Biomedical subjects

Z Schwartz

Publications and source records attributed to Z Schwartz.

At least 91 records · Page 5Linked to original sources

PubMed2000

Primary mineralization at the surfaces of implants.

Osteogenesis around implants is affected by the physical and chemical characteristics of the biomaterials used. The osteoprogenitor cells must migrate to the implant site and synthesize and secrete a mineralizable extracellular matrix. Because this is neo-bone formation, the mechanism by which the cells calcify their matrix involves extracellular organelles called matrix vesicles in a process termed "primary mineralization". Two different methods for assessing the effects of implant materials on primary mineralization are presented in this report. In the first approach, different implant materials used in dentistry and orthopedic surgery were placed in rat tibial bones after marrow ablation. Two groups of implants were used, bone-bonding and non-bonding materials. We examined the effects of the materials on calcification morphometrically by quantitating changes in matrix vesicle morphology and distribution in endosteal tissue around implants as compared with normal endosteal bone healing. In addition, matrix vesicles were isolated from the endosteal tissue around the implant as well as from the contralateral limb and were examined biochemically. The results demonstrated that bone-bonding materials induced a greater increase in matrix vesicle enzyme activity than did non-bonding materials. However, all materials caused changes in matrix vesicles that were different from those seen in normal endosteal bone formation following injury. The effects of implant materials on biochemical markers of mineralization, including specific activities of matrix vesicle alkaline phosphatase and phospholipase A2 and phosphatidylserine content, demonstrated a high correlation with the morphometric observations with regard to enhancement and/or delay of primary mineralization. In the other approach, we used a radioisotopic method to evaluate the effects of implant materials on primary mineralization. This analysis revealed that implants alter bone healing, as shown by the differential uptake of 99mTc and 32P in different bone compartments. Decreased 32P uptake by the organic phase in the presence of bone-bonding implants suggests that cleavage of 99mTcMD32P into its technetium and methylene diphosphonate moieties was inhibited by the presence of the implants. In summary, these approaches to evaluating the effects of materials on primary mineralization demonstrate that the marrow ablation model can easily distinguish between bone-bonding and non-bonding materials. The use of this model can be valuable in the development of new materials.

Animals↗

PubMed2000

Maturation state determines the response of osteogenic cells to surface roughness and 1,25-dihydroxyvitamin D3.

In this study we assessed whether osteogenic cells respond in a differential manner to changes in surface roughness depending on their maturation state. Previous studies using MG63 osteoblast-like cells, hypothesized to be at a relatively immature maturation state, showed that proliferation was inhibited and differentiation (osteocalcin production) was stimulated by culture on titanium (Ti) surfaces of increasing roughness. This effect was further enhanced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In the present study, we examined the response of three additional cell lines at three different maturation states: fetal rat calvarial (FRC) cells (a mixture of multipotent mesenchymal cells, osteoprogenitor cells, and early committed osteoblasts), OCT-1 cells (well-differentiated secretory osteoblast-like cells isolated from calvaria), and MLO-Y4 cells (osteocyte-like cells). Both OCT-1 and MLO-Y4 cells were derived from transgenic mice transformed with the SV40 large T-antigen driven by the osteocalcin promoter. Cells were cultured on Ti disks with three different average surface roughnesses (Ra): PT, 0.5 microm; SLA, 4.1 microm; and TPS, 4.9 microm. When cultures reached confluence on plastic, vehicle or 10(-7) M or 10(-8) M 1,25(OH)2D3 was added for 24 h to all of the cultures. At harvest, cell number, alkaline phosphatase-specific activity, and production of osteocalcin, transforming growth factor beta1 (TGF-beta1) and prostaglandin E2 (PGE2) were measured. Cell behavior was sensitive to surface roughness and depended on the maturation state of the cell line. Fetal rat calvarial (FRC) cell number and alkaline phosphatase-specific activity were decreased, whereas production of osteocalcin, TGF-beta1, and PGE2 were increased with increasing surface roughness. Addition of 1,25(OH)2D3 to the cultures further augmented the effect of roughness for all parameters in a dose-dependent manner; only TGF-beta1 production on plastic and PT was unaffected by 1,25(OH)2D3. OCT-1 cell number and alkaline phosphatase (SLA > TPS) were decreased and production of PGE2, osteocalcin, and TGF-beta1 were increased on SLA and TPS. Response to 1,25(OH)2D3 varied with the parameter being measured. Addition of the hormone to the cultures had no effect on cell number or TGF-beta1 production on any surface, while alkaline phosphatase was stimulated on SLA and TPS; osteocalcin production was increased on all Ti surfaces but not on plastic; and PGE2 was decreased on plastic and PT, but unaffected on SLA and TPS. In MLO-Y4 cultures, cell number was decreased on SLA and TPS; alkaline phosphatase was unaffected by increasing surface roughness; and production of osteocalcin, TGF-beta1, and PGE2 were increased on SLA and TPS. Although 1,25(OH)2D3 had no effect on cell number, alkaline phosphatase, or production of TGF-beta1 or PGE2 on any surface, the production of osteocalcin was stimulated by 1,25(OH)2D3 on SLA and TPS. These results indicate that surface roughness promotes osteogenic differentiation of less mature cells, enhancing their responsiveness to 1,25(OH)2D3. As cells become more mature, they exhibit a reduced sensitivity to their substrate but even the terminally differentiated osteocyte is affected by changes in surface roughness.

