24R,25-Dihydroxyvitamin D3 Protects against Articular Cartilage Damage following Anterior Cruciate Ligament Transection in Male Rats

Osteoarthritis (OA) in humans is associated with low circulating 25-hydroxyvitamin D3 [25 (OH)D3]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25 (OH)2D3] accumulates in articular cartilage following injection of [3 H]-25(OH)D3. Previously, we showed that 24R,25(OH)2D3 blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH)2D3 in maintaining cartilage health. We examined the ability of 24R,25(OH)2D3 to prevent degenerative changes in articular cartilage in an OAlike environment and the potential mechanisms involved. In vitro, rat articular chondrocytes were treated with IL-1β with and without 24R,25(OH)2D3 or 1α,25(OH)2D3. 24R,25(OH)2D3 but not 1α,25(OH)2D3 blocked the effects of IL-1β in a dose-dependent manner, and its effect was partially mediated through the TGF-β1 signaling pathway. In vivo, unilateral anterior cruciate ligament transections were performed in immunocompetent rats followed by intra-articular injections of 24R,25(OH)2D3 or vehicle (t = 0, 7, 14, 21 days). Tissues were harvested on day 28. Joints treated with vehicle had changes typical of OA whereas joints treated with 24R,25(OH)2D3 had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH)2D3 protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis

Amelogenin Peptide Extract Increases Differentiation and Angiogenic and Local Factor Production and Inhibits Apoptosis in Human Osteoblasts

Enamel matrix derivative (EMD), a decellularized porcine extracellular matrix (ECM), is used clinically in periodontal tissue regeneration. Amelogenin, EMD’s principal component, spontaneously assembles into nanospheres in vivo, forming an ECM complex that releases proteolytically cleaved peptides. However, the role of amelogenin or amelogenin peptides in mediating osteoblast response to EMD is not clear. Human MG63 osteoblast-like cells or normal human osteoblasts were treated with recombinant human amelogenin or a 5 kDa tyrosine-rich amelogenin peptide (TRAP) isolated from EMD and the effect on osteogenesis, local factor production, and apoptosis assessed. Treated MG63 cells increased alkaline phosphatase specific activity and levels of osteocalcin, osteoprotegerin, prostaglandin E2, and active/latent TGF-β1, an effect sensitive to the effector and concentration. Primary osteoblasts exhibited similar, but less robust, effects. TRAP-rich 5 kDa peptides yielded more mineralization than rhAmelogenin in osteoblasts in vitro. Both amelogenin and 5 kDa peptides protected MG63s from chelerythrine-induced apoptosis. The data suggest that the 5 kDa TRAP-rich sequence is an active amelogenin peptide that regulates osteoblast differentiation and local factor production and prevents osteoblast apoptosis

Implant osseointegration and the role of microroughness and nanostructures: Lessons for spine implants

The use of spinal implants for spine fusion has been steadily increasing to avoid the risks of complications and donor site morbidity involved when using autologous bone. A variety of fusion cages are clinically available, with different shapes and chemical compositions. However, detailed information about their surface properties and the effects of such properties on osteogenesis is lacking in the literature. Here we evaluate the role of surface properties for spinal implant applications, covering some of the key biological processes that occur around an implant and focusing on the role of surface properties, specifically the surface structure, on osseointegration, drawing examples from other implantology fields when required. Our findings revealed that surface properties such as microroughness and nanostructures can directly affect early cell behavior and long-term osseointegration. Microroughness has been well established in the literature to have a beneficial effect on osseointegration of implants. In the case of the role of nanostructures, the number of reports is increasing and most studies reveal a positive effect from the nanostructures alone and a synergistic effect when combined with microrough surfaces. Long-term clinical results are nevertheless necessary to establish the full implications of surface nanomodificationsThe use of spinal implants for spine fusion has been steadily increasing to avoid the risks of complications and donor site morbidity involved when using autologous bone. A variety of fusion cages are clinically available, with different shapes and chemical compositions. However, detailed information about their surface properties and the effects of such properties on osteogenesis is lacking in the literature. Here we evaluate the role of surface properties for spinal implant applications, covering some of the key biological processes that occur around an implant and focusing on the role of surface properties, specifically the surface structure, on osseointegration, drawing examples from other implantology fields when required. Our findings revealed that surface properties such as microroughness and nanostructures can directly affect early cell behavior and long-term osseointegration. Microroughness has been well established in the literature to have a beneficial effect on osseointegration of implants. In the case of the role of nanostructures, the number of reports is increasing and most studies reveal a positive effect from the nanostructures alone and a synergistic effect when combined with microrough surfaces. Long-term clinical results are nevertheless necessary to establish the full implications of surface nanomodification

