Bone Extracellular Matrix Proteins Research Articles

Use of the Phylobone database for the annotation of bone extracellular matrix proteins in reindeer (Rangifer tarandus).

Bone extracellular matrix (ECM) proteins play a key role in bone formation and regeneration, including structural and regulatory functions. The Phylobone database consists of 255 ECM protein groups from 39 species and can be used to support bone research. Here, we gathered bone ECM proteins from reindeer (Rangifer tarandus), a member of the Cervidae family. The importance of reindeer lies in their ability to regenerate their antlers, in both male and female individuals. Protein sequences were extracted from the National Center for Biotechnology Information's repository and selected by homology searches. We identified 215 proteins and their corresponding functional domains, which are putatively present in the bone ECM of reindeer. Protein sequence alignments have shown a high degree of conservation between R. tarandus and other members of the Cervidae family. This update expands the Phylobone database and shows that it is a useful resource for the preliminary annotation of bone ECM proteins in novel proteomes.

The Osteoblast Transcriptome in Developing Zebrafish Reveals Key Roles for Extracellular Matrix Proteins Col10a1a and Fbln1 in Skeletal Development and Homeostasis.

Zebrafish are now widely used to study skeletal development and bone-related diseases. To that end, understanding osteoblast differentiation and function, the expression of essential transcription factors, signaling molecules, and extracellular matrix proteins is crucial. We isolated Sp7-expressing osteoblasts from 4-day-old larvae using a fluorescent reporter. We identified two distinct subpopulations and characterized their specific transcriptome as well as their structural, regulatory, and signaling profile. Based on their differential expression in these subpopulations, we generated mutants for the extracellular matrix protein genes col10a1a and fbln1 to study their functions. The col10a1a-/- mutant larvae display reduced chondrocranium size and decreased bone mineralization, while in adults a reduced vertebral thickness and tissue mineral density, and fusion of the caudal fin vertebrae were observed. In contrast, fbln1-/- mutants showed an increased mineralization of cranial elements and a reduced ceratohyal angle in larvae, while in adults a significantly increased vertebral centra thickness, length, volume, surface area, and tissue mineral density was observed. In addition, absence of the opercle specifically on the right side was observed. Transcriptomic analysis reveals up-regulation of genes involved in collagen biosynthesis and down-regulation of Fgf8 signaling in fbln1-/- mutants. Taken together, our results highlight the importance of bone extracellular matrix protein genes col10a1a and fbln1 in skeletal development and homeostasis.

Phylobone: a comprehensive database of bone extracellular matrix proteins in human and model organisms

The bone extracellular matrix (ECM) contains minerals deposited on highly crosslinked collagen fibrils and hundreds of non-collagenous proteins. Some of these proteins are key to the regulation of bone formation and regeneration via signaling pathways, and play important regulatory and structural roles. However, the complete list of bone extracellular matrix proteins, their roles, and the extent of individual and cross-species variations have not been fully captured in both humans and model organisms. Here, we introduce the most comprehensive resource of bone extracellular matrix (ECM) proteins that can be used in research fields such as bone regeneration, osteoporosis, and mechanobiology. The Phylobone database (available at https://phylobone.com) includes 255 proteins potentially expressed in the bone extracellular matrix (ECM) of humans and 30 species of vertebrates. A bioinformatics pipeline was used to identify the evolutionary relationships of bone ECM proteins. The analysis facilitated the identification of potential model organisms to study the molecular mechanisms of bone regeneration. A network analysis showed high connectivity of bone ECM proteins. A total of 214 functional protein domains were identified, including collagen and the domains involved in bone formation and resorption. Information from public drug repositories was used to identify potential repurposing of existing drugs. The Phylobone database provides a platform to study bone regeneration and osteoporosis in light of (biological) evolution, and will substantially contribute to the identification of molecular mechanisms and drug targets.

