Biomedical subjects

C H Lohmann

Publications and source records attributed to C H Lohmann.

At least 37 records · Page 2Linked to original sources

PubMed2001

[Hemialloarthroplasty of the knee joint. Results of 2 different gliding prosthesis models].

The outcome of 2 different unicompartimental knee arthroplasty procedures (St. Georg, Link AG, and Wessinghage, Sulzer Medica AG) is presented. The clinical results were evaluated by use of the Insall Score (Knee Society Rating System). According to this system, the subjective opinion of the patients are presented as a "Function Score" and the clinical evaluations is shown as a "Knee Score". In this study, the median follow-up for the St. Georg implant was 7 years and 6 months with good results reported by both scores. The median follow-up for the Wessinghage implant was 3 years and 2 months and it also showed good results. Despite the high average age of the patients in this study as well as the high percentage of female patients, both types of prostheses produced good results. If any, partial or complete radioluciencies were mainly observed in the tibial component. However, they had no clinical consequence. It should be noted, however, that arthritis in the compartment not receiving arthroplasty surgery progressed in almost 2/3 of the patients. By implantation of both implants a physiological axis of the leg could be achieved. The survivorship of the St. Georg implant was 88.92% (10 years survivorship). For the Wessinghage implant it was 98.35% (5 years survivorship).

Aged↗

PubMed2001

Tissue response and osteoinduction of human bone grafts in vivo.

Freeze-dried human bone allograft is used clinically as an adjunct to autologous bone graft. When freeze-dried human bone allograft is demineralized, the allograft is osteoinductive, since it causes bone to form heterotopically. Both types of allograft are also used alone, such as in spinal fusions, critical size defects, and periodontal therapy. The purpose of this study was to determine the effect of demineralization on the osteoinductive potential of human bone grafts obtained from two different groups of patients. One group consisted of six patients younger than 42 years of age, while the other group consisted of six patients who were older than 70 years of age. The harvested material was lyophilized and divided into two portions, one of which was used directly while the other was demineralized. Osteoinductive ability was established using an in vivo assay for heterotopic bone formation. Activity in these bone grafts was compared with a batch of commercially prepared demineralized, freeze-dried human bone grafts that had been previously shown to be active and another batch that had been shown to display low ('inactive') osteoinductive ability. A bone induction score was determined for each group of grafts based on the number and size of any ossicles formed. In addition, the area of new bone formation and area of residual particles were determined histomorphometrically. Tissue response to the bone grafts varied with donor age and whether the samples had been demineralized or not. Only demineralized, freeze-dried bone graft from patients younger than 42 years of age was osteoinductive; all other batches displayed little or no osteoinductive activity. In the demineralized, freeze-dried bone from donors younger than 42 years of age, the bone induction score and new bone area were significantly higher than in the other batches of bone graft, and the area of residual particles was reduced. Both demineralized and nondemineralized bone graft from patients older than 70 years of age were encapsulated in dense, fibrous connective tissue. These results may help explain the observed differences in clinical outcome when demineralized, freeze-dried bone graft or nondemineralized, freeze-dried bone graft from different donors is used in bone regeneration applications.

Adult↗

PubMed2001

Local factor production by MG63 osteoblast-like cells in response to surface roughness and 1,25-(OH)2D3 is mediated via protein kinase C- and protein kinase A-dependent pathways.

