Biomedical subjects

D D Dean

Publications and source records attributed to D D Dean.

At least 109 records · Page 6Linked to original sources

PubMed1997

Interleukin-1alpha and beta in growth plate cartilage are regulated by vitamin D metabolites in vivo.

Matrix remodeling plays a prominent role in growth plate calcification. Since interleukin-1 (IL-1) has been implicated in stimulating proteinase production and inhibiting matrix synthesis in articular cartilage, we examined whether IL-1 was present in growth plate and whether the vitamin D metabolites, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 1,25) and 24,25(OH)2D3 (24,25), regulate the level of IL-1 found in this tissue. Sprague-Dawley rats were placed on normal (Normal rats) or rachitogenic diet (-VDP rats). The -VDP rats were either left untreated, injected 24 h prior to euthanasia with 24,25 (-VDP+24,25 rats) or 1,25 (-VDP+1,25 rats), or were given ergocalciferol (Ergo rats) orally, 48 h prior to euthanasia. Growth plates were harvested and extracted in buffer containing 1 M guanidine. IL-1 activity was measured by adding authentic cytokine or growth plate extracts to cultures of lapine articular cartilage and assaying release of glycosaminoglycans (GAGs) and changes in collagenase and neutral metalloproteinase activity. Neutralization of activity in the extracts was performed using polyclonal antisera to IL-1alpha or IL-1beta. An ELISA was used to determine levels of IL-1alpha and beta in the extracts. All extracts contained IL-1alpha and beta, as determined by ELISA. Levels of IL-1beta, but not IL-1alpha, were affected by the vitamin D status of the animal. Extracts from -VDP+24,25 animals contained significantly more IL-1beta than any of the other treatment groups, with the level found in these animals being 3-fold higher than normal and 2-fold higher than -VDP. Extracts were also tested in the bioassay to determine the level of active cytokine present. All growth plate extracts contained activity which altered GAG and proteinase release by lapine articular cartilage. Extracts from -VDP-, -VDP+1,25-, and -VDP+Ergo-treated rats stimulated a 40% increase in glycosaminoglycan release compared with extracts from normal rats. In contrast, extracts from -VDP+24,25-treated rats stimulated a 300% increase in glycosaminoglycan release. Both collagenase and neutral metalloproteinase activity of lapine cartilage were increased after incubation with the growth plate extracts. Collagenase activity was significantly increased 8- to 13-fold by the addition of extracts from -VDP-, -VDP+24,25-, or -VDP+1,25-treated animals. Neutral metalloproteinase activity was similarly increased by 4- to 10-fold. To characterize this activity further, growth plate extracts were incubated with neutralizing antibody to IL-1alpha or beta prior to addition to the lapine articular cartilage cultures. When antibodies were used separately, only partial inhibition was observed; incubation with both antibodies blocked 25% of the glycosaminoglycan release observed without antibody and greater than 80% of the enzyme activity released by the articular cartilage cultures. The results of this study show that growth plate cartilage contains both IL-1alpha and beta and indicate that vitamin D regulates the level of IL-1 in this tissue.

24,25-Dihydroxyvitamin D 3↗

PubMed1996

A mechanism of adaptation to hypergravity in the statocyst of Aplysia californica.

The gravity-sensing organ of Aplysia californica consists of bilaterally paired statocysts containing statoconia, which are granules composed of calcium carbonate crystals in an organic matrix. In early embryonic development, Aplysia contain a single granule called a statolith, and as the animal matures, statoconia production takes place. The objective of this study was to determine the effect of hypergravity on statoconia production and homeostasis and explore a possible physiologic mechanism for regulating this process. Embryonic Aplysia were exposed to normogravity or 3 x g or 5.7 x g and each day samples were analyzed for changes in statocyst, statolith, and body dimensions until they hatched. In addition, early metamorphosed Aplysia (developmental stages 7-10) were exposed to hypergravity (2 x g) for 3 weeks, and statoconia number and statocyst and statoconia volumes were determined. We also determined the effects of hypergravity on statoconia production and homeostasis in statocysts isolated from developmental stage 10 Aplysia. Since prior studies demonstrated that urease was important in the regulation of statocyst pH and statoconia formation, we also evaluated the effect of hypergravity on urease activity. The results show that hypergravity decreased statolith and body diameter in embryonic Aplysia in a magnitude-dependent fashion. In early metamorphosed Aplysia, hypergravity decreased statoconia number and volume. Similarly, there was an inhibition of statoconia production and a decrease in statoconia volume in isolated statocysts exposed to hypergravity in culture. Urease activity in statocysts decreased after exposure to hypergravity and was correlated with the decrease in statoconia production observed. In short, there was a decrease in statoconia production with exposure to hypergravity both in vivo and in vitro and a decrease in urease activity. It is concluded that exposure to hypergravity downregulates urease activity, resulting in a significant decrease in the formation of statoconia.