Alkaline Phosphatase↗

PubMed2000

Parathyroid hormone and transforming growth factor-beta1 coregulate chondrocyte differentiation in vitro.

Parathyroid hormone (1-34) (PTH(1-34) and transforming growth factor-beta1 (TGF-beta1) regulate chondrocyte proliferation, differentiation, and matrix synthesis. Both proteins mediate their effects in a dose- and time-dependent manner, and the effects are cell maturation specific. Moreover, similar signaling pathways are used, suggesting that there may be cross talk leading to coregulated cell response. To test this hypothesis, confluent cultures of rat costochondral resting zone and growth zone chondrocytes were treated with 0.22, 0.44, or 0.88 ng/mL of rhTGF-beta1 for 24 h, followed by treatment with 10(-11) to 10(-8) M PTH(1-34) for 10 min or 24 h. [3H]-Thymidine incorporation, specific activity of alkaline phosphatase (AP), and [35S]-sulfate incorporation were measured. PTH(1-34) had no effect on [3H]-thymidine incorporation by growth zone cells pretreated with 0.22 or 0.44 ng/mL of TGF-beta1, but in cultures treated with 0.88 ng/mL, PTH(1-34) caused a dose-dependent decrease that was maximal at the lowest concentration tested. By contrast, PTH(1-34) stimulated [3H]-thymidine incorporation by resting zone cells, and this effect was additive with the stimulation caused by 0.22 ng/mL of TGF-beta1. PTH(1-34) caused a synergistic increase in AP in growth zone cells treated with 0.44 or 0.88 ng/mL of TGF-beta1, but not in cells treated with 0.22 ng/mL of TGF-beta1. It had no effect on AP in resting zone cells pretreated with any concentration of TGF-beta1. PTH(1-34) increased [35S]-sulfate incorporation in growth zone and resting zone cell cultures treated with 0.22 ng/mL of TGF-beta1 to levels seen in cultures treated with 0.88 ng/mL of TGF-beta1 alone. These results support the hypothesis that PTH(1-34) and TGF-beta1 coregulate growth plate chondrocytes and that the effects are cell maturation dependent.

Alkaline Phosphatase↗

PubMed2000

Osteoblast proliferation and differentiation on dentin slices are modulated by pretreatment of the surface with tetracycline or osteoclasts.

BACKGROUND: Implant surface roughness and chemical composition, as well as other factors, affect the ability of osteogenic cells to form bone adjacent to an implant. The same principles may also apply to the tooth root and some reports have shown that surface modification of the root may lead to improved restoration of the periodontal apparatus. The most common of these surface modification techniques involves demineralization with citric acid or treatment with tetracycline to expose collagen fibrils. In addition, during normal bone remodeling, osteoclasts demineralize the extracellular matrix, leaving resorption pits and exposed collagen fibrils. In this study, the effect of different dentin surface-preparation techniques on osteoblasts were compared. METHODS: Slices of sperm whale dentin were mechanically polished and surfaces were treated with tetracycline-HCl (TCN) or were cultured with mouse bone marrow cells to create a surface with osteoclast (OC) resorption pits or left untreated. Profilometry, x-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) were used to evaluate the 3 different dentin surfaces. MG63 osteoblast-like cells were cultured on the 3 different surfaces and the effect of dentin surface preparation technique on MG63 cell proliferation (cell number), differentiaton (alkaline phosphatase specific activity of isolated cells and cell layer lysates; osteocalcin production), and local factor production (transforming growth factor (TGF)-beta1 and prostaglandin E2 (PGE2) compared. RESULTS: Profilometry showed the polished and TCN surfaces were smooth with comparable Ra values, whereas the OC surfaces were slightly rougher due to resorption pits which covered 3.7% of the surface. XPS measurements showed that TCN treatment reduced the Ca and P content of the surface, indicating that it had dissolved the mineral. Osteoclast-resorption also reduced the Ca and P content, but to a lesser extent. MG63 cell proliferation on polished dentin and tissue culture polystyrene was equivalent. In contrast, cells grown on the TCN- and OC-treated surfaces exhibited increased proliferation. No effect of surface treatment on cell alkaline phosphatase activity was observed, but activity in the cell layer lysates was increased on the TCN- and OC-treated surfaces. Osteocalcin production was reduced on all dentin surfaces, but the greatest reduction was found on the TCN-treated surface. Production of both TGF-beta1 and PGE2 was increased on the treated surfaces. All effects were greatest in cultures grown on the TCN-treated dentin. CONCLUSIONS: These data indicate that demineralization of the dentin surface promotes proliferation of osteoblasts and early differentiation events like production of alkaline phosphatase and autocrine mediators such as PGE2 and TGF-beta1. However, later differentiation events like osteocalcin production are decreased. Osteoclast-mediated bone resorption elicits similar responses; less than 4% of the dentin surface resulted in approximately 75% of the response caused by TCN treatment. These observations suggest that greater attention should be paid to the effects of osteoclastic resorption in designing methods for enhancing bone and cementum formation adjacent to root surfaces.

Alkaline Phosphatase↗

PubMed2000

Ability of deproteinized cancellous bovine bone to induce new bone formation.

