Osteocalcin Production Research Articles

Use of Polyphenol Tannic Acid to Functionalize Titanium with Strontium for Enhancement of Osteoblast Differentiation and Reduction of Osteoclast Activity.

Implant anchorage remains a challenge, especially in porous osteoporotic bone with high osteoclast activity. The implant surface is modified with osteogenic molecules to stimulate osseointegration. Strontium (Sr) is known for its osteogenic and anti-osteoclastogenic effects. In this study, Sr was immobilized on a titanium (Ti) surface using bioinspired polyphenol tannic acid (pTAN) coating as an ad-layer (Ti-pTAN). Two separate coating techniques were employed for comparative analysis. In the first technique, Ti was coated with a tannic acid solution containing Sr (Ti-pTAN-1Stp). In the second method, Ti was first coated with pTAN, before being immersed in a SrCl2 solution to immobilize Sr on Ti-pTAN (Ti-pTAN-2Stp). Ti-pTAN-1Stp and Ti-pTAN-2Stp augmented the alkaline phosphatase activity, collagen secretion, osteocalcin production and calcium deposition of MC3T3-E1 cells as compared to those of Ti and Ti-pTAN. However, osteoclast differentiation of RAW 264.7, as studied by TRAP activity, total DNA, and multinucleated cell formation, were decreased on Ti-pTAN, Ti-pTAN-1Stp and Ti-pTAN-2Stp as compared to Ti. Of all the substrates, osteoclast activity on Ti-pTAN-2Stp was the lowest. Hence, an economical and simple coating technique using pTAN as an adlayer preserved the dual biological effects of Sr. These results indicate a promising new approach to tailoring the cellular responses of implant surfaces.

Relationship of Iron Deposition to Calcium Deposition in Human Aortic Valve Leaflets

BackgroundIntraleaflet hematomas are associated with advanced stages of aortic valve calcification and suspected to be involved in disease progression. However, the mechanism by which the entry of blood cells into the valves affects the biology of aortic valvular interstitial cells (VICs) remains to be elucidated. ObjectivesThis study sought to evaluate the putative link between intraleaflet hematoma and aortic valve calcification and to assess its pathophysiological implications. MethodsThe spatial relationship between calcium deposits and intraleaflet hematomas was analyzed by whole-mount staining of calcified and noncalcified human aortic valves, obtained in the context of heart transplantation and from patients who underwent surgical valve replacement. Endothelial microfissuring was evaluated by en face immunofluorescence and scanning electron microscopic analyses of the fibrosa surface. Red blood cell (RBC) preparations were used in vitro to assess, by immunofluorescence microscopy and Alizarin red staining, the potential impact of intraleaflet hematomas on phenotypic changes in VICs. ResultsIntraleaflet hematomas, revealed by iron deposits and RBCs into the fibrosa, secondary to endothelial microfissuring, were consistently found in noncalcified valves. The contact of primary VICs derived from these valves with RBCs resulted in a global inflammatory and osteoblastic phenotype, reflected by the up-regulation of interleukin-6, interleukin-1β, bone sialoprotein, osteoprotegerin, receptor activator of nuclear factor kappa B, bone morphogenic protein 2, and muscle segment homeobox 2, the production of osteocalcin, and the formation of calcium deposits. ConclusionsThe acquisition of an osteoblastic phenotype in VICs that come into contact with the senescent RBCs of intraleaflet hematomas may play a critical role in the initiation of calcium deposition into the fibrosa of human aortic valves.

