Differential effects of bone graft substitutes on regeneration of bone marrow.

BACKGROUND: This study used a rat tibial marrow ablation model to test the hypothesis that bone remodeling within the medullary canal varies with bone graft materials of different chemical compositions and structural properties, impacting marrow cavity restoration. Bone graft materials were selected based on their relative resorption or degradation in vivo and their osteogenic properties. METHODS: Following ablation of the right tibial marrow in male Sabra-strain rats, materials were implanted in the proximal marrow cavity: poly-D,L-lactide-co-glycolide 75 : 25 (PLGA); coralline-hydroxyapatite (HA), calcium-sulfate (CaSO4), collagen-HA-tricalcium phosphate granules, anorganic bovine bone mineral, demineralized bone matrix (DBM), 45S5 Bioglass (BG), PLGA with BG 50 : 50, PLGA : BG 80 : 20, and PLGA and PLGA:BG 50 : 50 plus bone marrow (BM). Control tibias were ablated but received no implants. At 2 (endosteal bone healing), 4 (marrow cavity remodeling) and 8 weeks (marrow restoration), six to eight animals per group were euthanized and tibias processed for histomorphometry of proximal and distal medullary canals. RESULTS: Control tibias showed primary bone in proximal and distal medullary canals at 2 weeks, with trabeculae surrounded by cellular marrow. At 4 and 8 weeks, control trabeculae were thinned and marrow had more fat cells. In the treated tibias, trabecular bone volume (TBV) varied with time and was material specific. Most implants supported comparable TBV at 2 weeks. Sites with CaSO4 or DBM exhibited decreased TBV with time whereas trabecular bone was retained in proximal tibias containing other materials, closely juxtaposed to the implants. TBV did not always correlate directly with implant volume, but changes in BM volume were correlated inversely with TBV. Addition of BM increased marrow restoration in sites containing PLGA; however, BM reduced restoration of marrow when added to PLGA : BG. Although the presence of implants in the proximal tibia resulted in retention of trabecular bone, there was a time-dependent reduction in TBV in distal canals; the rate and extent of the distal TBV reduction were implant dependent. CONCLUSIONS: Thus, although many materials can support bone formation in the marrow cavity, bone quality, quantity, and physical relationship to the implant, and its rate of resorption differ in a material-dependent manner, resulting in differences in the restoration of marrow. CLINICAL RELEVANCE: Bone graft materials should be selected not only for their ability to support new bone formation but also for their impact on the remodeling phase of bone healing.

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

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

Growth-plate chondrocytes respond to 17beta-estradiol with sex-specific increases in IP3 and intracellular calcium ion signalling via a capacitative entry mechanism.

17Beta-estradiol (E(2)) regulates growth-plate chondrocyte differentiation in a gender and cell maturation-dependent manner via classic nuclear receptors ERalpha and ERbeta, and membrane-associated signalling. Here we show that sex-specific effects of E(2) involve changes in intracellular calcium concentration (ICCC). Resting-zone chondrocytes (RC) and growth-zone chondrocytes (GC) were isolated from costochondral cartilage of male and female rats. Confluent cultures were treated with 10(-8)M E(2) or 17alpha-estradiol in the presence of high and low extracellular Ca(2+) concentration. The ICCC was determined using laser scanning confocal microscopy to measure changes in Fluo-4 fluorescence every 5s for a total of 500s. E(2) increased ICCC in the cells from female rats but had no effect on ICCC in male cells. The effect was rapid (peak at 140s) and stereospecific. E(2) increased ICCC in RC and GC chondrocytes but the effect was greater in RC cells. Low Ca(2+) media did not abolish the E(2)-dependent ICCC elevation, nor did inclusion of verapamil, which inhibits Ca(2+) channels on the cell membrane. Thapsigargin reduced the effect of E(2) on ICCC, showing that Ca(2+) pumps on the endoplasmic reticulum were involved. Pre-treatment of the cells with the ER antagonist ICI 182780 did not alter the stimulatory effect of E(2), suggesting that traditional estrogen receptor mechanisms do not play a role. E(2) caused rapid production of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) but only in female cells, and the effect was greater in RC chondrocytes. These results indicate that E(2) regulates ICCC in a sex-specific and cell maturation state-dependent manner. The mechanism is membrane-associated and is mediated by PLC-dependent IP3 production and release of Ca(2+) from the endoplasmic reticulum.