BACKGROUND: Preclinical and clinical studies indicate that deproteinized cancellous bovine bone is osteoconductive and may be osteopromotive. Previous studies using commercial preparations failed to demonstrate the presence of protein, implicating bone-mineral composition and 3-dimensional structure as reasons for clinical success; however, these studies did not examine whether osteoinductive factors might be present in close association with the mineral phase. METHODS: Deproteinized cancellous bovine bone was decalcified and any protein present released by chaotropic solvents using the protocol described for purification of bone morphogenetic proteins (BMPs). Three extracts were obtained and tested for their ability to support osteoinduction in the calf muscle of nude mice. RESULTS: Protein content averaged 11 microg/g based on absorbance at 280 nm using bovine serum albumin as a standard. All extracts contained material that stained positively with silver stain after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blots of these gels indicated the presence of transforming growth factor-beta (TGF-beta) and BMP-2. All 3 extracts were osteoinductive in the nude mouse model when combined with inactive DFDBA, and bone formation was comparable to that induced by active DFDBA. Deproteinized cancellous bovine bone by itself was not osteoinductive in the nude mouse, but in a clinical case, exhibited osteoclastic resorption with adjacent new bone formation. CONCLUSIONS: The results suggest that small amounts of protein are present in deproteinized cancellous bovine bone in close association with the mineral phase. Some of the extracted material has osteoinductive potential and may contain growth factors. This may explain the osteopromotive ability of deproteinized cancellous bovine bone clinically.

Adult↗

PubMed2000

Porcine fetal enamel matrix derivative enhances bone formation induced by demineralized freeze dried bone allograft in vivo.

BACKGROUND: Embryonic enamel matrix proteins are involved in the formation of acellular cementum during development of the periodontal attachment apparatus, suggesting that these proteins might be used clinically to promote periodontal regeneration. At present, it is unknown if these proteins are osteoinductive, osteoconductive, or osteopromotive. To address this question, we examined the ability of a commercially prepared embryonic porcine enamel matrix derivative to induce new bone formation in nude mouse calf muscle, or to enhance the bone induction ability of a demineralized freeze-dried bone allograft (DFDBA). METHODS: Porcine fetal enamel matrix derivative (EMD) was implanted bilaterally in the calf muscle of 4 male Nu/Nu mice per treatment group (N = 8 implants): 2 mg EMD alone; 4 mg EMD alone; inactive human DFDBA alone; inactive DFDBA + 2 mg EMD; inactive DFDBA + 4 mg EMD; active DFDBA alone; active DFDBA + 2 mg EMD; and active DFDBA + 4 mg EMD. Implants were harvested after 56 days and examined histologically for bone induction using a semi-quantitative score and histomorphometrically for area of new bone, cortical bone, bone marrow, and residual DFDBA. RESULTS: Implants containing inactive DFDBA, 2 mg EMD, 4 mg EMD, and inactive DFDBA + 2 or 4 mg EMD did not induce new bone. Active DFDBA and active DFDBA + 2 mg EMD induced new bone to a similar extent. In contrast, active DFDBA + 4 mg EMD resulted in enhanced bone induction, area of new bone, and cortical bone. Residual DFDBA was also increased in this group. CONCLUSIONS: EMD is not osteoinductive. However, it is osteopromotive, due in part to its osteoconductive properties, but a threshold concentration is required.

Animals↗

PubMed2000

Porcine fetal enamel matrix derivative stimulates proliferation but not differentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-like MG63 cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells.

BACKGROUND: Embryonic enamel matrix proteins are hypothesized to be involved in the formation of acellular cementum during tooth development, suggesting that these proteins can be used to regenerate periodontal tissues. Enamel matrix protein derived from embryonic porcine tooth germs is used clinically, but the mechanisms by which it promotes the formation of cementum, periodontal ligament, and bone are not well understood. METHODS: This study examined the response of osteoblasts at 3 stages of osteogenic maturation to porcine fetal enamel matrix derivative (EMD). Proliferation (cell number and [3H]-thymidine incorporation), differentiation (alkaline phosphatase and osteocalcin), matrix synthesis ([35S]-sulfate incorporation; percentage of collagen production), and local factor production (prostaglandin E2 [PGE2] and transforming growth factor-beta 1 [TGF-beta1]) were measured in cultures of 2T9 cells (pre-osteoblasts which exhibit osteogenesis in response to bone morphogenetic protein-2 [BMP-2]), MG63 human osteoblast-like osteosarcoma cells, and normal human osteoblasts (NHOst cells). RESULTS: EMD regulated osteoblast proliferation and differentiation, but the effects were cell-specific. In 2T9 cell cultures, EMD increased proliferation but had no effect on alkaline phosphatase-specific activity. EMD decreased proliferation of MG63 cells and increased cellular alkaline phosphatase and osteocalcin production. There was no effect on collagen synthesis, proteoglycan sulfation, or PGE2 production; however, TGF-beta1 content of the conditioned media was increased. There was a 60-fold increase in cell number in third passage NHOst cells cultured for 35 days in the presence of EMD. EMD also caused a biphasic increase in alkaline phosphatase that was maximal at day 14. CONCLUSIONS: EMD affects early states of osteoblastic maturation by stimulating proliferation, but as cells mature in the lineage, EMD enhances differentiation.