Analysis of Variance↗

PubMed1996

Effect of 17 beta-estradiol on chondrocyte membrane fluidity and phospholipid metabolism is membrane-specific, sex-specific, and cell maturation-dependent.

In this study we examined the hypothesis that 17 beta-estradiol exerts both rapid and direct, nongenomic effects of cells in the endochondral pathway. To do this, we used a cell culture model in which chondrocytes at two distinct stages of cell maturation are isolated from the costochondral cartilage of male and female rats, and examined the short-term effect of 17 alpha- and 17 beta-estradiol on [14C]arachidonic acid turnover in the cell layer and phospholipase A2 specific activity in plasma membranes and extracellular matrix vesicles isolated from similarly prepared cultures. In addition, the effect of 17 alpha- and 17 beta-estradiol on plasma membrane and matrix vesicle membrane fluidity was assessed. The effect of hormone on arachidonic acid turnover was rapid, time- and concentration-dependent, stereo-specific, and cell maturation-specific. Only resting zone cells from female rats were affected, and only 17 beta-estradiol elicited a response. Similarly, only female rat resting zone chondrocytes exhibited a change in phospholipase A2 activity after a 24 h exposure to hormone, causing an increase in enzyme activity in the matrix vesicles, but not plasma membranes. When isolated membranes were incubated directly with hormone, membrane fluidity was decreased in both plasma membranes and matrix vesicles isolated from female rat resting zone chondrocyte cultures. This nongenomic effect was dose-dependent and stereo-specific and differentially expressed in the two membrane fractions with respect to time course and magnitude of response. These results support the hypothesis that 17 beta-estradiol has a rapid action on chondrocyte membrane lipid metabolism and suggest that specific membrane components, characteristic of a particular sex and state of cell maturation, are involved in the nongenomic effects of this sex hormone on isolated matrix vesicles and plasma membranes.

Animals↗

PubMed1996

Chondrocyte cultures express matrix metalloproteinase mRNA and immunoreactive protein; stromelysin-1 and 72 kDa gelatinase are localized in extracellular matrix vesicles.

Previous studies have shown that costochondral cartilage cell cultures produce extracellular matrix vesicles which contain metalloproteinase activity. In the present study, we examined whether two matrix metalloproteinases (MMPs) known to be present in cartilage, stromelysin-1 and 72 kDa gelatinase, are expressed by fourth passage resting zone and growth zone costochondral chondrocytes and whether they are specifically incorporated into matrix vesicles produced by the cells. We also examined whether the cells synthesize tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1 and TIMP-2). Oligonucleotide primers for stromelysin-1, 72 kDa gelatinase, tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2), and GAPDH were synthesized and optimized for use in the reverse transcription-polymerase chain reaction (RT-PCR). It was found that both resting zone and growth zone chondrocytes produced mRNA for both MMPs and the two TIMPs. Further, immunostaining of cell layers with antibodies to 72 kDa gelatinase and stromelysin-1 showed that both cell types produced these MMPs in culture. Substrate gel electrophoresis and Western analysis were used to characterize MMP activity in matrix vesicles, media vesicles, or plasma membranes as well as in conditioned media produced by the chondrocyte cultures. It was found that matrix vesicles but not plasma membranes or media vesicles were selectively enriched in stromelysin-1. Also, 72 kDa gelatinase was found in matrix vesicles, but to a lesser extent than seen in media vesicles. The relative activity of each enzyme detected was cell maturation-dependent. No MMP activity was detected in conditioned media produced by either cell type. The results of this study show that MMPs are expressed by resting zone and growth zone chondrocytes in culture and differentially distributed among three different membrane compartments. This suggests that, in addition to the well-known activators and inhibitors of MMP activity in the matrix, differential membrane distribution may enable more precise control over the site, rate, and extent of matrix degradation by the cell.

