal femur (femoral, femoral neck and trochanter) were assessed, using dual-energy X-ray absorptiometry.RESULTSCH (4 h of hypoxic exposure) inhibited extracellular matrix mineralization and lipid-droplet formation in MSC induced to differentiate into osteoblasts or adipocytes, respectively. However, both parameters were not significantly affected by the other shorter hypoxia times assessed. The longest periods of hypoxia downregulated the expression of genes related to extracellular matrix formation, in MSC induced to differentiate into osteoblasts. Interestingly, osteocalcin (associated to energy metabolism) was upregulated. Vascular endothelial growth factor an expression and low-density lipoprotein receptor related protein 5/6/dickkopf Wnt signaling pathway inhibitor 1 (associated to Wnt/β-catenin pathway activation) increased in osteoblasts. Yet, they decreased in adipocytes after CH treatments, mainly with the longest hypoxia times. However, the same CH treatments increased the osteoprotegerin/receptor activator for nuclear factor kappa B ligand ratio in both cell types. An increase in total bone mineral density was observed in elderly people exposed to CH, but not in specific regions. The percentage of fat did not vary between groups.CONCLUSIONCH may have positive effects on bone health in the elderly, due to its possible inhibitory effect on bone resorption, by increasing the osteoprotegerin / receptor activator for nuclear factor kappa B ligand ratio.

Notoginsenoside R1 functionalized gelatin hydrogels to promote reparative dentinogenesis

Pulp-capping materials are commonly adopted in the clinic to form reparative dentin and thus protect dental pulp tissues from cases of deep caries, accidentally exposed pulps or partial pulpotomy. Some traditional pulp capping materials used in the clinic include calcium hydroxide and mineral trioxide aggregates. However, there are limitations to thin restorative dentin, and a long period of time is needed to cause degenerative changes in dental pulp. In this paper, injectable colloidal gels were developed to induce the formation of reparative dentin through a simple UV method from methacrylic acid functionalized gelatin loaded with notoginsenoside R1 (Gel-MA/NGR1). The results of the physicochemical property examinations showed that the prepared Gel-MA/NGR1 hydrogel possessed an appropriate interconnected porous microarchitecture with a pore size of 10.5 micrometres and suitable mechanical properties with a modulus of 50-60 kPa, enabling cell adhesion and proliferation. The hydrogel remained hydrophilic with sustained drug release performance. In addition, Gel-MA/NGR1 significantly enhanced the odontogenetic differentiation of mouse dental papilla cells by elevating the expression levels of the dentinogenic markers ALP and OCN and extracellular matrix mineralization. In vivo stimulation was carried out by injecting the precursors into the predrilled alveolar cavity of Sprague-Dawley rats followed by immediate in situ UV crosslinking. The results showed that Gel-MA/NGR1 has a strong capacity to promote reparative dentin formation. Haematoxylin& eosin, Masson, and immunohistochemical staining (DMP-1, DSPP, OCN and RUNX2) and micro-CT were employed to illustrate the effectiveness of dentinogenesis, and the relative volumes of calcification were found to have increased ~175-fold. All of the results showed that the Gel-MA/NGR1 hydrogel promoted reparative dentin formation, which suggests that this hydrogel provides great potential as a pulp-capping material to induce dentin formation.

Effect of the immune responses induced by implants in a integrated three-dimensional micro-nano topography on osseointegration.

In order to explore the abilities of an integrated three-dimensional micro-nano topography in immunomodulation and promoting bone formation, present study focuses on the titanium sheets used in the micro-nano topography by treating them with the sandblasted, large-grit and acid-etched (SLA)and alkaline thermal reaction. Further, we characterized and obtained the surface morphology, roughness, and hydrophilicity of the titanium sheets. Moreover, we detected their in vitro cytocompatibility and cell proliferation as well. In addition, investigation was carried out for the immunomodulatory ability of the titanium sheets in a micro-nano topography by observing the expression of M1 (classical activated macrophage) and M2 (alternatively activated macrophage) type marker factors, inflammatory factors, and morphological changes of RAW264.7 cells cultured on the titanium sheets in different topographies. Through cell migration experiments and coculture, we observed the effects of different titanium sheet immune environments on osteoblast migration, extracellular matrix mineralization, and osteoblast gene expression. These results showed that the micro-nano topography constructed through SLA and alkaline thermal treatment improved the hydrophilicity and promoted the cell proliferation. Moreover, it promoted RAW264.7 cells to polarize as M2 phenotype, thereby leading to the anti-inflammatory effect of local microenvironments. This facilitated osteoblasts to secrete bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor. Nonetheless, these findings provided a theoretical basis for the molecular biological mechanism related to implants in a micro-nano topography which promoted the osteointegration while offering a meaningful theoretical basis for the clinical treatment of such implants.