Titanium (Ti) surface roughness affects bone formation in vivo and osteoblast attachment, proliferation and differentiation in vitro. MG63 cells exhibit decreased proliferation and increased differentiation when cultured on rough Ti surfaces (Ra > 2 microm) and response to 1,25-(OH)2D3 is enhanced, resulting in synergistic increases in TGF-beta1 and PGE2. To examine the hypothesis that surface roughness and 1,25-(OH)2D3 exert their effects on local factor production through independent, but convergent, signaling pathways, MG63 cells were cultured on tissue culture plastic or on smooth (PT, Ra = 0.60 microm) and rough (SLA, Ra = 3.97 microm; TPS, Ra = 5.21 microm) Ti disks. At confluence (5 days), cultures were treated for 24h with 10(-8) M 1alpha,25-(OH)2D3 and active and latent TGF-beta1 in the conditioned media measured by ELISA. Cell layers were digested with plasmin and released TGF-beta1 was also measured. 1,25-(OH)2D3 regulated the distribution of TGF-beta1 between the media and the matrix in a surface-dependent manner; the effect was greatest in the matrix of cells cultured on SLA and TPS. Inhibition of PKA with H8 for the last 24 h of culture increased PGE2 on SLA and TPS, but when present throughout the entire culture period H8 caused an increase in PGE2 on all surfaces. 1,25-(OH)2D3 reduced the effect of H8 on PGE2 production in cultures treated for 24 h. H8 had no effect on TGF-beta1 in the media by itself but caused a complete inhibition of the 1,25-(OH)2D3 dependent increase. Inhibition of PKC with chelerythrine increased PGE2 in a surface-dependent manner and 1,25-(OH)2D3 reduced the effect of the PKC inhibitor. Chelerythrine also increased TGF-beta1 but the effect was not surface dependent; however, 1,25-(OH)2D3 reduced the effects of chelerythrine with the greatest effects on the smooth surface. Thus, the distribution of TGF-beta1 between the media and the matrix is regulated by 1,25-(OH)2D3 in a surface-dependent manner. Surface roughness exerts its effects on TGF-beta1 production via PKC but not PKA. The effect of 1,25-(OH)2D3 on TGF-beta1 production is not via PKC. PKA is involved in the surface-dependent regulation of PGE2 but not in the regulation of PGE2 by 1,25-(OH)2D3 on rough surfaces. Regulation of PKC affects PGE2 production but it is not involved in the surface roughness-dependent response to 1,25-(OH)2D3. These results suggest two independent but interconnected pathways are involved.

Animals↗

PubMed2001

Effect of polymer molecular weight and addition of calcium stearate on response of MG63 osteoblast-like cells to UHMWPE particles.

Periprosthetic osteolysis and implant loosening is associated with the presence of ultrahigh molecular weight polyethylene (UHMWPE) wear debris particles. Osteoblast phenotypic expression in vitro is affected by UHMWPE particles, suggesting that bone formation may also be affected by wear debris. Here we tested the hypothesis that the response of osteoblasts to UHMWPE can be modified by changes in UHMWPE particle chemistry. We used four different commercially available preparations of GUR UHMWPE particles to determine if chemical composition (+/- Ca-stearate) or polymer molecular weight (3.1-4.2 million or 5.4-6.5 million g/mol) modulates osteoblast response. Particles were characterized by size distribution, morphology, and number of particles added to the culture medium. They had an average equivalent circle diameter ranging from 0.46-1.26 microm. MG63 cell response was assessed by measuring cell number, cellular and cell layer alkaline phosphatase, and prostaglandin E2 (PGE2) production. There were dose-dependent effects of the particles on cell response. Cell number and PGE, production were increased, while alkaline phosphatase specific activity was decreased. In addition, there was a marked difference between cultures treated with particles containing Ca-stearate and as a function of polymer molecular weight. Particles of higher molecular weight caused a greater stimulation of proliferation and inhibition of alkaline phosphatase than particles of lower molecular weight. The presence of Castearate exerted a more pronounced depression of osteoblast phenotype as well as a significantly greater increase in PGE2 release by the cells. The present study shows that chemical composition and polymer molecular weight of UHMWPE are capable of modulating osteoblast response to particles. The results suggest that osteoblast differentiation is inhibited by UHMWPE particles, whereas cell proliferation and PGE2 production are stimulated. This may have direct effects on osteoblasts and bone formation, but also paracrine effects on cells of the monocytic lineage inducing bone resorption and promoting inflammation which may lead to aseptic loosening. The present results suggest that the cellular events in aseptic loosening may be modulated or even accelerated by changes in the composition of the UHMWPE used to fabricate implants.

Alkaline Phosphatase↗

PubMed2001

Inhibition of cyclooxygenase by indomethacin modulates osteoblast response to titanium surface roughness in a time-dependent manner.