Bioactive diatomite and POSS silica cage reinforced chitosan/Na-carboxymethyl cellulose polyelectrolyte scaffolds for hard tissue regeneration

Recently, natural polymers are reinforced with silica particles for hard tissue engineering applications to induce bone regeneration. In this study, as two novel bioactive agents, effects of diatomite and polyhedral oligomeric silsesquioxanes (POSS) on chitosan (CS)/Na-carboxymethylcellulose (Na-CMC) polymer blend scaffolds are examined. In addition, the effect of silica reinforcements was compared with Si-substituted nano-hydroxyapatite (Si-Hap) particles. The morphology, physical and chemical structures of the scaffolds were characterized with SEM, liquid displacement, FT-IR, mechanical analysis, swelling and degradation studies. The particle size and the crystal structure of diatomite, POSS and Si-Hap particles were determined with DLS and XRD analyses. In vitro studies were performed to figure out the cytotoxicity, proliferation, ALP activity, osteocalcin production and biomineralization to demonstrate the promising use of natural silica particles in bone regeneration. Freeze-dried scaffolds showed 190–307 μm pore size range and 61–70% porosity. Both inorganic reinforcements increased the mechanical strength, enhanced the water uptake capacity and fastened the degradation rate. The nanocomposite scaffolds did not show any cytotoxic effect and enhanced the surface mineralization in osteogenic medium. Thus, diatomite and POSS cage structures can be potential reinforcements for nanocomposite design in hard tissue engineering applications.

Bioactive glass ions induce efficient osteogenic differentiation of human adipose stem cells encapsulated in gellan gum and collagen type I hydrogels

BackgroundDue to unmet need for bone augmentation, our aim was to promote osteogenic differentiation of human adipose stem cells (hASCs) encapsulated in gellan gum (GG) or collagen type I (COL) hydrogels with bioactive glass (experimental glass 2-06 of composition [wt-%]: Na2O 12.1, K2O 14.0, CaO 19.8, P2O5 2.5, B2O3 1.6, SiO2 50.0) extract based osteogenic medium (BaG OM) for bone construct development. GG hydrogels were crosslinked with spermidine (GG-SPD) or BaG extract (GG-BaG). MethodsMechanical properties of cell-free GG-SPD, GG-BaG, and COL hydrogels were tested in osteogenic medium (OM) or BaG OM at 0, 14, and 21 d. Hydrogel embedded hASCs were cultured in OM or BaG OM for 3, 14, and 21 d, and analyzed for viability, cell number, osteogenic gene expression, osteocalcin production, and mineralization. Hydroxyapatite-stained GG-SPD samples were imaged with Optical Projection Tomography (OPT) and Selective Plane Illumination Microscopy (SPIM) in OM and BaG OM at 21 d. Furthermore, Raman spectroscopy was used to study the calcium phosphate (CaP) content of hASC-secreted ECM in GG-SPD, GG-BaG, and COL at 21 d in BaG OM. ResultsThe results showed viable rounded cells in GG whereas hASCs were elongated in COL. Importantly, BaG OM induced significantly higher cell number and higher osteogenic gene expression in COL. In both hydrogels, BaG OM induced strong mineralization confirmed as CaP by Raman spectroscopy and significantly improved mechanical properties. GG-BaG hydrogels rescued hASC mineralization in OM. OPT and SPIM showed homogeneous 3D cell distribution with strong mineralization in BaG OM. Also, strong osteocalcin production was visible in COL. ConclusionsOverall, we showed efficacious osteogenesis of hASCs in 3D hydrogels with BaG OM with potential for bone-like grafts.

Megakaryocytes in Bone Metastasis: Protection or Progression?

Bone is the primary site where some cancers develop secondary growth, particularly those derived from breast and prostate tissue. The spread of metastasis to distant sites relies on complex mechanisms by which only cells endowed with certain characteristics are able to reach secondary growth sites. Platelets play a pivotal role in tumour growth, by conferring resistance to shear stress to the circulating tumour cells and protection against natural killer cell attack. Mature polyploid megakaryocytes (MKs) reside in close proximity to the vascular sinusoids of bone marrow, where their primary function is to produce platelets. Emerging evidence has demonstrated that MKs are essential for skeletal homeostasis, due to the expression and production of the bone-related proteins osteocalcin, osteonectin, bone morphogenetic protein, osteopontin, bone sialoprotein, and osteoprotegerin. Debate surrounds the role that MKs play in the development of bone metastasis, which is the topic of this mini-review.