Biofunctionalization of polyetheretherketone implant material by bone-forming peptide-2 immobilization

Polyetheretherketone (PEEK) has favorable biomechanical properties to be used as an implant material. Unfortunately, it is hydrophobic and does not promote cellular adhesion, which could result in poor integration with bone tissue. Bio-functionalization of PEEK surface with osteogenic peptides derived from bone extracellular matrix proteins is an excit-ing approach to encourage bone formation around the implant. In the current study, bone-forming peptide-2 was immo-bilized on PEEK surface using two different methods, using dopamine and a diglycidyl ether as conjugate compounds, respectively. Peptide quantification test revealed that the two strategies resulted in the most amount of peptides were attached with 0.5 mM concentration and no further peptides were grafted with a higher peptide concentration. Both methods showed good peptide stability after agitation in aqueous solution. Peptide grafting was confirmed with ATR-FTIR. Surface characterizations with AFM and wettability tests resulted in a significant increase in surface roughness and surface area ratio for the peptide-grafted PEEK compared to unmodified PEEK, which led to a signifi-cant enhancement in the wettability of the modified PEEK surface

PCL and PCL/bioactive glass biomaterials as carriers for biologically active polyphenolic compounds: Comprehensive physicochemical and biological evaluation

In this work, polymeric and bioactive glass (BG)-modified composite films were successfully loaded with polyphenols (PPh) extracted from sage. It was hypothesized that PPh, alone and in combination with BGs particles, would affect physicochemical and biological properties of the films. Furthermore, sol-gel-derived BG particles would serve as an agent for control the release of the polyphenolic compounds, and other important properties related to the presence of PPh. The results showed that polyphenolic compounds significantly modified numerous material properties and also acted as biologically active substances. On the one hand, PPh can be considered as plasticizers for PCL, on the other hand, they can act as coupling agent in composite materials, improving their mechanical performance. The presence of PPh in materials improved their hydrophilicity and apatite-forming ability, and also provided antioxidant activity. What is important is that the aforementioned properties and kinetics of PPh release can be modulated by the use of various concentrations of PPh, and by the modification of PCL matrix with sol-gel-derived BG particles, capable of binding PPh. The films containing the lowest concentration of PPh exhibited cytocompatibility, significantly increased alkaline phosphatase activity and the expression of bone extracellular matrix proteins (osteocalcin and osteopontin) in human normal osteoblasts, while they reduced intracellular reactive oxygen species production in macrophages. Furthermore, materials loaded with PPh showed antibiofilm properties against Gram positive and Gram negative bacteria. The results suggest that obtained materials represent potential multifunctional biomaterials for bone tissue engineering with a wide range of tunable properties.

Analysis of multiple protein detection methods in human osteoporotic bone extracellular matrix: From literature to practice.

The punctual analysis of bone Extracellular Matrix (ECM) proteins represents a pivotal point for medical research in bone diseases like osteoporosis. Studies in this field, historically done to appreciate bone biology, were mainly conducted on animal samples and, up to today, only a few studies on protein detection in human bone are present. The challenges in bone ECM protein extraction and quantitation protocols are related to both the separation of proteins from the mineral content (i.e. hydroxyapatite) and the difficulty of avoiding protein denaturation during the extraction processes. The aim of the present work was to define appropriate protocol(s) for bone ECM protein extraction that could be applied to investigate both normal and pathological conditions. We compared and optimised some of the most used protocols present in the literature, modifying the protein precipitation method, the buffer used for resuspension and/or the volume of reagent used. Bradford and BCA assays and Western Blotting were used to evaluate the variations in the total protein recovery and the amount of selected proteins (Type I Collagen, TGF-β, IGF-1, Decorin, Osteopontin, Bone Sialoprotein-2 and Osteocalcin). Collectively, we were capable to draw-up two single-extract protocols with optimal recovery and ideal protein content, that can be used for a detailed analysis of ECM proteins in pathological bone samples. Time-consuming multi-extract procedures, optimised in their precipitation methods, are however crucial for a precise detection of specific proteins, like osteocalcin. As the matter of fact, also the demineralization processes, commonly suggested and performed in several protocols, could hinder an accurate protein detection, thus inherently affecting the study of a pathological bone ECM. This study represents a starting point for the definition of appropriate strategies in the study of bone extracellular matrix proteins involved in the onset and maintenance of bone diseases, as well as a tool for the development of customized scaffolds capable to modulate a proper feedback loop in bone remodelling, altered in case of diseases like osteoporosis.

