Hyaluronic acid and chondrogenesis of murine bone marrow mesenchymal stem cells in chitosan sponges.

Objective: To establish the dose-dependent effects of high-molecular-weight hyaluronic acid (HA) supplementation on chondrogenesis by mesenchymal stem cells (MSCs) cultured on chitosan sponges and to determine the extent to which MSC matrix production (chondrogenesis) can be influenced by incorporation of high-molecular-weight HA into chitosan scaffolds.

Sample Population: Murine MSCs derived from a multipotent bone marrow stromal precursor.

Procedures: MSCs were seeded on chitosan and chitosan-HA scaffolds in chondrogenic medium with various HA concentrations.

Inorganic phosphate induces mammalian growth plate chondrocyte apoptosis in a mitochondrial pathway involving nitric oxide and JNK MAP kinase.

Chondrocytes in the hypertrophic zone of the growth plate undergo apoptosis during endochondral bone development via mechanisms that involve inorganic phosphate (Pi) and nitric oxide (NO). Recent evidence suggests that Pi-dependent NO production plays a role in apoptosis of cells in the resting zone as well. This study examined the mechanism by which Pi induces NO production and the signaling pathways by which NO mediates its effects on apoptosis in these cells.

17β-Estradiol regulates rat growth plate chondrocyte apoptosis through a mitochondrial pathway not involving nitric oxide or MAPKs.

Estrogens cause growth plate closure in both males and females, by decreasing proliferation and inducing apoptosis of postproliferative growth plate chondrocytes. In vitro studies using 17β-estradiol (E(2)) conjugated to bovine serum albumin (E(2)-BSA) show that rat costochondral growth plate resting zone chondrocytes also respond to E(2). Moreover, they are regulated by E(2)-BSA via a protein kinase C and ERK MAPK signaling pathway that is functional only in female cells.

Are bacterial load and synovitis related in dogs with inflammatory stifle arthritis?

It has been proposed that small quantities of microbial material within synovial joints may act as a trigger for development of synovitis. We have previously identified an association between intra-articular bacteria and development of inflammatory stifle arthritis and cranial cruciate ligament rupture (CCLR) in dogs, and now wished to quantify bacterial load and markers of synovitis in dogs with and without stifle arthritis and CCLR. Joint tissues were collected from dogs with CCLR (n=51) and healthy dogs with normal stifles (n=9).

Dendritic cell responses to surface properties of clinical titanium surfaces.

Dendritic cells (DCs) play pivotal roles in responding to foreign entities during the innate immune response and in initiating effective adaptive immunity as well as maintaining immune tolerance. The sensitivity of DCs to foreign stimuli also makes them useful cells to assess the inflammatory response to biomaterials. Elucidating material property-DC phenotype relationships using a well-defined biomaterial system is expected to provide criteria for immunomodulatory biomaterial design.

Protein-disulfide isomerase-associated 3 (Pdia3) mediates the membrane response to 1,25-dihydroxyvitamin D3 in osteoblasts.

Protein-disulfide isomerase-associated 3 (Pdia3) is a multifunctional protein hypothesized to be a membrane receptor for 1,25(OH)(2)D(3). In intestinal epithelium and chondrocytes, 1,25(OH)(2)D(3) stimulates rapid membrane responses that are different from genomic effects via the vitamin D receptor (VDR). In this study, we show that 1,25(OH)(2)D(3) stimulates phospholipase A(2) (PLA(2))-dependent rapid release of prostaglandin E(2) (PGE(2)), activation of protein kinase C (PKC), and regulation of bone-related gene transcription and mineralization in osteoblast-like MC3T3-E1 cells (WT) via a mechanism involving Pdia3.

Alginate microencapsulation technology for the percutaneous delivery of adipose-derived stem cells.

Background: Autologous fat is the ideal soft-tissue filler; however, its widespread application is limited because of variable clinical results and poor survival. Engineered fillers have the potential to maximize survival. Alginate is a hydrogel copolymer that can be engineered into spheres of <200 μm, thus facilitating mass transfer, allowing for subcutaneous injection, and protecting cells from shearing forces.

Use of molecular beacons to image effects of titanium surface microstructure on beta1 integrin expression in live osteoblast-like cells.

This study used molecular beacon technology to examine substrate-dependent changes in integrin subunit expression in living cells. Molecular beacons are oligonucleotide probes that can be delivered into live cells to allow for real-time imaging of mRNA. They have a stem-loop hairpin structure with a fluorophore-quencher pair, which opens when bound to the target mRNA sequence, resulting in a fluorescent signal upon excitation.

24R,25-Dihydroxyvitamin D3, lysophosphatidic acid, and p53: a signaling axis in the inhibition of phosphate-induced chondrocyte apoptosis.