Alkaline Phosphatase↗

PubMed2000

The relationship between peripheral levels of leukocytes and neutrophils and periodontal disease status in a patient with congenital neutropenia.

BACKGROUND: Congenital neutropenia is characterized by an almost total absence of neutrophils and increased susceptibility to infection. Oral manifestations include ulcerations of mucous membranes, acute gingival inflammation with focal necrosis, and rapid loss of attachment. Treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) increases neutrophil numbers and ameliorates the periodontal status. METHODS: We report the treatment of a 22-year-old male with congenital neutropenia (Kostman syndrome), referred to us due to periodontal disease, and the effect of treatment on peripheral neutropenia. Diagnosis of neutropenia was made at year 1; at age 15, the patient started to receive injections of rhG-CSF, reducing the occurrence of infection and improving neutrophil count, although levels remained below normal. The patient underwent extraction of a molar at age 8; scaling, root planing, and modified Widman flaps at age 9; and oral hygiene maintenance every 2 to 3 months from age 18 to 21. At age 23, he initiated treatment at our periodontal clinic. The patient's gingiva was severely inflamed, and the dentition was covered with plaque and calculus. Attachment loss was advanced, all teeth were mobile, and bone loss was approximately 75% in most sites. Neutrophil counts were below normal, but other hematologic parameters were normal. Scaling and root planing were performed and the patient received antibiotics and chlorhexidine rinses twice each day for 2 weeks. Extracoronal splinting was performed, fluoride varnish was used to desensitize cervical areas, and tooth FDI #46 was restored. Root planing and deplaquing were repeated, and the patient received subgingival chlorhexidine irrigation 13 times over one year. Assessments were made on presentation, after the initial treatment, and at 1 and 2 years post-treatment. RESULTS: Mean probing depth was reduced posttreatment with a further reduction during the maintenance period. This was correlated with an increase in attachment levels. Total white blood cells increased, due in part to an increase in neutrophils, reaching normal levels. CONCLUSIONS: This report demonstrates for the first time that periodontal therapy, resulting in decreased bacterial load, may result in restoration of normal levels of circulating neutrophils in individuals with congenital neutropenia under treatment with rhG-CSF. The results also suggest that periodontal pathogens may be associated with depressed neutrophil levels, even when patients receive treatment for neutropenia.

Adult↗

PubMed1999

Vitamin D3 metabolites regulate LTBP1 and latent TGF-beta1 expression and latent TGF-beta1 incorporation in the extracellular matrix of chondrocytes.

Growth plate chondrocytes make TGF-beta1 in latent form (LTGF-beta1) and store it in the extracellular matrix via LTGF-beta1 binding protein (LTBP1). 1,25-(OH)2D3 (1,25) regulates matrix protein production in growth zone (GC) chondrocyte cultures, whereas 24,25-(OH)2D3 (24,25) does so in resting zone (RC) cell cultures. The aim of this study was to determine if 24,25 and 1,25 regulate LTBP1 expression as well as the LTBP1 -mediated storage of TGF-beta1 in the extracellular matrix of RC and GC cells. Expression of LTBP1 and TGF-beta1 in the growth plate and in cultured RC and GC cells was determined by in situ hybridization using sense and antisense oligonucleotide probes based on the published rat LTBP1 and TGF-beta1 cDNA sequences. Fourth passage male rat costochondral RC and GC chondrocytes were treated for 24 h with 10(-7)-10(-9) M 24,25 and 10(-8)-10(-10) M 1,25, respectively. LTBP1 and TGF-beta1 mRNA levels were measured by in situ hybridization; production of LTGF-beta1, LTGF-beta2, and LTBP1 protein in the conditioned media was verified by immunoassays of FPLC-purified fractions. In addition, ELISA assays were used to measure the effect of 1,25 and 24,25 on the level of TGF-beta1 in the media and matrix of the cultures. Matrix-bound LTGF-beta1 was released by digesting isolated matrices with 1 U/ml plasmin for 3 h at 37 degrees C. LTBP1 and TGF-beta1 mRNAs are co-expressed throughout the growth plate, except in the lower hypertrophic area. Cultured GC cells express more LTBP1 and TGF-beta1 mRNAs than RC cells. FPLC purification of the conditioned media confirmed that RC cells produce LTGF-beta1, LTGF-beta2, and LTBP1. GC cells also produce LTGF-beta2, but at lower concentrations. 1,25 dose-dependently increased the number of GC cells with high LTBP1 expression, as seen by in situ hybridization. 24,25 had a similar, but less pronounced, effect on RC cells. 1,25 also caused a dose-dependent increase in the amount of TGF-beta1 protein found in the matrix, significant at 10(-8) and 10(-9) M, and a corresponding decrease in TGF-beta1 in the media. 24,25 had no effect on the level of TGF-beta1 in the matrix or media produced by RC cells. This indicates that 1,25 induces the production of LTBP1 by GC cells and suggests that the TGF-beta1 content of the media is reduced through the formation of latent TGF-beta1 -LTBP1 complexes which mediates storage in the matrix. Although 24,25 induced the expression of LTBP1 by RCs, TGF-beta1 incorporation into the matrix is not regulated by this vitamin D3 metabolite. Thus, vitamin D3 metabolites may play a role in regulating the availability of TGF-beta1 by modulating LTBP1 production.