Biocompatibility and biomineralization ability of Bio-C Pulpecto. A histological and immunohistochemical study.

To evaluate the inflammatory response, biomineralization and production of osteocalcin (OCN), osteopontin (OPN), and bone sialoprotein (BSP) of a new root filling material for primary teeth (Bio-C Pulpecto) compared to MTA. Polyethylene tubes containing Bio-C Pulpecto, MTA, and empty tubes were implanted into the dorsal connective tissue. After 7, 15, 30, 60, and 90days, the tubes with surrounding tissue were removed and histologically processed to be analysed using haematoxylin and eosin, von Kossa staining, or no staining for observation under polarized light and immunohistochemistry for the detection of OCN, OPN, and BSP. The Kruskal-Wallis test was used followed by Dunn's test. The significance level was set at 5%. The inflammatory response observed with MTA and Bio-C Pulpecto was more exacerbated until the 15th day and decreased from 30days on. No significant difference was found between control, MTA, and Bio-C Pulpecto (P>0.05). Bio-C Pulpecto and MTA showed positivity for von Kossa and birefringent to polarized light. The immunolabelling for OCN, OPN, and BSP was more intense for MTA and Bio-C Pulpecto on days 60 and 90 (P>0.05). Bio-C Pulpecto was biocompatible, induced biomineralization and was immunopositive for osteogenic markers such as OCN, OPN, and BSP, similarly to MTA.

Characterization of biomimetic silicate- and strontium-containing hydroxyapatite microparticles embedded in biodegradable electrospun polycaprolactone scaffolds for bone regeneration

A significant need exists today to develop novel alternatives to traditional bone grafts. Here, we report the potential use of mechanochemically synthesized hydroxyapatite (HA), silicate-, or strontium-containing HA microparticles and microparticle aggregates in combination with polycaprolactone (PCL) as hybrid scaffolds for filling bone defects. The detailed characterization of scaffolds was performed with high-resolution synchrotron radiation–based microcomputed laminography, XRD, EDX, and FTIR. An in vitro cell-scaffold interaction analysis showed a significant improvement of cell spreading in the case of hybrid scaffolds with silicate- and Sr-containing HA. Scaffolds with Sr- and silicate-containing HA affected the expression of several genes involved in morphogenesis and transcription. Scaffolds with Sr-containing HA increased the expression of markers of the primary component of the extracellular matrix, and scaffolds with Sr-containing HA facilitated cell mineralization via an increase in osteocalcin production. The hybrid scaffolds with silicate- and Sr-containing HA microparticles exerted the highest antibacterial activity against gram-positive bacterium Staphylococcus aureus compared to the unmodified PCL scaffolds. Based on these findings, the obtained scaffolds with Sr- or silicate-containing HA are believed to hold promise for bone tissue regeneration as compared to scaffolds containing pure HA.

Phosvitin and its hydrolysate promote differentiation and inhibit TNF-ɑ induced inflammation in MC3T3-E1 cells via ERK and AKT pathways

Phosvitin and its hydrolysate were reported to show antioxidant, anti-inflammation and antimicrobial activities. Given the critical role of inflammation on bone health, the work was designed to study the effects of phosvitin and its hydrolysate on osteoblast differentiation and inflammation in osteoblastic (MC3T3-E1) cells. Both phosvitin and its hydrolysate promoted osteoblast differentiation by upregulating expression of runt-related transcription factor 2, alkaline phosphatase (by phosvitin hydrolysate), osteocalcin production (by phosvitin) and collagen synthesis. They also inhibited TNF-ɑ induced inflammation by reducing the production of regulated on activation, normal T cell expressed and secreted and monocyte chemoattractant protein-1, which are crucial for recruiting osteoclast progenitor and maintaining osteoclast functions, and cyclooxygenase-2, a proinflammatory protein. Adding phosvitin and its hydrolysate activated both extracellular signal–regulated kinases (ERK) and protein kinase B (AKT) signaling pathways, indicating that both phosvitin and its hydrolysate promote osteoblast differentiation and exert antiinflammation effects via ERK and AKT pathways.