Maintenance of the pool of chondrocytes in the resting zone of the growth plate in the presence of the physiological apoptogen inorganic phosphate (Pi) is crucial for skeletal development. Costochondral resting zone chondrocytes are regulated by the vitamin D metabolite 24R,25-dihydroxyvitamin D3 [24R,25(OH)(2)D(3)], with increased production of sulfated glycosaminoglycan-rich extracellular matrix, and reduced matrix metalloproteinase activity. The effects of 24R,25(OH)(2)D(3) are mediated by activation of phospholipase D (PLD), resulting in increased production of lysophosphatidic acid (LPA) and LPA-mediated proliferation, maturation, inhibition of Pi-induced apoptosis, and reduction of p53.

Disruption of Pdia3 gene results in bone abnormality and affects 1alpha,25-dihydroxy-vitamin D3-induced rapid activation of PKC.

1,25-dihydroxy-vitamin D3 [1alpha,25(OH)2D3] is a critical regulator of bone development. Protein disulfide isomerase A3 (Pdia3) is a multifunctional protein that has been associated with rapid membrane-initiated signalling by 1alpha,25(OH)2D3 in several cell types. To identify the physiological roles of Pdia3 in skeletal development, we generated Pdia3-deficient mice.

The dependence of MG63 osteoblast responses to (meth)acrylate-based networks on chemical structure and stiffness.

The cell response to an implant is regulated by the implant's surface properties including topography and chemistry, but less is known about how the mechanical properties affect cell behavior. The objective of this study was to evaluate how the surface stiffness and chemistry of acrylate-based copolymer networks affect the in vitro response of human MG63 pre-osteoblast cells. Networks comprised of poly(ethylene glycol) dimethacrylate (PEGDMA; Mn approximately 750) and diethylene glycol dimethacrylate (DEGDMA) were photopolymerized at different concentrations to produce three compositions with moduli ranging from 850 to 60 MPa.

Regulating in vivo calcification of alginate microbeads.

Alginate calcification has been previously reported clinically and during animal implantation; however no study has investigated the mechanism, extensively characterized the mineral, or evaluated multiple methods to regulate or eliminate mineralization. In the present study, alginate calcification was first studied in vitro: calcium-crosslinked alginate beads sequestered surrounding phosphate while forming traces of hydroxyapatite. Calcification in vivo was then examined in nude mice using alginate microbeads with and without adipose stem cells (ASCs).

Regulation of angiogenesis during osseointegration by titanium surface microstructure and energy.

Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the alpha(2)beta(1) integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via alpha(2)beta(1) signaling to regulate the expression of angiogenic growth factors.

24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] controls growth plate development by inhibiting apoptosis in the reserve zone and stimulating response to 1alpha,25(OH)2D3 in hypertrophic cells.

Previously we showed that costochondral growth plate resting zone (RC) chondrocytes response primarily to 24R,25(OH)2D3 whereas prehypertrophic and hypertrophic (GC) cells respond to 1alpha,25(OH)2D3. 24R,25(OH)2D3 increases RC cell proliferation and inhibits activity of matrix processing enzymes, suggesting it stabilizes cells in the reserve zone, possibly by inhibiting the matrix degradation characteristic of apoptotic hypertrophic GC cells. To test this, apoptosis was induced in rat RC cells by treatment with exogenous inorganic phosphate (Pi).

The synergistic effects of 3-D porous silk fibroin matrix scaffold properties and hydrodynamic environment in cartilage tissue regeneration.

Autologous cell-based tissue engineering using three-dimensional porous scaffolds has provided a good option for the repair of cartilage defects. Silk fibroin-based scaffolds are naturally degradable materials with excellent biocompatibility and robust mechanical properties, indicating potential applications in cartilage tissue engineering. In this study, silk fibroin scaffolds prepared by freeze-drying (FD) and salt-leaching (SL300 and SL500) were fully characterized and used to study the effects of silk fibroin scaffold properties on chondrocyte attachment, proliferation and differentiation.

A new animal model for assessing cartilage repair and regeneration at a nonarticular site.

The aim of this study was to establish a critical-sized nonjoint chondral defect animal model and to evaluate its feasibility for testing cartilage regeneration strategies. Dermal biopsy punches 1-4 mm in diameter were used to create cylindrical full-thickness defects in the center of athymic rat xiphoids. The 3 and 4 mm defects remained unhealed 35 days postsurgery, with a large area in the center that had low proteoglycan content based on contrast-enhanced microCT (EPIC-microCT), radiographic, and histological analyses.

Characterization of a small animal growth plate injury model using microcomputed tomography.

Injuries to the growth plate remain a significant clinical challenge. The need to better understand mechanisms of growth disruption following transphyseal injuries and evaluate new therapeutic approaches to growth restoration motivates development of a well characterized model of growth plate injury. The goals of this study were to develop a growth plate defect model in the rat and to use microcomputed tomography (micro-CT) imaging to detect and quantify associated changes in growth plate morphology and mineralization over time following injury and in response to treatment.