Substrate Stiffness Controls Osteoblastic and Chondrocytic Differentiation of Mesenchymal Stem Cells without Exogenous Stimuli

Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs) could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA) polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffnesschondrocytes, MSCs on lower stiffness substrates showed elevated expression of ACAN, SOX9, and COL2 and proteoglycan content; COMP was elevated in MSCs but reduced in chondrocytes. Substrate stiffness altered levels of RUNX2 mRNA, alkaline phosphatase specific activity, osteocalcin, and osteoprotegerin in osteoblasts, decreasing levels on the least stiff substrate. Expression of integrin subunits α1, α2, α5, αv, β1, and β3 changed in a stiffness- and cell type-dependent manner. Silencing of integrin subunit beta 1 (ITGB1) in MSCs abolished both osteoblastic and chondrogenic differentiation in response to substrate stiffness. Our results suggest that substrate stiffness is an important mediator of osteoblastic and chondrogenic differentiation, and integrin β1 plays a pivotal role in this process

Impaired Bone Formation in Pdia3 Deficient Mice

1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)2D3initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3+/− heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality.Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3+/− mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3+/− mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3+/− mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3+/− heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)2D3’s actions in regulating skeletal development

A review on the wettability of dental implant surfaces I: Theoretical and experimental aspects

The surface wettability of biomaterials determines the biological cascade of events at the biomaterial/ host interface. Wettability is modulated by surface characteristics, such as surface chemistry and surface topography. However, the design of current implant surfaces focuses mainly on specific micro- and nanotopographical features, and is still far from predicting the concomitant wetting behavior. There is an increasing interest in understanding the wetting mechanisms of implant surfaces and the role of wettability in the biological response at the implant/bone or implant/soft tissue interface. Fundamental knowledge related to the influence of surface roughness (i.e. a quantification of surface topography) on titanium and titanium alloy surface wettability, and the different associated wetting regimes, can improve our understanding of the role of wettability of rough implant surfaces on the biological outcome. Such an approach has been applied to biomaterial surfaces only in a limited way. Focusing on titanium dental and orthopaedic implants, the present study reviews the current knowledge on the wettability of biomaterial surfaces, encompassing basic and applied aspects that include measurement techniques, thermodynamic aspects of wetting and models predicting topographical and roughness effects on the wetting behavior.The surface wettability of biomaterials determines the biological cascade of events at the biomaterial/ host interface. Wettability is modulated by surface characteristics, such as surface chemistry and surface topography. However, the design of current implant surfaces focuses mainly on specific micro- and nanotopographical features, and is still far from predicting the concomitant wetting behavior. There is an increasing interest in understanding the wetting mechanisms of implant surfaces and the role of wettability in the biological response at the implant/bone or implant/soft tissue interface. Fundamental knowledge related to the influence of surface roughness (i.e. a quantification of surface topography) on titanium and titanium alloy surface wettability, and the different associated wetting regimes, can improve our understanding of the role of wettability of rough implant surfaces on the biological outcome. Such an approach has been applied to biomaterial surfaces only in a limited way. Focusing on titanium dental and orthopaedic implants, the present study reviews the current knowledge on the wettability of biomaterial surfaces, encompassing basic and applied aspects that include measurement techniques, thermodynamic aspects of wetting and models predicting topographical and roughness effects on the wetting behavior

Developing a Model of Aged Decellularized Muscle Matrix with Advanced Glycation Cross-linking

Volumetric muscle loss (VML) has been found to overwhelm muscle regeneration, resulting in loss of long-term muscle functionality. Decellularized muscle matrices (DMMs) provide an effective environment for muscle regeneration; however, the age of their source has not been adequately explored for clinical translation. Advanced glycation end-products (AGEs) are chemical cross-links that contribute to the aging process by accumulating on collagen fibers, resulting in a stiffening of the collagenous matrix and an increase in inflammation via the receptor for advanced glycation end-products (RAGE). In previous experiments, we found increased levels of AGE-specific cross-links within DMMs in old mice compared to young as proven by ALT-711 treatment. In this study, we developed a model of aged rat DMMs using AGE cross-links and hypothesized that our AGE-DMM model will contain a higher number of collagen cross-links compared to the control. This AGE-DMM model aims to elucidate the effect of AGEs on muscle regeneration when used in vitro or implanted in a volumetric muscle loss model.https://scholarscompass.vcu.edu/uresposters/1424/thumbnail.jp