A Human Amelogenin-Derived Oligopeptide Enhances Osteogenic Differentiation of Human Periodontal Ligament Stem Cells

We have found newly synthesized peptide derived from commercially available Emdogain® for periodontal tissue regeneration therapies in our previous study. That synthesized peptide consisted of seven amino acid sequence, WYQNMIR, which is exon 5 of porcine amelogenin and had the same effects as Emdogain®. The amino acid sequence of human amelogenin exon 5 is WYQSIR which both N (asparagine) and M (methionine) in porcine amelogenin are replaced by S (serine). In the present study, we investigated the effect of a synthetic human amelogenin-derived peptide (HAP) consisting of WYQSIR on human periodontal ligament stem cells (PDLSCs) to be useful or not for the tissue regeneration. PDLSCs were isolated from third molars of adult donors. The effect of HAP on PDLSCs was investigated by culturing them in normal or osteogenic medium with and without HAP. Proliferation of the PDLSCs was evaluated using a cell proliferation assay after they had been treated with HAP (ranging from 1 ng/ml to 10,000 ng/ml) for 1, 3, 5, and 7 days. Osteogenic differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, calcium deposition, osteocalcin production, and mRNA expression of Runx2 and osteonectin after the PDLSCs had been treated with HAP (1,000 ng/ml) for 1, 7, 14, and 21 days. The number of viable cells was significantly increased in the presence of HAP in normal medium. Compared to cells cultured without HAP in osteogenic medium, cells cultured with HAP showed significantly increased ALP activity, calcium deposition, osteocalcin production, and Runx2 mRNA expression. The results show that HAP enhances proliferation and osteogenic differentiation of PDLSCs. The present study suggests that HAP may be a useful material for periodontal tissue regeneration.

Blood penetration into human aortic valves consecutive to endothelial micro-fissuring of the fibrosa determines the site of calcium deposition: Physiopathological implications

Intraleaflet hematoma has been associated with advanced stages of aortic valve stenosis. Its presence is suspected to be involved in faster disease progression. However, the mechanism whereby the entry and the presence of blood elements into the valves would have an impact on the biology of aortic valvular interstitial cells (VICs) remains to be deciphered. To evaluate the presence of intraleaflet hematoma, in respect to calcium deposition, at all stages of aortic valve calcification (AC) and to assess its putative pathophysiologic implication. The spatial relationship between calcium deposits and intraleaflet hematomas was analyzed by whole-mount staining of calcified and non-calcified human aortic valves, obtained in the context of heart transplantation and from patients who underwent surgical valve replacement. Endothelial micro-fissuring was evaluated by en face immunofluorescence and scanning electron microscope analyses of the fibrosa surface. Red blood cell preparations (RBCs) were used in vitro in order to assess, by immunofluorescence microscopy and Alizarin red staining, the potential impact of intraleaflet hematomas on the phenotype change of human VICs. Intraleaflet hematomas revealed by ferric iron deposits and extensive penetration of RBCs into the fibrosa, secondary to endothelial micro-fissuring, were found both in calcified and non-calcified valves. Primary VICs derived from non-calcified valves and cultured in the presence of senescent RBCs acquired an osteoblastic phenotype, as demonstrated by the production of osteocalcin and the formation of calcium deposits. Intraleaflet hematomas occurring through endothelial micro-fissuring may play a critical role in the initiation of calcium deposition into human aortic valves by driving the acquisition of an osteoblastic phenotype by the VICs that enter in contact with the infiltrated RBCs.