MicroRNA-204 regulates osteogenic induction in dental follicle cells

The dental follicle is an ectomesenchymal tissue surrounding developing tooth germ that contains osteoblastic-lineage-committed stem/progenitor cells. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression during stem cell growth, proliferation, and differentiation. The aim of this study was to investigate the key regulators of miRNA during osteogenic differentiation in human dental follicle cells (hDFC). We analyzed miRNA expression profiles in hDFC during osteoblastic differentiation. Expression of miR-204 was decreased in hDFC during osteogenic induction on microarray analysis. Real-time and RT-PCR analysis also showed that the expression of miR-204 was decreased in all three hDFC during osteogenic differentiation. To investigate whether miR-204 has an effect on osteogenic differentiation, miR-204 was predicted to target alkaline phosphatase (ALP), secreted protein acidic and rich in cysteine (SPARC), and Runx2 in the in the 3'-UTRs by in silico analysis. When miR-204 was transfected into hDFC, the activity of ALP and protein levels of SPARC and Runx2 were decreased. mRNA levels of ALP, SPARC and Runx2 were also decreased by miR-204 transfection. Our data suggest that miR-204 negatively regulates the osteogenic differentiation of hDFC by targeting the bone-specific transcription factor Runx2, the mineralization maker ALP and the bone extracellular matrix protein SPARC.

Bone extracts immunomodulate and enhance the regenerative performance of dicalcium phosphates bioceramics

Immunomodulation strategies are believed to improve the integration and clinical performance of synthetic bone substitutes. One potential approach is the modification of biomaterial surface chemistry to mimic bone extracellular matrix (ECM). In this sense, we hypothesized that coating synthetic dicalcium phosphate (DCP) bioceramics with bone ECM proteins would modulate the host immune reactions and improve their regenerative performance. To test this, we evaluated the in vitro proteomic surface interactions and the in vivo performance of ECM-coated bioceramic scaffolds. Our results demonstrated that coating DCP scaffolds with bone extracts, specifically those containing calcium-binding proteins, dramatically modulated their interaction with plasma proteins in vitro, especially those relating to the innate immune response. In vivo, we observed an attenuated inflammatory response against the bioceramic scaffolds and enhanced peri-scaffold new bone formation supported by the increased osteoblastogenesis and reduced osteoclastogenesis. Furthermore, the bone extract rich in calcium-binding proteins can be 3D-printed to produce customized hydrogels with improved regeneration capabilities. In summary, bone extracts containing calcium-binding proteins can enhance the integration of synthetic biomaterials and improve their ability to regenerate bone probably by modulating the host immune reaction. This finding helps understand how bone allografts regenerate bone and opens the door for new advances in tissue engineering and bone regeneration. Statement of SignificanceForeign-body reaction is an important determinant of in vivo biomaterial integration, as an undesired host immune response can compromise the performance of an implanted biomaterial. For this reason, applying immunomodulation strategies to enhance biomaterial engraftment is of great interest in the field of regenerative medicine. In this article, we illustrated that coating dicalcium phosphate bioceramic scaffolds with bone-ECM extracts, especially those rich in calcium-binding proteins, is a promising approach to improve their surface proteomic interactions and modulate the immune responses towards such biomaterials in a way that improves their bone regeneration performance. Collectively, the results of this study may provide a conceivable explanation for the mechanisms involved in presenting the excellent regenerative efficacy of natural bone grafts.

Vertebrate SLRP family evolution and the subfunctionalization of osteoglycin gene duplicates in teleost fish

BackgroundOsteoglycin (OGN, a.k.a. mimecan) belongs to cluster III of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). In vertebrates OGN is a characteristic ECM protein of bone. In the present study we explore the evolution of SLRP III and OGN in teleosts that have a skeleton adapted to an aquatic environment.ResultsThe SLRP gene family has been conserved since the separation of chondrichthyes and osteichthyes. Few gene duplicates of the SLRP III family exist even in the teleosts that experienced a specific whole genome duplication. One exception is ogn for which duplicate copies were identified in fish genomes. The ogn promoter sequence and in vitro mesenchymal stem cell (MSC) cultures suggest the duplicate ogn genes acquired divergent functions. In gilthead sea bream (Sparus aurata) ogn1 was up-regulated during osteoblast and myocyte differentiation in vitro, while ogn2 was severely down-regulated during bone-derived MSCs differentiation into adipocytes in vitro.ConclusionsOverall, the phylogenetic analysis indicates that the SLRP III family in vertebrates has been under conservative evolutionary pressure. The retention of the ogn gene duplicates in teleosts was linked with the acquisition of different functions. The acquisition by OGN of functions other than that of a bone ECM protein occurred early in the vertebrate lineage.