24,25-Dihydroxyvitamin D 3↗

PubMed1999

Potential of porous poly-D,L-lactide-co-glycolide particles as a carrier for recombinant human bone morphogenetic protein-2 during osteoinduction in vivo.

Several different biodegradable bone graft materials are in clinical or preclinical use for the repair of bone defects in orthopedics, maxillofacial surgery, and periodontics. This study tested the hypothesis that poly-D,L-lactide-co-glycolide copolymer (PLG) can be used as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) and that the composite has osteoinductive ability. Porous PLG rods were shredded to a particle size ranging from 250 to 850 microm. Active and inactive demineralized freeze-dried bone allografts (DFDBA) with a comparable particle size were used as positive and negative controls, respectively. PLG particles were treated with vehicle or with 5 or 20 microg rhBMP-2. DFDBA and PLG particles were placed in gelatin capsules, mixed with vehicle or rhBMP-2, and implanted at intramuscular sites in male Nu/Nu (nude) mice. Each mouse underwent bilateral implantation with implants of the same formulation, resulting in five groups of four mice per group: active DFDBA, inactive DFDBA, PLG, PLG + 5 microg rhBMP-2, and PLG + 20 microg rhBMP-2. After 56 days, the implants were recovered and processed for histology. Bone induction was assessed by use of a semiquantitative scoring system based on the amount of new bone formed in representative histological sections. Histomorphometry was also used to measure the area of new bone formed and the area of residual implant material. The results showed that active DFDBA induced the formation of ossicles containing new bone with bone marrowlike tissue, whereas inactive DFDBA or PLG particles alone did not induce new bone. The addition of rhBMP-2 to PLG particles resulted in new bone formation that had a greater bone induction score than active DFDBA. Moreover, the histomorphometric analysis showed that the addition of rhBMP-2 to PLG particles induced the formation of a greater area of new bone and bone marrowlike tissue than active DFDBA. The resorption of the PLG particles was markedly increased with the addition of rhBMP-2, suggesting that rhBMP-2 may attract and regulate resorptive cells at the implantation site. The results of the present study indicate that PLG copolymers are good carriers for BMP and promote the induction of new bone formation. Further, the PLG copolymers with rhBMP-2 had a greater effect in inducing new bone formation and resorbing the implanted material than active DFDBA alone.

Animals↗

PubMed1999

Surface roughness modulates the response of MG63 osteoblast-like cells to 1,25-(OH)(2)D(3) through regulation of phospholipase A(2) activity and activation of protein kinase A.

Implant surface roughness influences osteoblast proliferation, differentiation, and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones such as 1, 25-(OH)(2)D(3) is altered by the effects of surface roughness; on the roughest Ti surfaces the effects of roughness and 1, 25-(OH)(2)D(3) are synergistic. Prostaglandin E(2) (PGE(2)) appears to be involved in mediating the effects of surface roughness on the cells, as well as in the response to 1,25-(OH)(2)D(3). However, it is not yet known through which signaling pathways surface roughness exerts its effects on the response of osteoblasts to 1, 25-(OH)(2)D(3). The present study examined the potential role of protein kinase A (PKA), phospholipase A(2)(PLA(2)), and protein kinase C (PKC) in this process. MG63 osteoblast-like human osteosarcoma cells were cultured on cpTi disks with R(a) values of 0. 54 microm (PT), 4.14 microm (SLA), or 4.92 microm (TPS). PKA was inhibited by adding H8 to the cultures; similarly, PLA(2) was inhibited with quinacrine or activated with melittin, and PKC was inhibited with chelerythrine. Inhibitors or activators were included in the culture media through the entire culture period or for the last 24 h of culture. In addition, cultures were treated for 24 h with inhibitors or activators in the presence of 1,25-(OH)(2)D(3). The effects on cell number and alkaline phosphatase specific activity were determined after 24 h; PKC activity was determined after 9 min and at 24 h. Cell number was reduced on rough surfaces, and alkaline phosphatase activity was increased. 1,25-(OH)(2)D(3) had a synergistic effect with surface roughness on alkaline phosphatase. However, neither surface roughness nor 1,25-(OH)(2)D(3) had an effect on PKC. H8 treatment for 24 h inhibited cell number and alkaline phosphatase on all surfaces; however, when it was present throughout the culture period, the PKA inhibitor had no effect on cell number, but decreased alkaline phosphatase-specific activity. H8 reduced the 1,25-(OH)(2)D(3)-mediated effect on cell number and alkaline phosphatase. Quinacrine inhibited cell proliferation and alkaline phosphatase on all surfaces and further reduced the 1,25-(OH)(2)D(3)-dependent decreases in both parameters. Melittin had no effect when applied for 24 h and did not modify the 1,25-(OH)(2)D(3) effect; however, when present throughout the culture period, it caused a decrease in proliferation and an increase in enzyme activity. Chelerythrine, the PKC inhibitor, only inhibited cell proliferation when it was present throughout the entire culture period. However, it decreased alkaline phosphatase in cultures treated for 24 h, but increased enzyme activity when it was present for the entire culture period. The results indicate that surface roughness and 1,25-(OH)(2)D(3) both mediate their effects through PLA(2) which catalyzes the rate-limiting step in PGE(2) production. Further downstream, PGE(2) activates PKA. Surface roughness-dependent effects are also mediated through PKC, but only after the cells have reached confluence and are undergoing phenotypic maturation. The effect of surface roughness on responsiveness to 1,25-(OH)(2)D(3) is mediated through PLA(2)/PKA and not through PKC.