VEGF-A regulates angiogenesis during osseointegration of Ti implants via paracrine/autocrine regulation of osteoblast response to hierarchical microstructure of the surface.

Establishment of a patent vasculature at the bone-implant interface plays a significant role in determining overall success of orthopedic and dental implants. Osteoblasts produce vascular endothelial growth factor-A (VEGF-A), an important regulator of angiogenesis during bone formation and healing, and the amount secreted is sensitive to titanium (Ti) surface microtopography and surface energy. The purpose of this study was to determine if surface properties modulate cellular response to VEGF-A. MG63 osteoblast-like cells were transfected with shRNA targeting VEGF-A at >80% knockdown. Cells stably silenced for VEGF-A secreted reduced levels of osteocalcin, osteoprotegerin, FGF-2, and angiopoietin-1 when cultured on grit-blasted/acid-etched (SLA) and hydrophilic SLA (modSLA) Ti surfaces and conditioned media from these cultures caused reduced angiogenesis in an endothelial tubule formation assay. Treatment of MG63 cells with 20 ng/mL rhVEGF-A165 rescued production in silenced cells and increased production of osteocalcin, osteoprotegerin, FGF-2, and angiopoietin-1, with greatest effects on control cells cultured on modSLA. Addition of a neutralization antibody against VEGF receptor 2 (VEGFR2; Flk-1) resulted in a significant increase in VEGF-A production. Overall, this study indicates that VEGF-A has two roles in osseointegration: enhanced angiogenesis and an autocrine/paracrine role in maturation of osteoblast-like cells in response to Ti surface properties. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 423-433, 2019.

Multifunctional effects of a modification of SLA titanium implant surface with strontium‐containing nanostructures on immunoinflammatory and osteogenic cell function

This study investigated the effects of surface modification of clinically available sandblasted/acid-etched (SLA) titanium oral implants with strontium (Sr)-containing nanostructures on both early immunoinflammatory macrophage cell functions and osteogenic stem cell functions. The goal was to provide insight for future surface engineering of titanium implants with multifunctional effects, that is, tissue healing capacity at both the nonosteogenic cell centered initial stage and the subsequent osteogenic cell-governed later stage-osseointegration process. The Sr-containing nanostructure was prepared in on the SLA-type implant surface by wet chemical treatment. The results showed that Sr modification is favorable for early macrophage cell functions and increases osteogenic capacity of the SLA surface. Surface Sr modification notably upregulated regenerative macrophage phenotype expression and anti-inflammatory cytokine IL10 production while suppressing inflammatory cytokine TNFα. Sr incorporation enhanced certain early cellular events of ST2 stem cells such as early cellular spreading and critical integrin gene expression, which in turn notably increased osteogenic differentiation (osteogenesis-related phenotype gene expression and osteocalcin production) when combined with the microstructured SLA implant surface. Surface modification of SLA-type implants with Sr-containing nanostructures demonstrated the ability to favorably influence early immunoinflammatory macrophage cell functions and the functionality of osteogenesis cells, resulting in an enhanced osseointegration outcome. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3009-3020, 2018.

Glucose Tolerance Tests and Osteocalcin Responses in Healthy People.

Aim: Osteocalcin and undercarboxylated osteocalcin are suggested to be endocrine messengers from the bones and have been shown to stimulate insulin secretion from pancreatic β-cells. Insulin is hypothesized to increase the osteoblastic production of osteocalcin. The aim of the study was to investigate whether the route of glucose administration influence the circulating levels of osteocalcin and undercarboxylated osteocalcin.Methods: Twelve healthy males were enrolled in an acute cross-over study where they underwent an oral glucose tolerance test (OGTT), an isoglycemic intravenous glucose infusion (IIGI) and a fasting period (control). Blood samples were collected throughout 180 min and analyzed for osteocalcin and undercarboxylated osteocalcin and compared to insulin, glucose, and gastro-intestinal hormone responses.Results: Neither osteocalcin levels nor undercarboxylated osteocalcin levels over time differed between the OGTT, IIGI, and fasting. Baseline insulin levels and glucose levels were not associated with osteocalcin or undercarboxylated osteocalcin levels. Increases in insulin and glucose levels were neither associated with altered osteocalcin nor undercarboxylated osteocalcin levels.Conclusion: The route of glucose administration does not influence the circulating levels of osteocalcin and undercarboxylated osteocalcin despite the differential insulin and incretin responses. In the acute setting this suggests that insulin does not increase osteoblastic production of osteocalcin in healthy human males.