Gender-specific, maturation-dependent effects of testosterone on chondrocytes in culture.

This study examined the effects of testosterone on chondrocytes in vitro in order to determine whether the effects of testosterone were dependent on the stage of chondrocyte maturation and gender specific. Cells derived from male or female rat costochondral growth zone and resting zone cartilage were used as the cell culture model. [3H]Thymidine incorporation, cell number, alkaline phosphatase specific activity, and percent collagen production were used as indicators. Alkaline phosphatase specific activity in matrix vesicles and plasma membranes isolated from male and female chondrocyte cultures was measured to determine which membrane fraction was targeted by the hormone. The role of fetal bovine serum in the culture medium was also addressed. The results demonstrated that testosterone decreases cell number and [3H]thymidine incorporation in male chondrocytes, suggesting that it may promote differentiation of these cells. Alkaline phosphatase specific activity is stimulated in growth zone cells, with no effect on resting zone cells. The increase in enzyme activity is targeted to the matrix vesicles. Cells cultured in serum-free medium exhibit a dose-dependent inhibition of alkaline phosphatase activity when cultured with testosterone, even in the presence of phenol red. Testosterone-dependent stimulation of enzyme activity is seen only in the presence of serum, suggesting that serum factors are also necessary. Testosterone increased the percent collagen production in male cells only, regardless of the cartilage zone of origin. The results of this study indicate that the effects of testosterone are dependent on the time of exposure, presence of serum, and sex and stage of maturation of the chondrocytes. Testosterone-dependent stimulation of alkaline phosphatase specific activity is targeted to matrix vesicles.

Tumor necrosis factor alpha and interferon gamma--induced cell growth arrest is mediated via insulin-like growth factor binding protein-3.

Retinoic acid (RA), and the combination of TNFalpha and Interferon (IFN-gamma) inhibit human salivary gland tumor (HSG) cell growth with the combination of all three being even more inhibitory (P<0. 05). Previous studies have demonstrated that these inhibitory effects of RA, and the combination of TNFalpha and IFN-gamma are associated with increased accumulation of IGFBP-3 in the culture medium of HSG cells. Therefore, we set out to determine if the increase in IGFBP-3 was due to increased production of IGFBP-3 by the cells and whether IGFBP-3 played a causative role in the inhibition of cellular proliferation. TNFalpha and IFN-gamma induced a rise in IGFBP-3 mRNA levels between 4 and 8 h, which returned to control levels after 24 h. IGFBP-3 was shown to inhibit HSG cell growth at concentrations of >/=75 U (P<0.05). When antibodies to IGFBP-3 were used with TNFalpha and IFN-gamma, the inhibitory effect of the cytokines on cell growth was diminished. Retinoic acid with TNFalpha and IFN-gamma had a marked inhibitory effect (P<0.05) which was similarly reversed by increasing concentrations of IGFBP-3 antibody. The present data support the hypothesis that the combination of TNFalpha and IFN-gamma with retinoic acid exert their anti-proliferative effect on HSG cells by reducing the mitogenic effect of IGF-I due to a shift in IGF-I from the free to the IGFBP-3-bound form.

Coverage of previously carious roots is as predictable a procedure as coverage of intact roots.