Prostaglandin E2 (PGE2) and transforming growth factor-beta 1 (TGF-beta 1) production are increased in cultures of osteoblasts grown on rough surfaces and prostaglandins are involved in osteoblast response to surface roughness. In the present study, we examined the effect of inhibiting cyclooxygenase on this response. MG63 osteoblast-like cells were cultured on cpTi disks with Ra values of 0.60 micron (PT), 3.97 microns (SLA), and 5.21 microns (TPS) in the presence or absence of 10(-7) M indomethacin. Treatment was begun on days 1, 2, 3, or 4 after seeding, and all cultures were harvested on day 5. Indomethacin decreased PGE2 release by the cells to less than 50% of basal levels when the cells were cultured on plastic. Cell number decreased with increasing surface roughness and indomethacin treatment abrogated the surface roughness effect over time. Alkaline phosphatase specific activity (ALP) increased with surface roughness; after one day with indomethacin, ALP was decreased on smooth surfaces, but increased on rough surfaces. Over time, ALP decreased on all surfaces examined and remained greater than plastic only in cultures on TPS. Indomethacin also caused a time-dependent decrease in osteocalcin production on rough surfaces, eventually abrogating the increases due to surface roughness, but had no effect on osteocalcin production on smooth surfaces. TGF-beta 1 levels in the cell layer and media were sensitive to surface roughness; on rougher surfaces, TGF-beta 1 shifted from the media to the matrix. Indomethacin reduced TGF-beta 1 levels over time, but the surface roughness effect was still evident at 4 days. This indicates that prostaglandin production mediates the effects of surface roughness, since indomethacin causes a time-dependent abrogation of the response, but has no effect on proliferation, osteocalcin release, or TGF-beta 1 levels on smooth surfaces. Indomethacin's effect was not immediate, suggesting that clinical protocols could be designed that would reduce inflammation without preventing osteoblastic differentiation. The effect of indomethacin was not complete, since TGF-beta 1 and ALP remained elevated on rough surfaces, suggesting that pathways or factors other than prostanoids are involved. TGF-beta 1 is preferentially stored in the matrix, acting on the cells through autocrine signaling, and may contribute to ALP even in the presence of indomethacin. These results demonstrate the importance of local factors in the autocrine regulation of osteogenesis and the potential for factors released in response to surface morphology to act in a paracrine manner.

Alkaline Phosphatase↗

PubMed2001

Pulsed electromagnetic fields increase growth factor release by nonunion cells.

The mechanisms involved in pulsed electromagnetic field stimulation of nonunions are not known. Animal and cell culture models suggest endochondral ossification is stimulated by increasing cartilage mass and production of transforming growth factor-beta 1. For the current study, the effect of pulsed electromagnetic field stimulation on cells from human hypertrophic (n = 3) and atrophic (n = 4) nonunion tissues was examined. Cultures were placed between Helmholtz coils, and an electromagnetic field (4.5-ms bursts of 20 pulses repeating at 15 Hz) was applied to 1/2 of them 8 hours per day for 1, 2, or 4 days. There was a time-dependent increase in transforming growth factor-beta 1 in the conditioned media of treated hypertrophic nonunion cells by Day 2 and of atrophic nonunion cells by Day 4. There was no effect on cell number, [3H]-thymidine incorporation, alkaline phosphatase activity, collagen synthesis, or prostaglandin E2 and osteocalcin production. This indicates that human nonunion cells respond to pulsed electromagnetic fields in culture and that transforming growth factor-beta 1 production is an early event. The delayed response of hypertrophic and atrophic nonunion cells (> 24 hours) suggests that a cascade of regulatory events is stimulated, culminating in growth factor synthesis and release.

Adult↗

PubMed2000

Pulsed electromagnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production.