Human osteoblasts exhibit sexual dimorphism in their response to estrogen on microstructured titanium surfaces

BackgroundOsseointegration is dependent on the implant surface, surrounding bone quality, and the systemic host environment, which can differ in male and female patients. Titanium (Ti) implants with microstructured surfaces exhibit greater pullout strength when compared to smooth-surfaced implants and exhibit enhanced osteogenic cellular responses in vitro. Previous studies showed that 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] has a greater effect on rat osteoblast differentiation on microstructured Ti compared to smooth Ti surfaces and tissue culture polystyrene (TCPS). The stimulatory effect of 17β-estradiol (E2) on differentiation is observed in female osteoblasts on micro-rough Ti, but it is not known if male osteoblasts behave similarly in response to E2 and microtopography. This study assessed whether human male and female osteoblasts exhibit sex-specific differences in response to E2 and 1α,25(OH)2D3 when cultured on microstructured Ti surfaces.MethodsOsteoblasts from three male and three female human donors were cultured on Ti discs with varying surface profiles: a smooth pretreatment (PT), a coarse grit-blasted/acid-etched (SLA), and an SLA surface having undergone modification in a nitrogen environment and stored in saline to maintain hydrophilicity (modSLA). Cells cultured on these surfaces were treated with E2 or 1α,25(OH)2D3.ResultsMale and female human osteoblasts responded similarly to microstructure although there were donor-specific differences; cell number decreased, and osteocalcin (OCN), osteoprotegerin (OPG), and latent and active transforming growth factor 1 increased on SLA and modSLA compared to TCPS. Female osteoblasts had higher alkaline phosphatase activity and OCN production than male counterparts but produced less OPG. Both sexes responded similarly to 1α,25(OH)2D3. E2 treatment reduced cell number and increased osteoblast differentiation and factor production only in female cells.ConclusionsMale and female human osteoblasts respond similarly to microstructure and 1α,25(OH)2D3 but exhibit sexual dimorphism in substrate-dependent responses to E2. E2 affected female osteoblasts, suggesting that signaling is sex-specific and surface-dependent. Donor osteoblasts varied in response, demonstrating the need to test multiple donors when examining human samples. Understanding how male and female cells respond to orthopedic biomaterials will enable greater predictability post-implantation as well as therapies that are more patient-specific.

Influence of Inclusion of Apatite-based Microparticles on Osteogenic Cell Pheonotype and Behavior

ABSTRACTThe proximity of minerals found in human hard tissues may influence cell phenotype. Since cells respond to a range of environmental cues, this study sought to identify the influence of two apatite-based microparticles, hydroxyapatite (HA) and fluoroapatite (FA), upon dental and bone cells. After bone marrow stromal cells (BMSCs), 7F2 osteoblasts and dental pulp stem cells (DPSCs) were plated into media with or without HA or FA particles, the cells were analyzed for alkaline phosphatase (ALP) production, collagen I production, osteocalcin production, and mineralization for two weeks. The BMSCs and DPSCs in media without any microparticles produced more ALP compared to those with microparticles from Day 5 forward. In addition, the collagen I and osteocalcin production in cultures without microparticles was higher than in cultures containing either HA or FA particles. While some studies have shown increased osteogeonic differentiation in the presence of mineral particles, those studies used nanoparticles that were able to be internalized by the cells and were smaller than the microparticles used in this study.