Bioprosthesis↗

PubMed1999

Ultrahigh molecular weight polyethylene particles have direct effects on proliferation, differentiation, and local factor production of MG63 osteoblast-like cells.

Small particles of ultrahigh molecular weight polyethylene stimulate formation of foreign-body granulomas and bone resorption. Bone formation may also be affected by wear debris. To determine if wear debris directly affects osteoblasts, we characterized a commercial preparation of ultrahigh molecular weight polyethylene (GUR4150) particles and examined their effect on MG63 osteoblast-like cells. In aliquots of the culture medium containing ultrahigh molecular weight polyethylene, 79% of the particles were less than 1 microm in diameter, indicating that the cells were exposed to particles of less than 1 microm. MG63 cell response to the particles was measured by assaying cell number, [3H]thymidine incorporation, alkaline phosphatase specific activity, osteocalcin production, [35S]sulfate incorporation, and production of prostaglandin E2 and transforming growth factor-beta. Cell number and [3H]thymidine incorporation were increased in a dose-dependent manner. Alkaline phosphatase specific activity, a marker of cell differentiation for the cultures, was significantly decreased, but osteocalcin production was not affected. [35S]sulfate incorporation, a measure of extracellular matrix production, was reduced. Prostaglandin E2 release was increased, but transforming growth factor-beta production was decreased in a dose-dependent manner. This shows that ultrahigh molecular weight polyethylene particles affect MG63 proliferation, differentiation, extracellular matrix synthesis, and local factor production. These effects were direct and dose dependent. The findings suggest that ultrahigh molecular weight polyethylene wear debris particles with an average size of approximately 1 microm may inhibit bone formation by inhibiting cell differentiation and reducing transforming growth factor-beta production and matrix synthesis. In addition, increases in prostaglandin E2 production may not only affect osteoblasts by an autocrine pathway but may also stimulate the proliferation and activation of cells in the monocytic lineage. These changes favor decreased bone formation and increased bone resorption as occur in osteolysis.

Alkaline Phosphatase↗

PubMed1999

Osteochondral progenitor cells in acute and chronic canine nonunions.

This study examined the ability of cells isolated from early healing segmental defects and from tissue from chronic nonunions to support bone and cartilage formation in vivo and their response to transforming growth factor-beta1 in vitro. Ostectomies (3 mm) were created in the radial diaphysis of four dogs. The dogs were splinted 3-5 days postoperatively and then allowed to bear full weight. At 7 days, tissue in the defect was removed and any periosteum was discarded; cells in the defect tissue were released by enzymatic digestion. The dogs were splinted again and allowed to bear full weight for 12 weeks. Radiographs confirmed a persistent nonunion in each dog. Defect tissue was again removed, any periosteum was discarded, and cells were isolated. Cells were also obtained from the defect tissue by nonenzymatic means with use of explant cultures. One-half of the tissue and one-half of any preconfluent, first-passage cultures were shipped to Cleveland by overnight carrier. At second passage, cells were loaded into ceramic cubes and implanted into immunocompromised mice for 3 or 6 weeks. Harvested cubes were examined histologically for cartilage and bone with use of a semiquantitative scoring system. Confluent fourth-passage cultures of 7 and 84-day defect tissue cells were cultured with 0.03-0.88 ng/ml transforming growth factor-beta1 for 24 hours, and [3H]thymidine incorporation and alkaline phosphatase specific activity were determined. Donor-dependent differences were noted in the rate at which defect cells achieved confluence; in general, cells from 7-day tissue divided most rapidly. Seven-day defect cells formed less bone and at a slower rate than was seen in the ceramic cubes containing samples from day 84. Cells derived enzymatically behaved similarly to those from explant cultures. Ceramic cubes contained fibrous connective tissue, cartilage, bone, and fat, indicating that multipotent cells were present. Stimulation of [3H]thymidine incorporation in response to transforming growth factor-beta1 was donor dependent and variable; only two of six separate isolates of cells exposed to it had measurable alkaline phosphatase activity (which was relatively low), and none of the cultures exhibited an increase in response to transforming growth factor-beta1 for 24 hours. This indicates that mesenchymal progenitor cells are present in the healing defect tissue at 7 and 84 days and that the relative proportion of osteochondroprogenitor cells is greater at the later time. The response to transforming growth factor-beta1 is typical of multipotent mesenchymal cells but not of committed chondrocytes or osteoblasts, indicating that these committed and differentiated cells are not present in early stages of healing and suggesting that their differentiation is inhibited in chronic nonunion.

Alkaline Phosphatase↗

PubMed1999

Effect of surface roughness and composition on costochondral chondrocytes is dependent on cell maturation state.