α-hemihydrate calcium sulfate/octacalcium phosphate combined with sodium hyaluronate promotes bone marrow-derived mesenchymal stem cell osteogenesis in vitro and in vivo.

PurposeThe aims of this research were to combine α-hemihydrate calcium sulfate/octacalcium phosphate (α-CSH/OCP) with sodium hyaluronate (SH) or SH sulfate (SHS) to determine whether these composites can be used as a new type of bone repair material. This study may provide a theoretical basis and new ideas for the construction of active bone repair materials and their clinical application.MethodsIn this study, we combined α-CSH/OCP with SH or SHS. Scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), and the wettability test were performed, and porosity, setting time, in vitro degradation, and the mechanical properties of these composite materials were analyzed to evaluate the ultrastructural and physicochemical properties. We evaluated the histocompatibility of these composites by MTT assay, hemolysis, acute toxicity, and pyrogenic and intracutaneous stimulation tests. In addition, the osteogenic differentiation ability of these materials was detected in vitro using Western blot analysis and in vivo using an animal model of bone defect.ResultsThe α-CSH/OCP/SH composite had a compressive strength of 13.72 MPa, a porous rate of 27.45%, and the 28-day degradation rate of 64%. The MTT assay results showed that the relative proliferation rates of the α-CSH/OCP/SH group were greater than 90%. The results of the α-CSH/OCP/SH composite in the hemolysis, acute toxicity, pyrogenic, and intracutaneous stimulation tests were within the normal range. Western blot analysis indicated that the expression of bone extracellular matrix (ECM) proteins was notably upregulated and always higher in the α-CSH/OCP/SH group than in the other groups. XRD of the rabbit radius-defect model indicated that bone healing in the area implanted with α-CSH/OCP/SH was excellent approximately 9 weeks after repair.Conclusionα-CSH/OCP/SH has very good biocompatibility and exhibits clear advantages in the induction of bone regeneration and self-repair, and this compound shows promise in the field of bone tissue engineering.

Effects of Timing of Extracorporeal Shock Wave Therapy on Mandibular Distraction Osteogenesis: An Experimental Study in a Rat Model

Distraction osteogenesis (DO) is an established method for bone lengthening in the craniofacial skeleton. Its major drawback is the long consolidation period with attendant morbidity and possible complications. Several methods have been suggested to shorten the consolidation period. We evaluated the timing and effects of extracorporeal shock wave therapy (ESWT) on bone mineralization and extracellular bone matrix proteins during mandibular DO. Twenty-seven rats underwent mandibular DO (latency period, 3days; distraction period, 10days; 0.5mm/day) and were divided into 3 groups according to the timing of ESWT application: group I (control) received no treatment, whereas groups II and III received ESWT (0.18mJ/mm2) before and after the active distraction period, respectively. The distracted mandibles were harvested after 4weeks of consolidation and analyzed radiographically, histologically, and immunohistochemically. Group III showed significantly increased mineral density, enhanced bone formation, a higher collagen orientation index, and greater expression of type I collagen and osteocalcin proteins. Application of ESWT after active distraction enhances bone maturation and mineralization.

TRIP-1 in the extracellular matrix promotes nucleation of calcium phosphate polymorphs

ABSTRACTIn search for bone and dentin extracellular matrix (ECM) proteins, transforming growth factor beta receptor II interacting protein 1 (TRIP-1) was identified as a novel protein synthesized by osteoblasts and odontoblasts and exported to the ECM. TRIP-1 is a WD-40 (WD is Tryptophan-Aspartic acid dipeptide) protein that has been well recognized for its physiological role in the endoplasmic reticulum (ER). In the ER, TRIP-1 functions as an essential subunit of eukaryotic elongation initiation factor 3 and is involved in the protein translational machinery. Recently, we reported that TRIP-1 is localized in the ECM of bone and dentin. In this study, we demonstrate that varying concentrations of TRIP-1 can participate in the nucleation of calcium phosphate polymorphs. Nucleation studies performed with high calcium and phosphate concentration demonstrated that recombinant TRIP-1 could orchestrate the formation of hydroxyapatite crystals. Nucleation experiments performed on demineralized and deproteinized dentin wafer under physiological conditions and subsequent transmission electron microscope analysis of the deposits at the end of 7 and 14 days showed that TRIP-1 promoted the deposition of calcium phosphate mineral aggregates in the gap-overlap region of type I collagen. Taken together, we provide mechanistic insight into the role of this intracellular protein in matrix mineralization.