In-office tooth bleaching with 38% hydrogen peroxide promotes moderate/severe pulp inflammation and production of ll-1β, TNF-β, GPX, FGF-2 and osteocalcin in rats.

Objectives:To study the intensity of inflammatory infiltrate and production of interleukin-1β (ll-1β), tumor necrosis factor-β (TNF-β), fibroblast growth factor-2 (FGF-2), glutathione peroxidase (GPX), and osteocalcin in response to in-office tooth bleaching in rats.Material and Methods:Twenty male Wistar rats were randomized into four groups (n=5) according to the received treatment (tooth bleaching or no treatment - control) and the period of euthanasia after treatment (24 h or 10 days). We performed tooth bleaching using a 38% hydrogen peroxide gel on maxillary and mandibular incisors. After euthanasia, incisors (20 per group) were processed for histological analysis, immunohistochemistry staining of ll-1β, TNF-β, FGF-2 and GPX and osteocalcin by immunofluorescence. We analyzed data using the Mann-Whitney and Kruskal-Wallis/Dunn tests (p<0.05).Results:The bleached groups presented statistically significant differences regarding the pulp inflammation stage compared with the control groups. Bleached teeth showed moderate/severe inflammatory infiltrate and control groups presented absent inflammatory cells or a negligible number of mononuclear cells (p<0.001) at two times (24 h and 10 days). There was strong staining for ll-1β, TNF-β, and GPX in bleached groups at 24 h and strong staining for ll-1β, TNF-β, GPX and FGF-2 at 10 days. After 10 days of tooth bleaching, the bleached group showed a statistically superior amount of osteocalcin than the other groups (p<0.01).Conclusions:Tooth bleaching with 38% hydrogen peroxide causes severe pulp inflammation, but characteristics of tissue repair after 10 days.

In Vitro and In Vivo Osteogenesis of Human Orbicularis Oculi Muscle-Derived Stem Cells.

Cell-based therapies for treating bone defects require a source of stem cells with osteogenic potential. There is evidence from pathologic ossification within muscles that human skeletal muscles contain osteogenic progenitor cells. However, muscle samples are usually acquired through a traumatic biopsy procedure which causes pain and morbidity to the donor. Herein, we identified a new alternative source of skeletal muscle stem cells (SMSCs) without conferring morbidity to donors. Adherent cells isolated from human orbicularis oculi muscle (OOM) fragments, which are currently discarded during ophthalmic cosmetic surgeries, were obtained using a two-step plating method. The cell growth kinetics, immunophenotype and capabilities of in vitro multilineage differentiation were evaluated respectively. Moreover, the osteogenically-induced cells were transduced with GFP gene, loaded onto the porous β-tricalcium phosphate (β-TCP) bioceramics, and transplanted into the subcutaneous site of athymic mice. Ectopic bone formation was assessed and the cell fate in vivo was detected. OOM-derived cells were fibroblastic in shape, clonogenic in growth, and displayed phenotypic and behavioral characteristics similar to SMSCs. In particular, these cells could be induced into osteoblasts in vitro evidenced by the extracellular matrix calcification and enhanced alkaline phosphatase (ALP) activity and osteocalcin (OCN) production. New bone formation was found in the cell-loaded bioceramics 6weeks after implantation. By using the GFP-labeling technique, these muscle cells were detected to participate in the process of ectopic osteogenesis in vivo. Our data suggest that human OOM tissue is a valuable and noninvasive resource for osteoprogenitor cells to be used in bone repair and regeneration.

The Growth Proliferation, Apoptotic Prevention, and Differentiation Induction of the Gelatin Hydrolysates from Three Sources to Human Fetal Osteoblasts (hFOB 1.19 Cells).