Pulsed electromagnetic field stimulation has been used to promote the healing of chronic nonunions and fractures with delayed healing, but relatively little is known about its effects on osteogenic cells or the mechanisms involved. The purpose of this study was to examine the response of osteoblast-like cells to a pulsed electromagnetic field signal used clinically and to determine if the signal modulates the production of autocrine factors associated with differentiation. Confluent cultures of MG63 human osteoblast-like cells were placed between Helmholtz coils and exposed to a pulsed electromagnetic signal consisting of a burst of 20 pulses repeating at 15 Hz for 8 hours per day for 1, 2, or 4 days. Controls were cultured under identical conditions, but no signal was applied. Treated and control cultures were alternated between two comparable incubators and, therefore, between active coils; measurement of the temperature of the incubators and the culture medium indicated that application of the signal did not generate heat above the level found in the control incubator or culture medium. The pulsed electromagnetic signal caused a reduction in cell proliferation on the basis of cell number and [3H]thymidine incorporation. Cellular alkaline phosphatase-specific activity increased in the cultures exposed to the signal, with maximum effects at day 1. In contrast, enzyme activity in the cell-layer lysates, which included alkaline phosphatase-enriched extracellular matrix vesicles, continued to increase with the time of exposure to the signal. After 1 and 2 days of exposure, collagen synthesis and osteocalcin production were greater than in the control cultures. Prostaglandin E2 in the treated cultures was significantly reduced at 1 and 2 days, whereas transforming growth factor-beta1 was increased; at 4 days of treatment, however, the levels of both local factors were similar to those in the controls. The results indicate enhanced differentiation as the net effect of pulsed electromagnetic fields on osteoblasts, as evidenced by decreased proliferation and increased alkaline phosphatase-specific activity, osteocalcin synthesis, and collagen production. Pulsed electromagnetic field stimulation appears to promote the production of matrix vesicles on the basis of higher levels of alkaline phosphatase at 4 days in the cell layers than in the isolated cells, commensurate with osteogenic differentiation in response to transforming growth factor-beta1. The results indicate that osteoblasts are sensitive to pulsed electromagnetic field stimulation, which alters cell activity through changes in local factor production.

Alkaline Phosphatase↗

PubMed2000

[Degree of differentiation of chondrocytes and their pretreatment with platelet-derived-growth factor. Regulating induction of cartilage formation in resorbable tissue carriers in vivo].

Current methods for articular cartilage repair are unpredictable with respect to clinical success. In the present study, we investigated the ability of cells from articular cartilage, perichondrium, and costochondral resting zone to form new cartilage when loaded onto biodegradable scaffolds and implanted into calf muscle pouches of nu/nu mice. Prior in vitro studies showed that platelet derived growth factor-BB (PDGF-BB), but not transforming growth factor beta-1 (TGF-beta 1), basic fibroblast growth factor, or bone morphogenetic protein-2 promoted proliferation and extracellular matrix sulfation of resting zone chondrocytes without causing the cells to exhibit a hypertrophic chondrocyte phenotype. TGF-beta 1 has also been shown to stimulate chondrogenesis by multipotent chondroprogenitor cells like those in the perichondrium. In addition, PDGF-BB has been shown to modulate chondrogensis by resting zone cells implanted in poly(D,L-lactide-co-glycolide) (PLG) scaffolds. In the present study we examined whether the cartilage formation is dependent on state of chondrocyte maturation and whether the pretreatment of chondrocytes with growth factors has an influence on the cartilage formation. Scaffolds were manufactured from 80% PLG with a 75:25 lactide:glycolide ratio and 20% modified PLG with a 50:50 lactide:glycolide ratio (PLG-H scaffolds). For each experimental group, four nude mice received two identical implants, one in each calf muscle resulting in an N = 8 implants: PLG-H scaffolds alone; PLG-H scaffolds with cells derived from either the femoral articular cartilage, costochondral periochondrium, or costochondral resting zone cartilage of 125 g male Sprague-Dawley rats; PLG-H scaffolds with either articular chondrocytes or resting zone chondrocytes that were pretreated with 37.5 ng/ml rhPDGF-BB for 4 h or 24 h before implantation, or with perichondrial cells treated with PDGF-BB plus 0.22 ng/ml rhTGF beta-1 for 4 h and 24 h. At 4 or 8 weeks after implantation, samples were harvested and analyzed histomorphometrically for new cartilage formed, area of residual implant and area of fibrous connective tissue. Only resting zone cells showed the ability to form new cartilage at a heterotopic site in this study. There was no neocartilage found in nude mice with implants loaded with either articular chondrocytes or perichondrial cells. Pretreatment of resting zone chondrocytes for 4 h prior to implantation significantly increased the amount of newly formed cartilage after 8 weeks and suppressed chondrocyte hypertrophy. The amount of fibrous connective tissue around implants containing either articular chondrocytes or perichondrial cells decreased with time, whereas the amount of fibrous connective tissue around implants containing resting zone chondrocytes pretreated with PDGF-BB was increased. The results showed that resting zone cells can be successfully incorporated into biodegradable porous PLG scaffolds and can induce new cartilage formation in a nonweight-bearing site. Articular chondrocytes as well as perichondrial cells did not have the capacity for neochondrogenesis when implanted heterotopically in this model.