Citrate Supplementation Restores the Impaired Mineralisation Resulting from the Acidic Microenvironment: An In Vitro Study.

Chronic metabolic acidosis leads to bone-remodelling disorders based on excessive mineral matrix resorption and inhibition of bone formation, but also affects the homeostasis of citrate, which is an essential player in maintaining the acid–base balance and in driving the mineralisation process. This study aimed to investigate the impact of acidosis on the osteogenic properties of bone-forming cells and the effects of citrate supplementation in restoring the osteogenic features impaired by the acidic milieu. For this purpose, human mesenchymal stromal cells were cultured in an osteogenic medium and the extracellular matrix mineralisation was analysed at the micro- and nano-level, both in neutral and acidic conditions and after treatment with calcium citrate and potassium citrate. The acidic milieu significantly decreased the citrate release and hindered the organisation of the extracellular matrix, but the citrate supplementation increased collagen production and, particularly calcium citrate, promoted the mineralisation process. Moreover, the positive effect of citrate supplementation was observed also in the physiological microenvironment. This in vitro study proves that the mineral matrix organisation is influenced by citrate availability in the microenvironment surrounding bone-forming cells, thus providing a biological basis for using citrate-based supplements in the management of bone-remodelling disorders related to chronic low-grade acidosis.

Matrix Control of Periodontal Ligament Cell Activity Via Synthetic Hydrogel Scaffolds.

Rebuilding the tooth-supporting tissues (periodontium) destroyed by periodontitis remains a clinical challenge. Periodontal ligament cells (PDLCs), multipotent cells within the periodontal ligament (PDL), differentiate and form new PDL and mineralized tissues (cementum and bone) during native tissue repair in response to specific extracellular matrix (ECM) cues. Thus, harnessing ECM cues to control PDLC activity ex vivo, and ultimately, to design a PDLC delivery vehicle for tissue regeneration is an important goal. In this study, poly(ethylene glycol) hydrogels were used as a synthetic PDL ECM to interrogate the roles of cell-matrix interactions and cell-mediated matrix remodeling in controlling PDLC activity. Results showed that PDLCs within matrix metalloproteinase (MMP)-degradable hydrogels expressed key PDL matrix genes and showed a six to eightfold increase in alkaline phosphatase (ALP) activity compared with PDLCs in nondegradable hydrogel controls. The increase in ALP activity, commonly considered an early marker of cementogenic/osteogenic differentiation, occurred independent of the presentation of the cell-binding ligand RGD or soluble media cues and remained elevated when inhibiting PDLC-matrix binding and intracellular tension. ALP activity was further increased in softer hydrogels regardless of degradability and was accompanied by an increase in PDLC volume. However, scaffolds that fostered PDLC ALP activity did not necessarily promote hydrogel ECM mineralization. Rather, matrix mineralization was greatest in stiffer, MMP-degradable hydrogels and required the presence of soluble media cues. These divergent outcomes illustrate the complexity of the PDLC response to ECM cues and the limitations of current scaffold materials. Nevertheless, key biomaterial design principles for controlling PDLC activity were identified for incorporation into scaffolds for periodontal tissue regeneration. Impact statement Engineered scaffolds are an attractive approach for delivering periodontal ligament cells (PDLCs) to rebuild the tooth-supporting tissues. Replicating key extracellular matrix (ECM) cues within tissue engineered scaffolds may maximize PDLC potential. However, the identity of important ECM cues and how they can be harnessed to control PDLC activity is still unknown. In this study, matrix degradability, cell-matrix binding, and stiffness were varied using synthetic poly(ethylene glycol) hydrogels for three-dimensional PDLC culture. PDLCs exhibited dramatic and divergent responses to these cues, supporting further investigation of ECM-replicating scaffolds for control of PDLC behavior and periodontal tissue regeneration.