Gelatins from the skin of bovine, porcine, and tilapia were hydrolyzed to three degrees of hydrolysis (DH) by alcalase, neutrase, and papain, respectively. These hydrolysates at 0.02–0.1 g/L promoted the growth of human fetal osteoblasts by 101.4–135.7%, while higher DH or using papain and tilapia gelatins resulted in higher proliferation. The hydrolysates from porcine and tilapia gelatins at 0.05 g/L prevented induced apoptosis (decreasing total apoptotic proportions from 28.4% or 35.2% to 10.3–17.5% or 16.0–23.6%), and had differentiation induction (increasing alkaline phosphatase activity by 126.9–246.7% in early differentiation stage, or enhancing osteocalcin production by 4.1–22.5% in later differentiation stage). These hydrolysates had a similar amino acid profile; however, tilapia gelatin hydrolysates by papain with DH 15.4% mostly displayed higher activity than others. Tilapia gelatin hydrolysate could up-regulate β-catenin, Wnt 3a, Wnt 10b, cyclin D1, and c-Myc expression at mRNA levels by 1.11–3.60 folds, but down-regulate GSK 3β expression by 0.98 fold. Of note, β-catenin in total cellular and nuclear protein was up-regulated by 1.14–1.16 folds but unchanged in cytoplasmic protein, Wnt 10b, cyclin D1, and c-Myc expression were up-regulated by 1.27–1.95 folds, whilst GSK 3β expression was down-regulated by 0.87 fold. Activation of Wnt/β-catenin pathway is suggested to mediate cell proliferation and differentiation.

Osteogenic Differentiation Modulates the Cytokine, Chemokine, and Growth Factor Profile of ASCs and SHED.

Great efforts have been made to improve bone regeneration techniques owing to a growing variety of sources of stem cells suitable for autologous transplants. Specifically, adipose-derived stem cells (ASCs) and stems cells from human exfoliated deciduous teeth (SHED) hold great potential for bone tissue engineering and cell therapy. After a preliminary characterization of the main biomolecules ASCs and SHED released in their conditioned media, cells were kept both in normal and osteo-inducing conditions. Conventional assays were performed to prove their osteogenic potential such as quantitative real-time polymerase chain reaction (qRT-PCR) (for RUNX-2, collagen type I, osteopontin and osteonectin), alkaline phosphatase activity, osteocalcin production, and von Kossa staining. Conditioned media were tested again after the osteogenic induction and compared to maintaining condition both at base line and after 14 days of culture. The osteogenic condition inhibited the release of all the biomolecules, with the exception, concerning SHED, of growth-regulated alpha protein precursor (GROα), and, to a lesser extent, interleukin (IL)-8. In conclusion, our data support that undifferentiated ASCs and SHED may be preferable to committed ones for general cell therapy approaches, due to their higher paracrine activity. Osteoinduction significantly affects the cytokine, chemokine, and growth factor profile in a differential way, as SHED kept a more pronounced pro-angiogenic signature than ASCs.

In Vitro and In Vivo Osteogenic Activity of Titanium Implants Coated by Pulsed Laser Deposition with a Thin Film of Fluoridated Hydroxyapatite.

To enhance biocompatibility, osteogenesis, and osseointegration, we coated titanium implants, by krypton fluoride (KrF) pulsed laser deposition, with a thin film of fluoridated hydroxyapatite (FHA). Coating was confirmed by scanning electron microscopy (SEM) and scanning probe microscopy (SPM), while physicochemical properties were evaluated by attenuated reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Calcium deposition, osteocalcin production, and expression of osteoblast genes were significantly higher in rat bone marrow mesenchymal stem cells seeded on FHA-coated titanium than in cells seeded on uncoated titanium. Implantation into rat femurs also showed that the FHA-coated material had superior osteoinductive and osseointegration activity in comparison with that of traditional implants, as assessed by microcomputed tomography and histology. Thus, titanium coated with FHA holds promise as a dental implant material.