Animals↗

PubMed2000

Pretreatment with platelet derived growth factor-BB modulates the ability of costochondral resting zone chondrocytes incorporated into PLA/PGA scaffolds to form new cartilage in vivo.

Optimal repair of chondral defects is likely to require both a suitable population of chondrogenic cells and a biodegradable matrix to provide a space-filling structural support during the early stages of cartilage formation. This study examined the ability of chondrocytes to support cartilage formation when incorporated into biodegradable scaffolds constructed from copolymers (PLG) of polylactic acid (PLA) and polyglycolic acid (PGA) and implanted in the calf muscle of nude mice. Scaffolds were fabricated to be more hydrophilic (PLG-H) or were reinforced with 10% PGA fibers (PLG-FR), increasing the stiffness of the implant by 20-fold. Confluent primary cultures of rat costochondral resting zone chondrocytes (RC) were loaded into PLG-H foams and implanted intramuscularly. To determine if growth factor pretreatment could modulate the ability of the cells to form new cartilage, RC cells were pretreated with recombinant human platelet derived growth factor-BB IPDGF-BB) for 4 or 24 h prior to implantation. To assess whether scaffold material properties could affect the ability of chondrogenic cells to form cartilage, RC cells were also loaded into PLG-FR scaffolds. To determine if the scaffolds or treatment with PDGF-BB affected the rate of chondrogenesis, tissue at the implant site was harvested at four and eight weeks post-operatively, fixed, decalcified and embedded in paraffin. Sections were obtained along the transverse plane of the lower leg, stained with haematoxylin and eosin, and then assessed by morphometric analysis for area of cartilage, area of residual implant, and area of fibrous connective tissue formation (fibrosis). Whether or not the cartilage contained hypertrophic cells was also assessed. The amount of residual implant did not change with time in any of the implanted tissues. The area occupied by PLG-FR implants was greater than that occupied by PLG-H implants at both time points. All implants were surrounded by fibrous connective tissue, whether they were seeded with RC cells or not. The amount of fibrosis was reduced at eight weeks for both implant types. When RC cells were present, the amount of fibrosis was less than seen in cell-free scaffolds. Pretreatment with PDGF-BB caused a slightly greater degree of fibrosis at four weeks than was seen if untreated cells were used in the implants. However, at eight weeks, if the cells had been exposed to PDGF-BB for 24 h, fibrosis was comparable to that seen associated with cell-free scaffolds. The cells supported an equivalent area of cartilage formation in both scaffolds. PDGF-BB caused a time-dependent decrease in cartilage formation at four weeks, but at eight weeks, there was a marked increase in cartilage formation in PDGF-BB-treated cells that was greatest in cells exposed for 4 h compared to those exposed for 24 h. Moreover, PDGF-BB decreased the formation of hypertrophic cells. The results indicate that in this model, RC cells produce cartilage; pretreatment of the RC cells with PDGF-BB promotes retention of a hyaline-like chondrogenic phenotype; and the material properties of the implant do not negatively impact on the ability of the cells to support chondrogenesis.

Animals↗

PubMed2000

Phagocytosis of wear debris by osteoblasts affects differentiation and local factor production in a manner dependent on particle composition.