Animals↗

PubMed1996

Effect of titanium surface roughness on chondrocyte proliferation, matrix production, and differentiation depends on the state of cell maturation.

Although it is well accepted that implant success is dependent on various surface properties, little is known about the effect of surface roughness on cell metabolism or differentiation, or whether the effects vary with the maturational state of the cells interacting with the implant. In the current study, we examined the effect of titanium (Ti) surface roughness on chondrocyte proliferation, differentiation, and matrix synthesis using cells derived from known stages of endochondral development. Chondrocytes derived from the resting zone (RCs) and growth zone (GCs) of rat costochondral cartilage were cultured on Ti disks that were prepared as follows: HF-HNO3-treated and washed (PT); PT-treated and electropolished (EP); fine sand-blasted, HCl-H2SO4-etched, and washed (FA); coarse sand-blasted, HCl-H2SO4-etched, and washed (CA); or Ti plasma-sprayed (TPS). Based on surface analysis, the Ti surfaces were ranked from smoothest to roughest: EP, PT, FA, CA, and TPS. Cell proliferation was assessed by cell number and [3H]-thymidine incorporation, and RNA synthesis was assessed by [3H]-uridine incorporation. Differentiation was determined by alkaline phosphatase specific activity (AL-Pase). Matrix production was measured by [3H]-proline incorporation into collagenase-digestible (CDP) and noncollagenase-digestible (NCP) protein and by [35S]-sulfate incorporation into proteoglycan. GCs required two trypsinizations for complete removal from the culture disks; the number of cells released by the first trypsinization was generally decreased with increasing surface roughness while that released by the second trypsinization was increased. In RC cultures, cell number was similarly decreased on the rougher surfaces; only minimal numbers of RCs were released by a second trypsinization. [3H]-thymidine incorporation by RCs decreased with increasing surface roughness while that by GCs was increased. [3H]-Uridine incorporation by both GCs and RCs was greater on rough surfaces. Conversely, ALPase in the cell layer and isolated cells of both cell types was significantly decreased. GC CDP and NCP production was significantly decreased on rough surfaces while CDP production by RC cells was significantly decreased on smooth surfaces. [35S]-sulfate incorporation by RCs and GCs was decreased on all surfaces compared to tissue culture plastic. The results of this study indicate that surface roughness affects chondrocyte proliferation, differentiation, and matrix synthesis, and that this regulation is cell maturation dependent.

Animals↗

PubMed1996

Surface roughness modulates the local production of growth factors and cytokines by osteoblast-like MG-63 cells.

Titanium (Ti) surface roughness affects proliferation, differentiation, and matrix production of MG-63 osteoblast-like cells. Cytokines and growth factors produced in the milieu surrounding an implant may also be influenced by its surface, thereby modulating the healing process. This study examined the effect of surface roughness on the production of two factors known to have potent effects on bone, prostaglandin E2 (PGE2) and transforming growth factor beta 1 (TGF-beta 1). MG-63 cells were cultured on Ti disks of varying roughness. The surfaces were ranked from smoothest to roughest: electropolished (EP), pretreated with hydrofluoric acid-nitric acid (PT), fine sand-blasted, etched with HCl and H2SO4, and washed (EA), coarse sand-blasted, etched with HCl and H2SO4, and washed (CA), and Ti plasma-sprayed (TPS). Cells were cultured in 24-well polystyrene (plastic) dishes as controls and to determine when confluence was achieved. Media were collected and cell number determined 24 h postconfluence. PGE2 and TGF-beta 1 levels in the conditioned media were determined using commercial radioimmunoassay and enzyme-linked immunosorbent assay kits, respectively. There was an inverse relationship between cell number and Ti surface roughness. Total PGE2 content in the media of cultures grown on the three roughest surfaces (FA, CA, and TPS) was significantly increased 1.5-4.0 times over that found in media of cultures grown on plastic or smooth surfaces. When PGE2 production was expressed per cell number, CA and TPS cultures exhibited six- to eightfold increases compared to cultures on plastic and smooth surfaces. There was a direct relationship between TGF-beta 1 production and surface roughness, both in terms of total TGF-beta 1 per culture and when normalized for cell number. TGF-beta 1 production on rough surfaces (CA and TPS) was three to five times higher than on plastic. These studies indicate that substrate surface roughness affects cytokine and growth factor production by MG-63 cells, suggesting that surface roughness may modulate the activity of cells interacting with an implant, and thereby affect tissue healing and implant success.