BACKGROUND: Coverage of roots exposed by gingival recession is one of the main objectives of periodontal reconstructive surgery. A variety of highly predictable and esthetically acceptable mucogingival grafting procedures are available for treating intact root surfaces. One of the indications for root coverage procedures is prevention of root caries. However, little is known about the ability to treat previously carious roots by mucogingival grafting, and even less is known about the outcome of such treatment. METHODS: Sixty patients with gingival recession were treated with subepithelial connective tissue grafts. During initial examination, 33 intact teeth with longstanding gingival recessions of 4.09 +/- 0.16 mm (mean +/- SEM) and 27 teeth with carious roots (restored and unrestored) and longstanding gingival recessions of 3.44 +/- 0.22 mm (mean +/- SEM) were found. Prior to grafting, any carious dentin and plastic restorations were removed. The exposed roots were thoroughly planed and covered by a subepithelial connective tissue graft without any further root treatment or conditioning. Patients were evaluated periodically between 1 and 6 years. Residual recession, defect coverage, and caries incidence were assessed. RESULTS: Recession was reduced to a similar extent on all teeth whether root caries was present (0.31 +/- 0.11 mm) or not (0.15 +/- 0.04 mm). This resulted in comparable defect coverages of 92.41 +/- 2.38% for previously carious roots and 97.46 +/- 0.79% for intact roots. In 44 cases, complete coverage of the recession was achieved. The results were stable, and no further recessions or recurrent caries were found during the periodic evaluations. CONCLUSIONS: These results indicate that coverage of previously carious roots with subepithelial connective tissue grafts is a very predictable procedure with results similar to those found on intact roots. This procedure may provide a definitive biological alternative for conventional restorative treatment modalities for root caries. Moreover, the results are more esthetic, biologically acceptable, and maintainable.

Bell's palsy associated with herpes simplex gingivostomatitis. A case report.

Bell's palsy is a sudden, isolated, peripheral facial paralysis caused by various known and sometimes unknown factors. The case of an 18-year-old man who developed Bell's palsy after onset of primary herpetic gingivostomatitis is presented. Although Bell's palsy has already been associated with herpes simplex virus type 1, the described case is the first in the literature in which enzyme-linked immunosorbent assays for immunoglobulin G to herpes simplex virus type 1 and herpes simplex virus type 1 culture were both positive. The recent literature regarding the possible relationship between herpes simplex virus type 1 and Bell's palsy is reviewed and discussed.

[The role of surface roughness in promoting osteointegration].

Scientific evidence that has been gathered in the past 20 years established that certain endosseous dental implants--primarily screw-type implants made of commercially pure titanium can be successfully utilized as anchorage for dental prostheses. In recent years, an effort has been made to simplify the surgical procedure, in order to modify clinical treatment modalities. One of the trends is to increasingly utilize microrough titanium implants. Roughened implant surfaces have a long history in implant dentistry, and the most prominent surface is titanium plasma-sprayed (TPS). In recent years new implant surfaces have emerged, so-called microrough titanium surfaces produced with reducing techniques such as grit-blasting with Al2O3 or TiO2 particles, sandblasting and acid-etching, or acid-etching alone. These different titanium surfaces have been tested in numerous in-vivo studies utilizing different animal models. Summarizing the results of these studies, it can be concluded that there is currently sufficient evidence that titanium implants with a microrough surfaces achieve a faster bone integration, a higher percentage of Bone implant Contact (BIC), and a higher resistance to shear documented with higher Removal Torque Values (RTV) when compared with titanium implants with a polished or machined surface. In order to understand the mechanism through which surface roughness modulates its effects mentioned above, recent studies used in-vitro experimental methods to study cell response to implant surface topography. These studies have shown that osteoblasts are sensitive to surface roughness, exhibiting decreased proliferation and a more differentiated phenotype on rougher surfaces. PGE2 production is enhanced on rough surfaces, as is the production of TGF beta 1, suggesting that surface roughness can mediate autocrine and paracrine regulation of osteogenesis. Moreover, surface roughness was found to modulate the effect of systemic hormones like 1,25-(OH)2D3 on osteoblasts. The clinical advantages of implants with rough surface were observed in recently conducted clinical trials. It was found, in humans, that roughened titanium implants need shorter healing period before loading, 6-8 (SLA and Osseotite respectively) weeks instead of 12 weeks. The clinical advantages of shorter healing periods are obvious. Moreover, it was found that certain roughened implants can be used in shorter sizes (6-8 mm) then accepted today. The utilization of shorter implants offers the avoidance of extensive surgical procedures such as nerve lateralization in the mandible or sinus grafting in the maxilla. However, sufficient long term documentation is still lacking, and the predictability of such modalities has yet to be examined in long term prospective clinical trials.