Improving the Intercellular Uptake and Osteogenic Potency of Calcium Phosphate via Nanocomplexation with the RALA Peptide.

Calcium phosphate-base materials (e.g., alpha tri-calcium phosphate (α–TCP)) have been shown to promote osteogenic differentiation of stem/progenitor cells, enhance osteoblast osteogenic activity and mediate in vivo bone tissue formation. However, variable particle size and hydrophilicity of the calcium phosphate result in an extremely low bioavailability. Therefore, an effective delivery system is required that can encapsulate the calcium phosphate, improve cellular entry and, consequently, elicit a potent osteogenic response in osteoblasts. In this study, collagenous matrix deposition and extracellular matrix mineralization of osteoblast lineage cells were assessed to investigate osteogenesis following intracellular delivery of α-TCP nanoparticles. The nanoparticles were formed via condensation with a novel, cationic 30 mer amphipathic peptide (RALA). Nanoparticles prepared at a mass ratio of 5:1 demonstrated an average particle size of 43 nm with a zeta potential of +26 mV. The average particle size and zeta potential remained stable for up to 28 days at room temperature and across a range of temperatures (4–37 °C). Cell viability decreased 24 h post-transfection following RALA/α-TCP nanoparticle treatment; however, recovery ensued by Day 7. Immunocytochemistry staining for Type I collagen up to Day 21 post-transfection with RALA/α-TCP nanoparticles (NPs) in MG-63 cells exhibited a significant enhancement in collagen expression and deposition compared to an untreated control. Furthermore, in porcine mesenchymal stem cells (pMSCs), there was enhanced mineralization compared to α–TCP alone. Taken together these data demonstrate that internalization of RALA/α-TCP NPs elicits a potent osteogenic response in both MG-63 and pMSCs.

Antagonism Between PEDF and TGF-β Contributes to Type VI Osteogenesis Imperfecta Bone and Vascular Pathogenesis.

Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder of bone and connective tissue, also known as brittle bone disease. Null mutations in SERPINF1, which encodes pigment epithelium-derived factor (PEDF), cause severe type VI OI, characterized by accumulation of unmineralized osteoid and a fish-scale pattern of bone lamellae. Although the potent anti-angiogenic activity of PEDF has been extensively studied, the disease mechanism of type VI OI is not well understood. Using Serpinf1(-/-) mice and primary osteoblasts, we demonstrate that loss of PEDF delays osteoblast maturation as well as extracellular matrix (ECM) mineralization. Barium sulfate perfusion reveals significantly increased vessel density in the tibial periosteum of Serpinf1(-/-) mouse compared with wild-type littermates. The increased bone vascularization in Serpinf1(-/-) mice correlated with increased number of CD31(+)/Endomucin(+) endothelial cells, which are involved in the coupling angiogenesis and osteogenesis. Global transcriptome analysis by RNA-Seq of Serpinf1(-/-) mouse osteoblasts reveals osteogenesis and angiogenesis as the biological processes most impacted by loss of PEDF. Intriguingly, TGF-β signaling is activated in type VI OI cells, and Serpinf1(-/-) osteoblasts are more sensitive to TGF-β stimulation than wild-type osteoblasts. TGF-β stimulation and PEDF deficiency showed additive effects on transcription suppression of osteogenic markers and stimulation of pro-angiogenic factors. Furthermore, PEDF attenuated TGF-β-induced expression of pro-angiogenic factors. These data suggest that functional antagonism between PEDF and TGF-β pathways controls osteogenesis and bone vascularization and is implicated in type VI OI pathogenesis. This antagonism may be exploited in developing therapeutics for type VI OI utilizing PEDF and TGF-β antibody. © 2022 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Strontium ranelate incorporated 3D porous sulfonated PEEK simulating MC3T3-E1 cell differentiation.