Biocompatible Materials↗

PubMed1996

Nongenomic regulation of protein kinase C isoforms by the vitamin D metabolites 1 alpha,25-(OH)2D3 and 24R,25-(OH)2D3.

Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell maturation-dependent. In the present study, we examined the membrane distribution of PKC and whether 1 alpha,25-(OH)2D3 and 24R,25-(OH)2D3 can directly regulate enzyme activity in isolated plasma membranes and extracellular matrix vesicles. Matrix vesicle PKC was activated by bryostatin-1 and inhibited by a PKC-specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform-specific anti-PKC antibodies suggested that PKC alpha is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKC zeta is the predominant form in matrix vesicles. This was confirmed in Western blots of immunoprecipitates as well as in studies using control peptides to block binding of the isoform specific antibody to the enzyme and using a PKC zeta-specific pseudosubstrate inhibitor peptide. The presence of PKC zeta in matrix vesicles was further verified by immunoelectron microscopy. Enzyme activity in the matrix vesicle was insensitive to exogenous lipid, whereas that in the plasma membrane required lipid for full activity. 1,25-(OH)2D3 and 24,25-(OH)2D3 inhibited matrix vesicle PKC, but stimulated plasma membrane PKC when added directly to the isolated membrane fractions. PKC activity in the matrix vesicle was calcium-independent, whereas that in the plasma membrane required calcium. Moreover, the vitamin D-sensitive PKC in matrix vesicles was not dependent on calcium, whereas the vitamin D-sensitive enzyme in plasma membranes was calcium-dependent. It is concluded that PKC isoforms are differentially distributed between matrix vesicles and plasma membranes and that enzyme activity is regulated in a membrane-specific manner. This suggests the existence of a nongenomic mechanism whereby the effects of 1,25-(OH)2D3 and 24,25-(OH)2D3 may be mediated via PKC. Further, PKC zeta may be important in nongenomic, autocrine signal transduction at sites distal from the cell.

24,25-Dihydroxyvitamin D 3↗

PubMed1996

Vitamin D3 regulation of stromelysin-1 (MMP-3) in chondrocyte cultures is mediated by protein kinase C.

Matrix metalloproteinases (MMPs) are a group of enzymes with the potential to degrade extracellular matrix proteins. One of the MMPs, stromelysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enzyme in remodeling of the extracellular matrix during endochondral development, a process which is regulated by the vitamin D metabolites, 1,25-(OH)2D3 and 24,25-(OH)2D3. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derived from growth zone (GC) and resting zone (RC) rat costochondral cartilage were treated with 1 alpha, 25-(OH)2D3 (1,25) and 24R,25-(OH)2D3 (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activity was enriched in the matrix vesicle fraction, only the plasma membrane enzyme was affected by the treatment; 1, 25 and 24,25 caused a marked decrease in plasma membrane stromelysin-1 activity in their target cells. Since plasma membrane protein kinase C (PKC) activity is stimulated by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylation. To test this, membrane fractions (containing endogenous PKC alpha and zeta as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [gamma 32 P]-ATP and anti-stromelysin-1 immunoprecipitates were analyzed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Selective inhibitors of PKC activity demonstrated that phosphorylation was inhibited by H7 and low concentrations of H8, but not by HA1004, indicating that PKC, not PKA, was responsible. Protein phosphatase 2A1 (PP2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone cell plasma membranes with 24,25 but not 1, 25, resulted in phosphorylation of stromelysin-1, demonstrating that the nongenomic effect was metabolite-specific. This suggests that this may be one mechanism by which vitamin D metabolites regulate stromelysin-1 activity and that PKC-dependent phosphorylation inhibits the metalloproteinase.