[The role of Matrix Metaloproteinases in the progression of periodontitis, and the use of specific inhibitors to these enzymes in the treatment of the periodontal disease].

Matrix Metaloproteinases (MMPs) play a crucial role in the pathogenesis of the periodontal disease. This group of enzymes can degrade most of the proteins that compose the extracellular matrix. Recently a new concept of treatment, the "host modulating therapy" (HMT) was developed. The rationale behind this concept is to inhibit the production of the inflammatory mediators and/or to block the production of the host MMPs and thus inhibiting disease production. In the recent years it was found that Tetracyclines (TC) decrease the levels of TNF-alpha, IL-1, Nitric Oxide, and prostaglandins, and via intra and extra cellular mechanisms inhibit the production and activation of the MMPs. A capsule of 20 mg Doxycycline, called later Low Dose Doxycycline (LDD), was the first HMT in the periodontal disease. In a 9 month study it was found that LDD bid after supragingival scaling statistically increased pocket reduction and attachment gain compared to supragingival scaling alone. However, this improvement was found only in pockets of 7 mm or more, and was not compared yet to periodontal supportive therapy every three month. There is a need for more basic and clinical research in the field of HMT in the treatment of periodontal disease, to expand our knowledge and clinical experience that will enable us to decide when and how to use medications to modulate host response.

Sexual dimorphism of growth plate prehypertrophic and hypertrophic chondrocytes in response to testosterone requires metabolism to dihydrotestosterone (DHT) by steroid 5-alpha reductase type 1.

Rat costochondral growth plate chondrocytes exhibit sex-specific and cell maturation dependent responses to testosterone. Only male cells respond to testosterone, although testosterone receptors are present in both male and female cells, suggesting other mechanisms are involved. We examined the hypothesis that the sex-specific response of rat costochondral cartilage cells to testosterone requires further metabolism of the hormone to dihydrotestosterone (DHT). Resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) chondrocytes from male and female Sabra strain rats exhibited sex-specific responses to testosterone and DHT: only male cells were responsive. Testosterone and DHT treatment for 24 h caused a comparable dose-dependent increase in [3H]-thymidine incorporation in quiescent preconfluent cultures of male GC cells, and a comparable increase in alkaline phosphatase specific activity in confluent cultures. RC cells responded in a differential manner to testosterone and DHT. Testosterone decreased DNA synthesis in male RC cells but DHT had no effect and alkaline phosphatase specific activity of male RC cells was unaffected by either hormone. Inhibition of steroid 5alpha-reductase activity with finasteride (1, 5, or 10 microg/ml), reduced the response of male GC cells to testosterone in a dose-dependent manner, indicating that metabolism to DHT was required. RT-PCR showed that both male and female cells expressed mRNAs for steroid 5alpha-reductase type 1 but lacked mRNAs for the type 2 form of the enzyme. Male cells also exhibited 5alpha-reductase activity but activity of this enzyme was undetectable in female cells. These observations show that sex-specific responses of rat growth zone chondrocytes to testosterone requires the further metabolism of the hormone to DHT and that the effect of DHT in the male growth plate is maturation-state dependent. Failure of female chondrocytes to respond to testosterone may reflect differences in testosterone metabolism, since these cells possess greater ability to aromatize the hormone to estradiol.

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.

Transforming growth factor-beta1 modulates chondrocyte responsiveness to 17beta-estradiol.