During endochondral bone formation, as occurs in fracture healing, chondrocytes are one of the first cells to see an implant surface. We tested the hypothesis that chemical composition and surface roughness affect chondrocyte differentiation, matrix synthesis, and local factor production and that the nature of the response is dependent on the state of maturation of the cells. To do this, we harvested rat growth zone and resting zone chondrocytes and examined their response to smooth and rough disk surfaces manufactured from either commercially pure titanium or titanium alloy. Profilometry, scanning electron microscopy, Auger spectroscopy, and Fourier transform infrared spectroscopy were used to characterize the surfaces. Average roughness values were 0.22 microm for smooth titanium surfaces, 0.23 microm for smooth titanium alloy surfaces, 4.24 microm for rough titanium surfaces, and 3.20 microm for rough titanium alloy surfaces. Cells were grown on the different disk surfaces until the cultures had reached confluence on plastic. The effect of the surfaces was determined by assaying cell number and [3H]thymidine incorporation as measures of cell proliferation, cell layer and cell alkaline phosphatase specific activity as markers of differentiation, and collagen production and [35S]sulfate incorporation as indicators of extracellular matrix production. In addition, the synthesis of prostaglandin E2 and transforming growth factor-beta were examined to measure changes in local factor synthesis. In growth zone and resting zone cultures, cell number and [3H]thymidine incorporation were decreased on rough surfaces; however, this effect was greater on commercially pure titanium surfaces. Cell layer and cell alkaline phosphatase specific activity were decreased in resting zone cells grown on rough surfaces. Cell alkaline phosphatase specific activity in growth zone cells was decreased on rough surfaces, whereas cell layer alkaline phosphatase specific activity was increased only in growth zone cells grown on rough commercially pure titanium surfaces. Resting zone cell collagen production was decreased only on rough commercially pure titanium, whereas in growth zone cells, collagen production was increased. Increased prostaglandin E2 release into the media was found for growth zone and resting zone cell cultures on the disks with rough surfaces. The observed effect was greater on rough commercially pure titanium. Production of transforming growth factor-beta by resting zones was similarly affected, whereas an increase in its production by growth zone cells was measured only on rough commercially pure titanium. These results indicate that surface roughness affects chondrocyte proliferation, differentiation, matrix synthesis, and local factor production and that these parameters are also affected by chemical composition. Furthermore, the nature and extent of the cell response is dependent on cell maturation. The overriding variable in response to an implant material, however, appears to be roughness of the surface.

Alloys↗

PubMed1999

TGFbeta1 regulates 25-hydroxyvitamin D3 1alpha- and 24-hydroxylase activity in cultured growth plate chondrocytes in a maturation-dependent manner.

Chondrocytes metabolize 25-(OH)D3 to the two active dihydroxylated forms of the secosteroid, 1,25-(OH)2D3 and 24,25-(OH)2D3. The aim of the present study was to examine the activity of the enzymes responsible for this metabolism, 1alpha-hydroxylase and 24R-hydroxylase, and their regulation by TGFbeta1. Basal 1alpha- and 24R-hydroxylase activities were measured in homogenates of confluent, fourth passage rat costochondral resting zone and growth zone chondrocytes and mouse cortico-tubular cells (MCT) were used as a positive control. The cells were harvested and homogenized in buffer optimized to maintain the activity and stability of the hydroxylases. Homogenates were incubated for 90 minutes and 1alpha- and 24R-hydroxylase activities determined by measuring the conversion of [3H]-25-(OH)D3 to [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3 using an HPLC with an inline radioisotope detector. Resting zone cells were also treated with various concentrations of recombinant human TGFbeta1 for 24 hours, and enzyme activity in total cell homogenates as well as 24-hydroxylase mRNA levels were determined. In addition, [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3 released into the conditioned media by resting zone chondrocyte cultures in response to TGFbeta1 were measured. In culture, all three cell types were found to contain 1alpha- and 24R-hydroxylase activities. Basal 1alpha-hydroxylase specific activity was significantly higher than 24R-hydroxylase specific activity in all cells. RT-PCR confirmed that resting zone and growth zone cells expressed mRNA for 24R-hydroxylase. Treatment of resting zone cells with TGFbeta1 increased 24R-hydroxylase mRNA levels in a dose-dependent manner. TGFbeta1 also increased 24R-hydroxylase activity 2- to 5-fold and decreased 1alpha-hydroxylase activity by 20-30%. Similar changes were observed with MCT cells, but not growth zone cells. Production of [3H]-24,25-(OH)2D3 by resting zone cells increased with TGFbeta1 treatment, while [3H]-1,25-(OH)2D3 production decreased. The effect was time- and dose-dependent, correlating with hydroxylase activity and 24-hydroxylase gene expression. These results demonstrate that growth plate chondrocytes contain the necessary enzymes to produce 1, 25-(OH)2D3 and 24,25-(OH)2D3 from 25-(OH)D3. In addition, the activity of these enzymes in resting zone cells, but not growth zone cells, is regulated by TGFbeta1 by increasing gene transcription, indicating that cell maturation-dependent autocrine/paracrine pathways exist for regulating vitamin D metabolite production.

Animals↗

PubMed1999

Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2.