24,25-Dihydroxyvitamin D 3↗

PubMed1996

24,25-(OH)2D3 regulates protein kinase C through two distinct phospholipid-dependent mechanisms.

We have previously shown that 24,25-(OH)2D3 plays a major role in resting zone (RC) chondrocyte differentiation and that this vitamin D metabolite regulates protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 24,25-(OH)2D3 to stimulate PKC activation. Confluent, fourth passage RC cells from rat costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of RC cultures with 24,25-(OH)2D3 for 90 min produced a dose-dependent increase in diacylglycerol (DAG). Addition of R59022, a diacylglycerol kinase inhibitor, significantly increased PKC activity in cultures treated with 24,25-(OH)2D3. Addition of dioctanoylglycerol (DOG) to plasma membranes isolated from RC increased PKC activity 447-fold. Addition of pertussis toxin or cholera toxin to control cultures elevated basal PKC activity. When added together with 10(-9) M 24,25-(OH)2D3, there was an additive effect on PKC activity but in cultures treated with 10(-8) M 24,25-(OH)2D3, only the hormone-dependent stimulation of PKC was observed. The phospholipase C inhibitor, U73-122, had no effect on PKC activity, indicating that the DAG produced in response to 24,25-(OH)2D3 is not derived from phosphatidylinositol. Addition of the tyrosine kinase inhibitor, genistein, also had no effect on 24,25-(OH)2D3-stimulated PKC, further supporting the hypothesis that phospholipase C is not involved in the mechanism and that phospholipase D is responsible for the increase in DAG production. Phospholipase A2 inhibitors, quinacrine and AACOCF3, and the cyclooxygenase inhibitor indomethacin increased PKC activity in the RC cultures. Exogenous PGE2, one of the downstream products of phospholipase A2 action, inhibited PKC activity. These results suggest that 24,25-(OH)2D3 regulates PKC activity by two distinct phospholipid-dependent mechanisms: production of DAG via phospholipase D and inhibition of the production of PGE2 via inhibition of phospholipase A2 and cyclooxygenase.

24,25-Dihydroxyvitamin D 3↗

PubMed1996

Evidence that interleukin-1, but not interleukin-6, affects costochondral chondrocyte proliferation, differentiation, and matrix synthesis through an autocrine pathway.

Although the effects of interleukin-1 (IL-1) and interleukin-6 (IL-6) on articular cartilage chondrocytes have been reported, little is known concerning the effects of these cytokines on growth plate chondrocytes. In this study, we examined the effect of IL-1 alpha, IL-1 beta, and IL-6 on growth plate chondrocyte proliferation, differentiation, and matrix production as a function of cell maturation and examined the ability of these cells to produce IL-1 alpha and IL-1 beta. Confluent fourth passage cultures of rat costochondral resting zone and growth zone chondrocytes were treated with 0-100 ng/ml of IL-1 alpha, IL-1 beta, or IL-6 for 24 h and then assayed for [3H]-thymidine incorporation, alkaline phosphatase specific activity, [35S]-sulfate incorporation, and percent collagen production. Neutralizing polyclonal antibodies were used to confirm the specificity of response to each cytokine. Treatment of resting zone cells with IL-1 alpha produced a significant, dose-dependent decrease in [3H]-thymidine incorporation, while similarly treated growth zone cells were unaffected by treatment with this cytokine. IL-1 alpha also stimulated alkaline phosphatase specific activity and inhibited [35S]-sulfate incorporation by resting zone chondrocytes, but had no affect on growth zone chondrocytes. When collagen production was examined, it was observed that IL-1 alpha had a stimulatory affect on growth zone cells but no affect on resting zone cells. When the effect of IL-1 beta was examined, it was observed that this cytokine inhibited [3H]-thymidine incorporation by resting zone cells and stimulated isotope incorporation in growth zone cells. IL-1 beta also stimulated alkaline phosphatase specific activity and inhibited [35S]-sulfate incorporation by resting zone chondrocytes but had no affect on growth zone chondrocytes. In contrast to IL-1 alpha, IL-1 beta stimulated collagen production by resting zone cells but not growth zone cells. IL-6 had no affect on any of the parameters measured in either cell type. When cytokine production was measured, it was found that IL-1 alpha was produced by both cell types, while IL-1 beta was produced only by resting zone cells. Resting zone cells secreted both IL-1 alpha and IL-1 beta into the media, but 75% of the total cytokine produced by these cells was retained in the cell layer. In contrast, growth zone cells did not secrete measurable IL-1 alpha into the media. These results suggest that IL-1 alpha and IL-1 beta target resting zone cells, inducing them to differentiate and acquire a phenotype characteristic of the more mature growth zone cells. Moreover, resting zone chondrocytes produce both IL-1 alpha and IL-1 beta, suggesting the possibility of an autocrine effect of these cytokines on the cells.