This study examined the interrelationship between transforming growth factor-beta1 (TGF-beta1) and 17beta-estradiol (E2) in the regulation of growth plate chondrocytes. To determine whether TGF-beta1 modulates chondrocyte response to E2, we used cells isolated from the resting zone (RC) and growth zone (GC) of costochondral cartilage. Confluent, fourth-passage cultures were pretreated with rhTGF-beta1 for 24 h, followed by treatment with E2 for 24 h. The effect of TGF-beta1 and E2 alone, or the sequential combination, were examined by measuring [3H]-thymidine incorporation (proliferation), alkaline phosphatase (AP) specific activity (differentiation), and [35S]-sulfate incorporation (matrix synthesis). TGF-beta1 alone increased [3H]-thymidine incorporation in both female and male RC and GC cells, but E2 affected this parameter only in RC cells, causing a dose-dependent decrease. At the highest concentration of TGF-beta1 and E2, [3H]-thymidine incorporation in female GC cells was the same as seen in untreated control cultures. In male GC cells, [3H]-thymidine incorporation in cultures treated with TGF-beta1 and E2 exhibited a comparable increase, as was seen in cultures treated with TGF-beta1 alone. TGF-beta1 caused a biphasic stimulation in AP that was maximal at 0.22 ng/mL, in both female and male RC and GC cells. E2, however, affected only female cells. Whereas the effect of TGF-beta1 predominated in RC and GC male cells, the biphasic stimulation caused by E2, maximal at 109 M, predominated in female RC cells. In female GC cells, however, TGF-beta1 caused a synergistic response, resulting in enhanced AP specific activity in cultures pretreated with 0.22 ng/mL of TGF-beta1 and 10(-8) M E2. TGF-beta1 alone caused dose-dependent increases in [35S]-sulfate incorporation in female RC and GC cells, as well as in male GC cells, but had no effect on male RC cells. E2 affected only female cells. TGF-beta1 potentiated the effect of E2 on this parameter, resulting in synergistic increases in the female cells. This is the first demonstration of a gender-specific response to TGF-beta1 in chondrocytes. These results suggest that chondrocyte response to a systemic hormone such as E2 can be modulated by local regulatory agents such as TGF-beta1.

Sex-dependent effects of 17-beta-estradiol on chondrocyte differentiation in culture.

This study examined the effects of 17-beta-estradiol (E2) on chondrocyte differentiation in vitro. Cells derived from male or female rat costochondral growth zone and resting zone cartilage were used to determine whether the effects of E2 were dependent on the stage of chondrocyte maturation and whether they were sex-specific. [3H]-Thymidine incorporation, cell number, alkaline phosphatase specific activity, and percent collagen production were used as indicators of differentiation. Alkaline phosphatase specific activity in matrix vesicles and plasma membranes isolated from female chondrocyte cultures was measured to determine which membrane fraction was targeted by the hormone. Specificity of the E2 effects was assessed using 17-alpha-estradiol. The role of fetal bovine serum and phenol red in the culture medium was also addressed. The results demonstrated that E2 decreases cell number and [3H]-thymidine incorporation in female chondrocytes, indicating that it promotes differentiation of these cells. Alkaline phosphatase specific activity is stimulated in both growth zone and resting zone cells, but the effect is greater in the less mature resting zone chondrocytes. The increase in enzyme activity is targeted to the matrix vesicles in both cell types, but the fold increase is greater in the growth zone cells. In male chondrocytes, there was a decrease in [3H]-thymidine incorporation at high E2 concentrations in resting zone cells at the earliest time point examined (12 hours) and a slight stimulation in alkaline phosphatase activity in growth zone cells at 24 hours. Cells cultured in serum-free medium exhibited a dose-dependent inhibition in alkaline phosphatase activity when cultured with E2, even in the presence of phenol red. E2-dependent stimulation of enzyme activity is seen only in the presence of serum, suggesting that serum factors are also necessary. E2 increased percent collagen production in female cells only; the magnitude of the effect was greatest in the resting zone chondrocyte cultures. The results of this study indicate that the effects of E2 are dependent on time of exposure, presence of serum, and the sex and state of maturation of the chondrocytes. E2-dependent stimulation of alkaline phosphatase specific activity is targeted to matrix vesicles.

The effects of 17 beta-estradiol on chondrocyte differentiation are modulated by vitamin D3 metabolites.