Polyetheretherketone (PEEK) has been used as an implant material because it has similar mechanical properties to natural bone. However, inferior osseointegration and bioinertness hamper the clinical application of PEEK. In this study, the surfaces of sulfonated three-dimensional (3D) PEEK porous structures were loaded with different concentrations of strontium ranelate, a compound commonly used in the treatment or prevention of osteoporosis by promoting bone formation and inhibiting bone resorption. Field-emission scanning electron microscopy was used to characterize the topography of the structures, elemental carbon, oxygen and strontium contents were measured by X-ray photoelectron spectroscopy, and surface zeta potentials and water-contact angle were also measured. The results indicated that strontium ranelate was successfully loaded onto the 3D porous structures. In vitro cellular results showed that strontium ranelate-treated sulfonated PEEK (SP-SR) strengthened the adhesion of MC3T3-E1 cells. The activity of alkaline phosphatase, collagen secretion and extracellular matrix mineralization deposition of MC3T3-E1 cells were also improved on the surface of SP-SR. These results indicate that SP-SR could serve a new implant candidate for surgical treatment.

Fluorosilicic acid induces DNA damage and oxidative stress in bone marrow mesenchymal stem cells

Excess fluoride in water can produce changes in tooth enamel mineralization and lead to diseases such as dental or skeletal fluorosis. The present study aimed to assess the genotoxic effects, oxidative stress, and osteoblastic mineralization induced by fluorosilicic acid (FA) in murine bone marrow-derived mesenchymal stem cells (BM-MSCs). BM-MSCs were isolated from the femurs and tibias of rats and cultured under standard conditions. Cells exposure occurred for 3, 7, 14, and 21 days to different concentrations of FA (0.6–9.6 mg/L). Cytotoxicity was observed in 14 and 21 days of exposure for all concentrations of FA (cell proliferation below 60%), and for 3 and 7 days, in which the proliferation was above 80%. Alkaline comet assay results demonstrated significant increased damage at concentrations of 0.3–2.4 mg/L, and the micronucleus test showed increased rates for micronucleus (1.2–2.4 mg/L) and nuclear buds (NBUDs) (0.3–2.4 mg/L) (P < 0.05/Dunnett’s test). An alkaline comet assay modified by repair endonuclease (FPG) was used to detect oxidized nucleobases, which occurred at 0.6 mg/L. The oxidative stress was evaluated by lipid peroxidation (TBARS) and antioxidant activity (TAC). Only lipid peroxidation was increased at concentrations of 0.6 mg/L and 1.2 mg/L (P < 0.001/Tukey’s test). The osteogenesis process determined the level of extracellular matrix mineralization. The mean concentration of Alizarin red increased significantly in 14 days at the 0.6 mg/L concentration group (P < 0.05/Tukey’s test) compared to the control group, and a significant difference between the groups regarding the activity of alkaline phosphatase (ALP) was observed. Unlike other studies, our results indicated that FA in BM-MSCs at concentrations used in drinking water induced genotoxicity, oxidative stress, and acceleration of bone mineralization.

Hydroxyapatite-modified micro/nanostructured titania surfaces with different crystalline phases for osteoblast regulation

Surface structures and physicochemical properties critically influence osseointegration of titanium (Ti) implants. Previous studies have shown that the surface with both micro- and nanoscale roughness may provide multiple features comparable to cell dimensions and thus efficiently regulate cell-material interaction. However, less attention has been made to further optimize the physicochemical properties (e.g., crystalline phase) and to further improve the bioactivity of micro/nanostructured surfaces. Herein, micro/nanostructured titania surfaces with different crystalline phases (amorphous, anatase and anatase/rutile) were prepared and hydroxyapatite (HA) nanorods were deposited onto the as-prepared surfaces by a spin-assisted layer-by-layer assembly method without greatly altering the initial multi-scale morphology and wettability. The effects of crystalline phase, chemical composition and wettability on osteoblast response were investigated. It is noted that all the micro/nanostructured surfaces with/without HA modification presented superamphiphilic. The activities of MC3T3-E1 cells suggested that the proliferation trend on the micro/nanostructured surfaces was greatly influenced by different crystalline phases, and the highest proliferation rate was obtained on the anatase/rutile surface, followed by the anatase; but the cell differentiation and extracellular matrix mineralization were almost the same among them. After ultrathin HA modification on the micro/nanostructured surfaces with different crystalline phases, it exhibited similar proliferation trend as the original surfaces; however, the cell differentiation and extracellular matrix mineralization were significantly improved. The results indicate that the introduction of ultrathin HA to the micro/nanostructured surfaces with optimized crystalline phase benefits cell proliferation, differentiation and maturation, which suggests a favorable biomimetic microenvironment and provides the potential for enhanced implant osseointegration in vivo.