Animals↗

PubMed1996

Vitamin D metabolites regulate matrix vesicle metalloproteinase content in a cell maturation-dependent manner.

Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcification in vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-beta. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1, 25(OH)2D3 and 24,25(OH)2D3. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 10(-10)-10(-7) M24, 25(OH)2D3 or growth zone chondrocytes incubated with 10(-11)-10(-8) M 1,25(OH)2D3, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)2D3 increased alkaline phosphatase by 35-60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)2D3 increased alkaline phosphatase by 35-60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)2D3 regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A2 specific activities as well as metalloproteinases which degrade proteoglycans.

24,25-Dihydroxyvitamin D 3↗

PubMed1996

Role of material surfaces in regulating bone and cartilage cell response.

Tissue engineering in vitro and in vivo involves the interaction of cells with a material surface. The nature of the surface can directly influence cellular response, ultimately affecting the rate and quality of new tissue formation. Initial events at the surface include the orientated adsorption of molecules from the surrounding fluid, creating a conditioned interface to which the cell responds. The gross morphology, as well as the microtopography and chemistry of the surface, determine which molecules can adsorb and how cells will attach and align themselves. The focal attachments made by the cells with their substrate determine cell shape which, when transduced via the cytoskeleton to the nucleus, result in expression of specific phenotypes. Osteoblasts and chondrocytes are sensitive to subtle differences in surface roughness and surface chemistry. Studies comparing chondrocyte response to TiO2 of differing crystallinities show that cells can discriminate between surfaces at this level as well. Cellular response also depends on the local environmental and state of maturation of the responding cells. Optimizing surface structure for site-specific tissue engineering is one option; modifying surfaces with biologicals is another.

Adsorption↗

PubMed1996

Proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63) cultured on previously used titanium surfaces.

This study compared osteoblasts proliferation, differentiation, and protein synthesis on new and used titanium (Ti) disks to test the hypothesis that cleaning and resterilization of previously used Ti disks does not alter cell response to a particular surface. Ti disks of varying roughness were prepared by one of five different treatment regimens. Standard tissue culture plastic was used as a control. Human osteoblast-like cells (MG63) were cultured on the Ti disks and cell proliferation, cell differentiation, RNA synthesis and matrix production (collagen and noncollagen protein; proteoglycans) measured. After their first use, the disks were cleaned, resterilized by autoclaving, and MG63 cells cultured on them as before. At confluence, the same parameters were measured and cell behavior on new and used disks compared. When confluent cultures of cells on plastic were compared to those cultured on new Ti surfaces, cell number was reduced on the roughest surfaces and equivalent to plastic on the other surfaces. Cell number was further reduced when disks with the roughest surfaces were re-used; no differences in cell number could be discerned after cleaning and resterilization. Cell proliferation was inversely related to surface roughness and was less than seen on tissue culture plastic. Re-use of the Ti disks resulted in no change in cell proliferation rate. Alkaline phosphatase specific activity in isolated cells was lowest on the rougher surfaces; no differences between new and used disks were observed. Similarly, enzyme activity in the cell layer was decreased in cultures grown on rougher surfaces, with no effect of prior disk use being noted. RNA synthesis was decreased with respect to plastic in cultures on smoother surfaces and increased on rougher surfaces; prior disk use did not alter RNA synthesis. Collagen production by the cells was decreased on smoother surfaces, but was comparable to tissue culture plastic when grown on rougher surfaces. Non-collagen protein production was unaffected by culture surface and whether or not the disk had been previously used. Proteoglycan synthesis by cells was decreased on all surfaces studied and comparable on both new and used disks. The results of this study indicate that Ti implant surfaces are unaffected by cleaning and resterilization, although rougher surfaces may require more extensive cleaning than smoother ones. This suggests the possibility that implants, in the same patient, could be safely reused. In vivo studies in animals, however, need to be performed before clinical application can be considered.