Both 17 beta-estradiol (17 beta) and the vitamin D metabolites, 1,25-(OH)2D3(1,25) and 24,25-(OH)2D3(24,25), regulate endochondral bone formation in vivo and in vitro. The effects of 17 beta are sex-specific and cell maturation-dependent. Similarly, the effects of 1,25 and 24,25 are cell maturation-dependent, with 1,25 affecting growth zone chondrocytes (GC) and 24,25 affecting resting zone chondrocytes (RC). This study examined whether the response of chondrocytes to 17 beta is altered after pretreatment with 1,25 or 24,25. Cells were isolated from the costochondral cartilage of male or female rats. Confluent, fourth-passage GC and RC cultures were pretreated with 1,25 or 24,25, respectively, for 24 or 48 h followed by treatment with 17 beta for an additional 24 h. At harvest, cell proliferation ([3H]-thymidine incorporation), differentiation (alkaline phosphatase specific activity [ALPase]), general metabolism ([3H]-uridine incorporation), and proteoglycan production ([35S]-sulfate incorporation) were determined. 1,25 enhanced the inhibitory effect of 17 beta on [3H]-thymidine incorporation by female GC cells; in contrast, no effect was observed in GC cells obtained from male rats. When male RC cells were treated with 17 beta, [3H]-thymidine incorporation was inhibited; however, when these cells were pretreated with 24,25 for 48 h, 17 beta stimulated [3H]-thymidine incorporation 24,25 had no effect on 17 beta-dependent [3H]-thymidine incorporation by female RC cells. 17 beta stimulated ALPase in female GC cells, but had no effect on male GC cells. 1,25 pretreatment of female GC cells inhibited the stimulatory effect of 17 beta on ALPase, but had no effect on ALPase in male GC cultures. 17 beta had no effect on male RC cell ALPase and stimulated ALPase in female RC cells. This was not affected by pretreatment with 24,25. Pretreatment with 1,25 increased the basal level of sulfate incorporation only in female GC. No effect was found in RC cells. These results indicate that pretreatment of rat costochondral chondrocytes with vitamin D metabolites modulate the effect of 17 beta. Although the effect of vitamin D metabolites alone on these chondrocytes is maturation-dependent and not sex-specific, the influence of preincubation with vitamin D metabolites on the effect of 17 beta is hormone-specific, sex-specific, and maturation-dependent.

Evidence for receptors specific for 17 beta-estradiol and testosterone in chondrocyte cultures.

Recently, sex hormones were shown to stimulate chondrocyte differentiation and matrix protein synthesis in vitro in a sex-specific and maturation-dependent manner. The aim of the present study was to determine whether cytosolic receptors in these cells would specifically bind 17 beta-estradiol and testosterone, and if so, whether binding was gender- and maturation-dependent. Confluent, fourth passage cultures of cells derived from male or female rat costochondral growth zone and resting zone cartilage were homogenized and specific binding of 17 beta-estradiol or testosterone measured in the cytosolic fraction. Scatchard analysis indicated the presence of a high-affinity 17 beta-estradiol receptor (Kd = 4.5 to 8.7 x 10(-11) M), with low binding capacity (3.9 to 11.2 fmol/mg protein). Chondrocytes from female rats were found to have a significantly greater binding capacity for 17 beta-estradiol than chondrocytes from male rats. However, cells from both sexes had binding capacities that were independent of cell maturation. A high-affinity testosterone receptor (Kd = 4.3 to 6.3 x 10(-11) M) with low binding capacity (4.1 to 5.9 fmol/mg protein) was found in both males and females, but no difference in binding capacity was noted, either as a function of gender or stage of cell maturation. Immunohistochemistry using antibodies against 17 beta-estradiol and testosterone and the 17 beta-estradiol nuclear receptor (D-75) confirmed that 17 beta-estradiol and testosterone receptors were present in chondrocytes from both male and female rats. These data demonstrate that chondrocytes from growth zone and resting zone cartilage are capable of binding both 17 beta-estradiol and testosterone. This suggests that these hormones mediate their direct effects on chondrocytes via receptors specific for their appropriate ligand. The sex-specific effects of 17 beta-estradiol may be due to differences in receptor number between chondrocytes derived from female and male rats. In contrast, the sex-specific effects of testosterone may be regulated at the post receptor level since no differences in binding capacity were found between males and females.