Osteopontin as a novel substrate for the proprotein convertase 5/6 (PCSK5) in bone

Seven proprotein convertases cleave the basic amino acid consensus sequence K/R-Xn-K/R↓ (where n=0, 2, 4 or 6 variable amino acids) to activate precursor proteins. Despite similarities in substrate specificity, basic amino acid-specific proprotein convertases have a distinct tissue distribution allowing for enzymatic actions on tissue-resident substrates. Proprotein convertase 5/6 (PC5/6) has two splice variants – soluble PC5/6A and membrane-bound PC5/6B – and is expressed during mouse development in many tissues including bone and tooth, but little is known about the substrates for PC5/6 therein. Osteopontin (OPN) is an abundant bone extracellular matrix protein with roles in mineralization, cell adhesion and cell migration, and it has putative consensus sequence sites for cleavage by PC5/6, which may modify its function in bone. Since PC5/6-knockout mouse embryos show developmental abnormalities, and reduced overall mineralization, we designed this study to determine whether OPN is a substrate of PC5/6. In silico analysis of OPN protein sequences identified four potential PC5/6 consensus cleavage sites in human OPN, and three sites – including a noncanonical sequence – in mouse OPN. Ex vivo co-transfections with human OPN revealed complete OPN cleavage reducing full-length OPN (~70kDa) to an N-terminal fragment migrating at ~50kDa and two C-terminal fragments at ~18kDa and ~16kDa. Direct cleavage of OPN by PC5/6A – the predominant isoform expressed in human osteoblast cells – was confirmed by cell-free enzyme-substrate assays and by mass spectrometry. The latter was also used to investigate potential cleavage sites. Co-transfections of PC5/6 and mouse OPN showed partial cleavage of OPN into a C-terminal OPN fragment migrating at ~30kDa and an N-terminal fragment migrating at ~29kDa. Micro-computed tomography of PC5/6-knockout embryos at E18.5 confirmed a reduction in mineralized bone, and in situ hybridization performed on cryo-sections of normal mouse bone using Pcsk5 and Opn anti-sense and control-sense cRNA probes indicated the co-localization of the expression of these genes in bone cells. This mRNA expression profile was supported by semi-quantitative RT-PCR using osteoblast primary cultures, and cultured MC3T3-E1 osteoblast and MLO-Y4 osteocyte cell lines. Immunoblotting for OPN from mouse bone extracts showed altered OPN processing in PC5/6-knockout mice compared to wildtype mice. OPN fragments migrated at ~25kDa and ~16kDa in wildtype bone and were not present in PC5/6-deficient bone. In conclusion, this study demonstrates that Pcsk5 is expressed in bone-forming cells, and that OPN is a novel substrate for PC5/6. Cleavage of OPN by PC5/6 may modify the function of OPN in bone and/or modulate other enzymatic cleavages of OPN, leading to alterations in the bone phenotype.

Collagen type XV and the 'osteogenic status'.

We have previously demonstrated that collagen type XV (ColXV) is a novel bone extracellular matrix (ECM) protein. It is well known that the complex mixture of multiple components present in ECM can help both to maintain stemness or to promote differentiation of stromal cells following change in qualitative characteristics or concentrations. We investigated the possible correlation between ColXV expression and mineral matrix deposition by human mesenchymal stromal cells (hMSCs) with different osteogenic potential and by osteoblasts (hOBs) that are able to grow in culture medium with or without calcium. Analysing the osteogenic process, we have shown that ColXV basal levels are lower in cells less prone to osteo‐induction such as hMSCs from Wharton Jelly (hWJMSCs), compared to hMSCs that are prone to osteo‐induction such as those from the bone marrow (hBMMSCs). In the group of samples identified as ‘mineralized MSCs’, during successful osteogenic induction, ColXV protein continued to be detected at substantial levels until early stage of differentiation, but it significantly decreased and then disappeared at the end of culture when the matrix formed was completely calcified. The possibility to grow hOBs in culture medium without calcium corroborated the results obtained with hMSCs demonstrating that calcium deposits organized in a calcified matrix, and not calcium ‘per se’, negatively affected ColXV expression. As a whole, our data suggest that ColXV may participate in ECM organization in the early‐phases of the osteogenic process and that this is a prerequisite to promote the subsequent deposition of mineral matrix.