Earlier studies have shown that implant surface roughness influences osteoblast proliferation, differentiation, matrix synthesis and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones, such as 1,25-(OH)2D3, at the implant surface is also influenced by surface roughness and this effect is mediated by changes in prostaglandins. At present, it is not known which signaling pathways are involved in mediating cell response to surface roughness and how 1,25-(OH)2D3 treatment alters the activation of these pathways. This paper reviews a series of studies that have addressed this question. MG63 osteoblast-like cells were cultured on commercially pure titanium (cpTi) surfaces of two different roughnesses (Ra 0.54 and 4.92 microm) in the presence of control media or media containing 1,25-(OH)2D3 or 1,25-(OH)2D3 plus H8 (a protein kinase A inhibitor) or quinacrine (a phospholipase A2 inhibitor). At harvest, the effect of these treatments on cell number and alkaline phosphatase specific activity was measured. Compared to cultures grown on the smooth surface, cell number was reduced on the rough surface. 1,25-(OH)2D3 inhibited cell number on both surfaces and inhibition of protein kinase A in the presence of 1,25-(OH)2D3 restored cell number to that seen in the control cultures. Inhibition of phospholipase A2 in the presence of 1,25-(OH)2D3 caused a further reduction in cell number on the smooth surface, and partially reversed the inhibitory effects of 1,25-(OH)2D3 on the rough surface. Alkaline phosphatase specific activity was increased in cultures grown on the rough surface compared with those grown on the smooth surface; 1,25-(OH)2D3 treatment increased enzyme specific activity on both surfaces. Cultures treated with H8 and 1,25-(OH)2D3 displayed enzyme specific activity that approximated that seen in control cultures. Inhibition of phospholipase A2 also inhibited the 1,25-(OH)2D3-dependent effect on the smooth surface, but on the rough surface there was an inhibition of the 1,25-(OH)2D3 effect as well as a partial inhibition of the surface roughness-dependent effect. The results indicate that surface roughness and 1,25-(OH)2 D3 mediate their effects through phospholipase A2, which catalyzes one of the rate-limiting steps in prostaglandin E2 production. Further downstream, prostaglandin E2 activates protein kinase A.

Alkaline Phosphatase↗

PubMed1999

Prostaglandins mediate the effects of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes in a metabolite-specific and cell maturation-dependent manner.

Prior studies have shown that 1,25-(OH)2D3 stimulates alkaline phosphatase, phospholipase A2 (PLA2), and protein kinase C (PKC)-specific activities, and production of prostaglandin E2 (PGE2) in growth zone chondrocytes. In contrast, 24,25-(OH)2D3 stimulates alkaline phosphatase and PKC-specific activities but inhibits PLA2-specific activity and PGE2 production in resting zone cells. This indicates that different mechanisms are involved in the action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on their respective target cells. In this study, we examined the hypothesis that differential regulation of prostaglandin production modulates the activity of PKC and alkaline phosphatase. To do this, we examined the effect of the cyclooxygenase inhibitor indomethacin (Indo) on alkaline phosphatase, PLA2, and PKC-specific activities in growth plate chondrocytes treated with these two vitamin D metabolites. In addition, we examined whether inhibition of PKC altered PGE2 production. In growth zone cells, Indo inhibited basal alkaline phosphatase and blocked the 1,25-(OH)2D3-dependent increase in alkaline phosphatase. This effect was due to inhibition of both plasma membrane and matrix vesicle alkaline phosphatase. In resting zone cells, Indo increased basal alkaline phosphatase activity in a dose-dependent manner, but it did not further enhance the 24,25-(OH)2D3-dependent stimulation of this enzyme. The effect of Indo was found in both plasma membranes and matrix vesicles. These data indicate that 1,25-(OH)2D3-dependent increases in alkaline phosphatase-specific activity in growth zone cells are mediated through increased prostaglandin production, whereas 24,25-(OH)2D3-mediated changes in enzyme activity in resting zone cells are mediated through decreased prostaglandin production. Regulation of PLA2 by either 1,25-(OH)2D3 or 24,25-(OH)2D3 in their target cells was unaffected by Indo, indicating that the effect of the vitamin D metabolites on this enzyme is not dependent on changes in PGE2 production. The rapid increase in 1,25-(OH)2D3-dependent PKC-specific activity in growth zone cells was inhibited by Indo, whereas there was a potentiation of the effect of 24,25-(OH)2D3 on PKC activity in resting zone cells. In addition, inhibition of PKC blocked the 1,25-(OH)2D3-dependent increase in PGE2 production in growth zone cells and the 24,25-(OH)2D3-dependent decrease in PGE2 production by resting zone cells. These data indicate that prostaglandins are involved in mediating the rapid effects of 1,25-(OH)2D3 on growth zone cells, and contribute to the effects of 24,25-(OH)2D3 on resting zone cells; in both instances, the vitamin D metabolites exert their effects on PKC through changes in arachidonic acid via the action of PLA2. In addition, PKC by itself may mediate the production of PGE2.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

Implant surface characteristics modulate differentiation behavior of cells in the osteoblastic lineage.

This paper reviews the role of surface roughness in the osteogenic response to implant materials. Cells in the osteoblast lineage respond to roughness in cell-maturation-specific ways, exhibiting surface-dependent morphologies and growth characteristics. MG63 cells, a human osteoblast-like osteosarcoma cell line, respond to increasing surface roughness with decreased proliferation and increased osteoblastic differentiation. Alkaline phosphatase activity and osteocalcin production are increased. Local factor production is also affected; production of both TGF-beta 1 and PGE2 is increased. On rougher surfaces, MG63 cells exhibit enhanced responsiveness to 1,25-(OH)2D3. Prostaglandins mediate the effects of surface roughness, since indomethacin prevents the increased expression of differentiation markers in these cells.

Animals↗