Wear debris is considered to be one of the main factors responsible for aseptic loosening of orthopaedic endoprostheses. Whereas the response of cells in the monocytic lineage to foreign materials has been extensively studied, little is known about cells at the bone formation site. In the present study, we examined the hypothesis that the response of osteoblasts to wear debris depends on the chemical composition of the particles. We produced particles from commercially pure titanium (cpTi), Ti-6Al-4V (Ti-A), and cobalt-chrome (CoCr) and obtained ultrahigh molecular weight polyethylene (UHMWPE; GUR 4150) particles from a commercial source. The equivalent circle diameters of the particles were comparable: 1.0 +/- 0.96 microm for UHMWPE; 0.84 +/- 0.12 microm for cpTi; 1.35 +/- 0.09 microm for Ti-A, and 1.21 +/- 0.16 microm for CoCr. Confluent primary human osteoblasts and MG63 osteoblast-like cells were incubated in the presence of particles for 24 h. Harvested cultures were examined by transmission electron microscopy to determine if the cells had phagocytosed the particles. Particles were found intracellularly, primarily in the cytosol, in both the primary osteoblasts and MG63 cells. The chemical composition of the particles inside the cells was confirmed by energy-dispersive X-ray analysis. Morphologically, both cell types had extensive ruffled cell membranes, less-developed endoplasmic reticulum, swollen mitochondria, and vacuolic inclusions compared with untreated cells. CpTi, Ti-A, and CoCr particles were also added to cultures of MG63 cells to assess their effect on proliferation (cell number) and differentiation (alkaline phosphatase activity), and PGE2 production. All three types of particles had effects on the cells. The effect on cell number was dependent on the chemical composition of the particles; Ti-A and CoCr caused a dose-dependent increase, while cpTi particles had a biphasic effect with a maximal increase in cell number observed at the 1:10 dilution. Alkaline phosphatase specific activity was also affected and cpTi was more inhibitory than Ti-A or CoCr. PGE2 production was increased by all particles, but the magnitude of the effect was particle-dependent: CoCr > cpTi > Ti-A. This study demonstrates clearly that human osteoblast-like cells and MG63 cells can phagocytose small UHMWPE, CoCr, Ti-A, and cpTi particles. Phagocytosis of the particles is correlated with changes in morphology, and analysis of MG63 response shows that cell proliferation, differentiation, and prostanoid production are affected. This may have negative effects on bone formation adjacent to an orthopaedic implant and may initiate or contribute to the cellular events that cause aseptic loosening by inhibiting bone formation. The effects on alkaline phosphatase and PGE2 release are dependent on the chemical composition of the particles, suggesting that both the type and concentration of wear debris at an implant site may be important in determining clinical outcome.

Aged↗

PubMed2000

Re-use of implant coverscrews changes their surface properties but not clinical outcome.

This study examined the effect of re-using coverscrews for dental implants (Brånemark) and the influence of re-use on clinical outcome. Nine patients, each receiving 3 implants in either the maxilla or the mandible, received 1 new coverscrew, 1 re-used coverscrew, and a third coverscrew that had been used multiple times. In all cases, the re-used coverscrews had been washed, mechanically cleaned, and steam-sterilized prior to re-implantation. Clinical outcome was assessed by X-ray analysis of the mandible/maxilla and light microscopy of histologically prepared sections of the overlying tissue. The surfaces of the coverscrews were characterized by profilometry, scanning electron microscopy (SEM), Auger electron spectroscopy (AES), and atomic force microscopy (AFM). There was no difference in clinical outcome whether the coverscrews were new or re-used multiple times. Histological evaluation showed no influence of re-use on the overlying epithelial and connective tissues at the time the coverscrew was removed. Surface topography and roughness changed with increasing number of uses, but surface chemistry was virtually unchanged. SEM and AFM analyses revealed the presence of machining marks, as well as deep scratches, across the surface of the re-used coverscrews. This study shows that coverscrews can be cleaned and re-used without any apparent adverse affect on clinical outcome. However, the cleaning procedures, as well as the surgical procedure, change the surface characteristics. If this approach were applied to the implant itself, it might affect osseointegration.

Adult↗

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

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↗

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↗