Glycosylation of Dentin Matrix Protein 1 is critical for osteogenesis.

Proteoglycans play important roles in regulating osteogenesis. Dentin matrix protein 1 (DMP1) is a highly expressed bone extracellular matrix protein that regulates both bone development and phosphate metabolism. After glycosylation, an N-terminal fragment of DMP1 protein was identified as a new proteoglycan (DMP1-PG) in bone matrix. In vitro investigations showed that Ser89 is the key glycosylation site in mouse DMP1. However, the specific role of DMP1 glycosylation is still not understood. In this study, a mutant DMP1 mouse model was developed in which the glycosylation site S89 was substituted with G89 (S89G-DMP1). The glycosylation level of DMP1 was down-regulated in the bone matrix of S89G-DMP1 mice. Compared with wild type mice, the long bones of S89G-DMP1 mice showed developmental changes, including the speed of bone remodeling and mineralization, the morphology and activities of osteocytes, and activities of both osteoblasts and osteoclasts. These findings indicate that glycosylation of DMP1 is a key posttranslational modification process during development and that DMP1-PG functions as an indispensable proteoglycan in osteogenesis.

Osteoblast differentiation is enhanced by a nano-to-micro hybrid titanium surface created by Yb:YAG laser irradiation.

The aim of this study was to analyze the capacity of a new modified laser surface to stimulate calvarial osteoblasts isolated from neonatal mouse bones to differentiate and form mineralized nodules. Titanium discs were subjectezd or not to laser irradiation according to specific parameters and characterized. Osteoblasts isolated from neonatal mouse calvaria were cultured over the discs, and the capacity of these cells to proliferate (MTT assay), form mineralized nodules (Alizarin red assay), and enhance alkaline phosphatase activity (ALPase activity) was analyzed. Real-time PCR was used for quantification of gene expression. Laser-irradiated titanium discs (L) presented a rough nano-to-micrometric oxidized surface contrasting with the smooth pattern on polished discs (P). The Ra on the micrometric level increased from 0.32 ± 0.01 μm on P surfaces to 10.57 ± 0.39 μm on L surfaces. When compared with P, L promoted changes in osteoblast morphology, increased mineralized nodule formation in osteoblasts cultured on the surfaces for 14 days, and enhanced ALPase activity at days 7 and 14. Transcription factors triggering osteoblast differentiation (Runx2 and Sp7) and genes encoding the bone extracellular matrix proteins collagen type-1 (Col1a1), osteopontin (Spp1), and osteocalcin (Bglap) were upregulated in cells on L surfaces compared with those on P surfaces at days 1-14. Laser treatment of titanium surfaces created a rough surface that stimulated osteoblast differentiation. Laser treatment of titanium generates a reproducible and efficient surface triggering osteoblast differentiation that can be of importance for osteointegration.

Modulation of human valve interstitial cell phenotype and function using a fibroblast growth factor 2 formulation.

Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the VICs back to a fibroblastic phenotype with phenotypic and functional characteristics ascribed to cells in the intact valve. This methodology is fundamental in the study of normal valve biology, pathology and in the field of tissue engineering.

Osteogenic differentiation of adipose tissue-derived mesenchymal stem cells on nanostructured Ti6Al4V and Ti13Nb13Zr.

Bone tissue engineering and nanotechnology enable the design of suitable substitutes to restore and maintain the function of human bone tissues in complex fractures and other large skeletal defects. Long-term stability and functionality of prostheses depend on integration between bone cells and biocompatible implants. Human adipose tissue-derived mesenchymal stem cells (hAMSCs) have been shown to possess the same ability to differentiate into osteoblasts and to produce bone matrix of classical bone marrow derived stem cells (BMMSCs). Ti6A14V and Ti13Nb13Zr are two different biocompatible titanium alloys suitable for medical bone transplantation. Preliminary results from our Research Group demonstrated that smooth Ti6Al4V surfaces exhibit an osteoconductive action on hAMSCs, granting their differentiation into functional osteoblasts and sustaining bone matrix synthesis and calcification. The purpose of this study is to assay the ability of nanostructured Ti6Al4V and Ti13Nb13Zr alloys to preserve the growth and adhesion of hAMSCs and, mostly, to sustain and maintain their osteogenic differentiation and osteoblast activity. The overall results showed that both nanostructured titanium alloys are capable of sustaining cell adhesion and proliferation, to promote their differentiation into osteoblast lineage, and to support the activity of mature osteoblasts in terms of calcium deposition and bone extracellular matrix protein production.