Alkaline Phosphatase↗

PubMed1996

The role of implant surface characteristics in the healing of bone.

The surface of an implant determines its ultimate ability to integrate into the surrounding tissue. The composite effect of surface energy, composition, roughness, and topography plays a major role during the initial phases of the biological response to the implant, such as protein adsorption and cellular adherence, as well as during the later and more chronic phases of the response. For bone, the successful incorporation (and hence rigid fixation) of an alloplastic material within the surrounding bony bed is called osteointegration. The exact surface characteristics necessary for optimal osteointegration, however, remain to be elucidated. This review will focus on how surface characteristics, such as composition and roughness, affect cellular response to an implant material. Data from two different culture systems suggest that these characteristics play a significant role in the recruitment and maturation of cells along relevant differentiation pathways. In the case of osteointegration, if the implant surface is inappropriate or less than optimal, cells will be unable to produce the appropriate complement of autocrine and paracrine factors required for adequate stimulation of osteogenesis at the implant site. In contrast, if the surface is appropriate, cells at the implant surface will stimulate interactions between cells at the surface and those in distal tissues. This, in turn, will initiate a timely sequence of events which include cell proliferation, differentiation, matrix synthesis, and local factor production, thereby resulting in the successful incorporation of the implant into the surrounding bony tissue.

Adsorption↗

PubMed1996

Ability of commercial demineralized freeze-dried bone allograft to induce new bone formation.

Demineralized freeze-dried bone allograft (DFDBA) has been used extensively in periodontal therapy. The rationale for use of DFDBA includes the fact that proteins capable of inducing new bone; i.e., bone morphogenetic proteins, can be isolated from bone grafts. Commercial bone banks have provided DFDBA to the dental practitioner for many years; however, these organizations have not verified the osteoinductive capacity of their DFDBA preparations. The aim of this study was to determine the ability of commercial DFDBA preparations to induce new bone formation. DFDBA with particle sizes ranging from 200 to 500 microns was received from six bone banks using various bone production methods. Different lots of DFDBA from the same tissue bank were sometimes available. A total of 14 lots were examined. The surface area of bone particles in each sample was measured morphometrically and the pH of a solution containing the particles after suspension in distilled water determined. Samples from each DFDBA lot were implanted intramuscularly (10 mg) or subcutaneously (20 mg) into three different animals and tissue biopsies harvested after 4 weeks. One sample from each tissue bank was implanted and harvested after 8 weeks. At harvest, each area where DFDBA had been implanted was excised and examined by light microscopy. The ability of DFDBA to produce new bone was evaluated and the amount of residual bone particles measured. The results show that bone particles from all tissue banks had a variety of shapes and sizes, both before implantation and after 1 or 2 months of implantation. The pH of particle suspensions also varied between batches, as well as between tissue banks. None of the DFDBA induced new bone formation when implanted subcutaneously. Intramuscular implants from three banks induced new bone formation after 1 and 2 months. DFDBA from two banks caused new bone formation only after 2 months. However, DFDBA from one bank did not induce new bone at all. Particle size before implantation correlated with particle size after implantation. However, particle size did not correlate with ability to induce bone. The results show that commercial DFDBA differs in both size and ability to induce new bone formation, but that the two are not related. The study also indicates that wide variation in commercial bone bank preparations of DFDBA exist and that ability to induce new bone formation also varies widely. Furthermore, the results suggest that methods or assays for evaluating the ability of DFDBA to induce new bone should be developed and standardized.