Electrical Polarization of Titanium Surfacesfor the Enhancement of Osteoblast Differentiation

Electrical stimulation has been used clinically to promote bone regeneration in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports suggesting its beneficial effects on bone formation. However, the use of electrical stimulation of titanium (Ti) implants to enhance osseointegration is less understood, in part because of the few in vitro models that attempt to represent the in vivo environment. In this article, the design of a new in vitro system that allows direct electrical stimulation of osteoblasts through their Ti substrates without the flow of exogenous currents through the media is presented, and the effect of applied electrical polarization on osteoblast differentiation and local factor production was evaluated. A custom-made polycarbonate tissue culture plate was designed to allow electrical connections directly underneath Ti disks placed inside the wells, which were supplied with electrical polarization ranging from 100 to 500 mV to stimulate MG63 osteoblasts. Our results show that electrical polarization applied directly through Ti substrates on which the cells are growing in the absence of applied electrical currents may increase osteoblast differentiation and local factor production in a voltage-dependent manner. Bioelectromagnetics © 2013 Wiley Periodicals, IncElectrical stimulation has been used clinically to promote bone regeneration in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports suggesting its beneficial effects on bone formation. However, the use of electrical stimulation of titanium (Ti) implants to enhance osseointegration is less understood, in part because of the few in vitro models that attempt to represent the in vivo environment. In this article, the design of a new in vitro system that allows direct electrical stimulation of osteoblasts through their Ti substrates without the flow of exogenous currents through the media is presented, and the effect of applied electrical polarization on osteoblast differentiation and local factor production was evaluated. A custom-made polycarbonate tissue culture plate was designed to allow electrical connections directly underneath Ti disks placed inside the wells, which were supplied with electrical polarization ranging from 100 to 500 mV to stimulate MG63 osteoblasts. Our results show that electrical polarization applied directly through Ti substrates on which the cells are growing in the absence of applied electrical currents may increase osteoblast differentiation and local factor production in a voltage-dependent manner. Bioelectromagnetics © 2013 Wiley Periodicals, In

Effects of structural properties of electrospun TiO2 nanofiber meshes on their osteogenic potential

Ideal outcomes in the field of tissue engineering and regenerative medicine involve biomaterials that can enhance cell differentiation and production of local factors for natural tissue regeneration without the use of systemic drugs. Biomaterials typically used in tissue engineering applications include polymeric scaffolds that mimic the three-dimensional structural environment of the native tissue, but these are often functionalized with proteins or small peptides to improve their biological performance. For bone applications, titanium implants, or more appropriately the TiO2 passive oxide layer formed on their surface, have been shown to enhance osteoblast differentiation in vitro and to promote osseointegration in vivo. In this study we evaluated the effect on osteoblast differentiation of pure TiO2 nanofiber meshes with different surface microroughness and nanofiber diameters, prepared by the electrospinning method. MG63 cells were seeded on TiO2 meshes, and cell number, differentiation markers and local factor production were analyzed. The results showed that cells grew throughout the entire surfaces and with similar morphology in all groups. Cell number was sensitive to surface microroughness, whereas cell differentiation and local factor production was regulated by both surface roughness and nanofiber diameter. These results indicate that scaffold structural cues alone can be used to drive cell differentiation and create an osteogenic environment without the use of exogenous factorsIdeal outcomes in the field of tissue engineering and regenerative medicine involve biomaterials that can enhance cell differentiation and production of local factors for natural tissue regeneration without the use of systemic drugs. Biomaterials typically used in tissue engineering applications include polymeric scaffolds that mimic the three-dimensional structural environment of the native tissue, but these are often functionalized with proteins or small peptides to improve their biological performance. For bone applications, titanium implants, or more appropriately the TiO2 passive oxide layer formed on their surface, have been shown to enhance osteoblast differentiation in vitro and to promote osseointegration in vivo. In this study we evaluated the effect on osteoblast differentiation of pure TiO2 nanofiber meshes with different surface microroughness and nanofiber diameters, prepared by the electrospinning method. MG63 cells were seeded on TiO2 meshes, and cell number, differentiation markers and local factor production were analyzed. The results showed that cells grew throughout the entire surfaces and with similar morphology in all groups. Cell number was sensitive to surface microroughness, whereas cell differentiation and local factor production was regulated by both surface roughness and nanofiber diameter. These results indicate that scaffold structural cues alone can be used to drive cell differentiation and create an osteogenic environment without the use of exogenous factor