Successful revision of a femoral head ostectomy (complicated by postoperative sciatic neurapraxia) to a total hip replacement in a cat.

A three-year-old neutered male Domestic Medium Hair cat was referred for evaluation of a right hindlimb monoparesis of one month duration following a femoral head ostectomy (FHO) performed elsewhere. Examination of the limb revealed muscle atrophy and decreased sensory perception over the sciatic nerve distribution with conscious proprioception and postural reaction deficits. The tentative diagnosis was sciatic neurapraxia.

Direct and indirect effects of microstructured titanium substrates on the induction of mesenchymal stem cell differentiation towards the osteoblast lineage.

Microstructured and high surface energy titanium substrates increase osseointegration in vivo. In vitro, osteoblast differentiation is increased, but effects of the surface directly on multipotent mesenchymal stem cells (MSCs) and consequences for MSCs in the peri-implant environment are not known. We evaluated responses of human MSCs to substrate surface properties and examined the underlying mechanisms involved.

The roles of Wnt signaling modulators Dickkopf-1 (Dkk1) and Dickkopf-2 (Dkk2) and cell maturation state in osteogenesis on microstructured titanium surfaces.

Osteoblast differentiation on tissue culture polystyrene (TCPS) requires Wnt/beta-catenin signaling, regulating modulators of the Wnt pathway like Dickkopf-1 (Dkk1) and Dkk2. Osteoblast differentiation is increased on microstructured titanium (Ti) surfaces compared to TCPS; therefore, we hypothesized that surface topography and hydrophilicity affect Dkk1 and Dkk2 expression and that their roles in osteoblast differentiation on Ti differs depending on cell maturation state. Human osteoblast-like MG63 cells, normal human osteoblasts (HOBs), and human mesenchymal stem cells (MSCs), as well as MG63 cells stably silenced for Dkk1 or Dkk2 were grown for 6 days on TCPS and Ti surfaces (PT [Ra<0.

Regeneration of bone marrow after tibial ablation in immunocompromised rats is age dependent.

Injuries to the marrow cavity result in rapid endosteal bone formation followed by remodeling and regeneration of the marrow. It is not known whether this process is affected by age, although marrow quality is markedly different in young and old animals. To test if marrow regeneration differs with age, we used a bone marrow ablation model that has been used to examine calcification, osteointegration of metal implants, and remodeling of bone graft substitutes.

The role of phospholipase D in osteoblast response to titanium surface microstructure.

Biomaterial surface properties such as microtopography and energy can change cellular responses at the cell-implant interface. Phospholipase D (PLD) is required for the differentiation of osteoblast-like MG63 cells on machined and grit-blasted titanium surfaces. Here, we determined if PLD is also required on microstructured/high-energy substrates and the mechanism involved.

Formation of tethers linking the epiphysis and metaphysis is regulated by vitamin d receptor-mediated signaling.

Rat tibial growth plates have X-ray opaque tethers that link the epiphysis and metaphysis and increase with age as the growth plate (GP) becomes thinner. To determine if tether formation is a regulated process of GP maturation, we tested the hypotheses that tether properties and distribution can be quantified by micro-computed tomography (microCT), that rachitic GPs typical of vitamin D receptor knockout (VDR(-/-)) mice have fewer tethers and altered tether distribution, and that tether formation is regulated by signaling via the VDR. Distal femoral GPs from VDR(+/+) and VDR(-/-) 8-week-old mice were analyzed with microCT and then processed for decalcified and undecalcified histomorphometry.

Mechanisms regulating increased production of osteoprotegerin by osteoblasts cultured on microstructured titanium surfaces.

Osteoblasts grown on microstructured Ti surfaces enhance osteointegration by producing local factors that regulate bone formation as well as bone remodeling, including the RANK ligand decoy receptor osteoprotegerin (OPG). The objective of this study was to explore the mechanism by which surface microstructure and surface energy mediate their stimulatory effects on OPG expression. Titanium disks were manufactured to present different surface morphologies: a smooth pretreatment surface (PT, Ra<0.

Inorganic phosphate modulates responsiveness to 24,25(OH)2D3 in chondrogenic ATDC5 cells.

Chondrogenic ATDC5 cells were used as a model of in vitro endochondral maturation to study the role of inorganic phosphate (Pi) in the regulation of growth plate chondrocytes by vitamin D3 metabolites. ATDC5 cells that were cultured for 10 days post-confluence in differentiation media and then treated for 24 h with Pi produced a type II collagen matrix based on immunohistochemistry and expressed mRNAs for several chondrocytic markers, including aggrecan, collagen types II and X, cartilage oligomeric matrix protein, and SOX9. Pi also caused a decrease in [(35)S]-sulfate incorporation and stimulated apoptosis, as evidenced by increased DNA fragmentation and caspase-3 activity.