Adolescent↗

PubMed1996

Cell biology of calcified tissues: experimental models of differentiation and mechanisms by which local and systemic factors exert their effects.

Interpretation of the cell biology literature, as it relates to formation and mineralization of calcifying tissues, is complicated by the plethora of models available. Some culture models use heterogeneous populations of cells while others use relatively homogeneous populations. The issues are further confused by comparison of monolayer and three dimensional cultures. In addition, transformed and nontransformed cell lines are also used. As little clinical data about the age and sex of the original donor for many of these cell lines is lacking, it is impossible to know where in the cell lineage the cells were when they were isolated, yet this information can have a direct impact on the data obtained and their interpretation. Furthermore, many responses are attributed to the cell, while much of the effect observed may be targeted to the matrix. These issues are discussed and a potential mechanism explaining how cells can modulate events in the matrix nongenomically is presented.

Animals↗

PubMed1996

The synergistic effect of TGF beta and 24,25-(OH)2D3 on resting zone chondrocytes is metabolite-specific and mediated by PKC.

Transforming growth factor-beta (TGF beta) and 24,25-(OH)2D3 play a major role in chondrocyte maturation during endochondral bone formation. TGF beta and vitamin D metabolites when added separately to resting zone (RC) or growth zone (GC) chondrocyte cultures, activate protein kinase C (PKC). The present study determined whether there is an additive or synergistic effect of vitamin D3 metabolites and TGF beta on alkaline phosphatase and PKC specific activities and whether this effect is cell maturation-dependent. GC and RC chondrocytes were isolated from rat costochondral cartilage and cultured to fourth passage. The cells were incubated with vitamin D3 metabolites and TGF beta alone or in combination, and the specific activity of alkaline phosphatase, as well as the specific activity of PKC, were measured. The addition of 24,25-(OH)2D3 with TGF beta to RC cells caused a synergistic effect on alkaline phosphatase activity; this result was not found if the vitamin D3 metabolite was replaced by 1,25-(OH)2D3. The addition of 1,25-(OH)2D3 or 24,25-(OH)2D3 with TGF beta on GC cells had no synergistic or additive effect. The addition of 24,25-(OH)2D3 and TGF beta for 12 hours caused a synergistic effect on PKC activity; this effect was also observed if TGF beta was added first for 12 hours and 24,25-(OH)2D3 for the last 90 min. However, the addition of 24,25-(OH)2D3 for 90 min followed by the addition of TGF beta for an additional 10.5 hours had no synergistic effect. This study indicates that TGF beta and 24,25-(OH)2D3 have a synergistic effect on chondrocyte differentiation as well as on PKC activity, suggesting that the synergistic effect of 24,25-(OH)2D3 and TGF beta on chondrocyte differentiation may be mediated through activation of PKC. The synergistic effect of 24,25-(OH)2D3 and TGF beta was cell maturation dependent.

24,25-Dihydroxyvitamin D 3↗

PubMed1996

Regulation of statoconia mineralization in Aplysia californica in vitro.

Statoconia are calcium carbonate inclusions in the lumen of the gravity-sensing organ, the statocyst, of Aplysia californica. The aim of the present study was to examine the role of carbonic anhydrase and urease in statoconia mineralization in vitro. The experiments were performed using a previously described culture system (Pedrozo et al., J. Comp. Physiol. (A) 177:415-425). Inhibition of carbonic anhydrase by acetazolamide decreased statoconia production and volume, while inhibition of urease by acetohydroxamic acid reduced total statoconia number, but had no affect on statoconia volume. Inhibition of carbonic anhydrase initially increased and then decreased the statocyst pH, whereas inhibition of urease decreased statocyst pH at all times examined; simultaneous addition of both inhibitors also decreased pH. These effects were dose and time dependent. The results show that carbonic anhydrase and urease are required for statoconia formation and homeostasis, and for regulation of statocyst pH. This suggests that these two enzymes regulate mineralization at least partially through regulation of statocyst pH.

Animals↗