Short-term glucocorticoid treatment causes spinal osteoporosis in ovariectomized rats.

In humans, glucocorticoid-induced osteoporosis is the most common cause of medication-induced osteoporosis. Recent clinical data suggest that glucocorticoid therapy increases the risk of vertebral fractures within a short treatment period. Therefore, this study aimed at investigating vertebral bone in a rat model of glucocorticoid-induced postmenopausal osteoporosis. Fifty Sprague-Dawley rats were randomly assigned into three groups: 1) untreated controls, 2) Sham-operated group, and 3) ovariectomized rats treated with glucocorticoid (dexamethasone) for 3 months (3M) after recovery from bilateral ovariectomy. Osteoporotic bone status was determined by means of the gold standard dual energy X-ray absorptiometry (DEXA) scan. Vertebral bodies were examined using µCT, histological analysis, mRNA expression analysis, and biomechanical compression testing. Further systemic effects were studied biochemically using serum marker analysis. Dexamethasone treatment showed at 3M a significantly lower bone mineral density in ovariectomized rats compared to Sham-operated control (p < 0.0001) as analyzed in vivo by DEXA. Furthermore, Z scores reached levels of -5.7 in the spine indicating sever osteoporotic bone status. Biomechanical testing of compression stability indicated a lower functional competence (p < 0.0001) in the spine of treated rats. µCT analysis showed significant reduction of bone volume density (BV/TV%; p < 0.0001), significantly enhanced trabecular spacing (Tb.Sp; p < 0.0001) with less trabecular number (Tb.N; p < 0.001) and complete loss of trabecular structures in glucocorticoid-treated ovariectomized rats. Histological analysis by osteoblast and osteoclast activities reflected a higher bone catabolism reflected by osteoclast counts by TRAP (p < 0.019) and lower bone catabolism indicated by ALP-stained area (p < 0.035).Serum analysis showed a significant increase in osteocalcin (p < 0.0001), osteopontin (p < 0.01) and insulin (p < 0.001) at 3M. Expression analysis of molecular markers in the vertebral body revealed lower expression in tenascin C in the OVX-steroid animals at 3M. Short-term glucocorticoid treatment of ovariectomized rats indicates according to DEXA standards a severe osteoporotic bone status in vertebral bone. Nonetheless, dysfunctional bone anabolism and enhanced bone catabolism are observed. Alterations of bone extracellular matrix proteins that correlate to inferior mechanical stability and affected microstructure were noticed and suggest further investigation. Treatment with dexamethasone was also seen to affect insulin and osteopontin levels and thus osteoblast function and maturation. This described animal model presents a recapitulation of clinically obtained data from early phase glucocorticoid-induced osteoporosis observed in patients.

Bone-like mineral nucleating peptide nanofibers induce differentiation of human mesenchymal stem cells into mature osteoblasts.

A bone implant should integrate to the tissue through a bone-like mineralized interface, which requires increased osteoblast activity at the implant-tissue boundary. Modification of the implant surface with synthetic bioinstructive cues facilitates on-site differentiation of progenitor stem cells to functional mature osteoblasts and results in subsequent mineralization. Inspired by the bioactive domains of the bone extracellular matrix proteins and the mussel adhesive proteins, we synthesized peptide nanofibers to promote bone-like mineralization on the implant surface. Nanofibers functionalized with osteoinductive collagen I derived Asp-Gly-Glu-Ala (DGEA) peptide sequence provide an advantage in initial adhesion, spreading, and early commitment to osteogenic differentiation for mesenchymal stem cells (hMSCs). In this study, we demonstrated that this early osteogenic commitment, however, does not necessarily guarantee a priority for maturation into functional osteoblasts. Similar to natural biological cascades, early commitment should be further supported with additional signals to provide a long-term effect on differentiation. Here, we showed that peptide nanofibers functionalized with Glu-Glu-Glu (EEE) sequence enhanced mineralization abilities due to osteoinductive properties for late-stage differentiation of hMSCs. Mussel-inspired functionalization not only enables robust immobilization on metal surfaces, but also improves bone-like mineralization under physiologically simulated conditions. The multifunctional osteoinductive peptide nanofiber biointerfaces presented here facilitate osseointegration for long-term clinical stability.