Modulation of endothelial cell phenotype by cyclic stretch: inhibition of collagen production

Endothelial cells (EC) are constantly subjected to pulsatile stretch in vivo, but most studies of EC are performed under stationary tissue culture conditions. The aim of this study was to examine the effect of repetitive mechanical stretching on EC collagen production. Bovine EC were seeded in 35-mm flexible-bottom culture wells, allowed to attach for 24 hr, and then subjected to up to 24% elongation cyclic deformation, 3 cycles/min, 10 sec of elongation alternating with 10 sec of relaxation, for 5 days. Twenty-four hours prior to harvesting, serum-free media containing 50 microCi [3H]proline (PRO), an amino acid hydroxylated (OH) in collagen, and 50 micrograms/ml ascorbate were added per well. On Days 1, 3, and 5, the media and cells were collected, precipitated with TCA, sedimented, lyophilized, and analyzed by HPLC for OH-PRO and PRO. The results of this study indicate that EC production of collagen is inhibited with repetitive deformation. Since previous studies have shown that EC proliferation is increased after 3 cycles/min stretching, this supports the theory that collagen gene expression varies inversely with the proliferative state.     

Response of porcine aortic smooth muscle cells to cyclic tensional deformation in culture

The vascular wall is continuously subjected to pulsatile hydrostatic pressures. However, most studies of smooth muscle cells (SMC) are done under static tissue culture conditions. We have utilized a flexible-bottom culture plate and applied cyclic tensional deformation to cultured SMC. Recent studies have shown that endothelial cells (EC) proliferate but remain randomly oriented when subjected to a force regimen of 3 cycle/min of up to 24% elongation. The current experiments were performed to study the effects of a similar force regimen on SMC. Porcine SMC were seeded at 200,000 cells/35-mm2 well and permitted to adhere for 24 hr. After 24 hr, the plates were placed on a vacuum-operated stress-providing unit that exerted a 24% elongation on the culture plate substratum at maximum downward deflection of the plate bottom. The stretched cells (n = 6 wells/day) were subjected to cycles of 10 sec of 24% elongation and 10 sec of relaxation for 7 days. The control cells (n = 6 wells/day) were subjected to similar incubations as the control group but without cyclic deformation. Media were changed every 24 hr. [3H]Thymidine (THY), a precursor for DNA synthesis, was added (2 microCi/well) to plates 24 hr prior to harvesting. On Days 0, 1, 3, 5, and 7 cells were counted and analyzed for THY incorporation. While previous results indicate that pulsatile stretching stimulates EC, the present study indicates that a similar regimen of stress inhibits SMC proliferation. THY incorporation into SMC paralleled the observed changes in cell count. Morphologically, SMC aligned in an annular pattern in response to applied strain, whereas EC maintained a random orientation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical stress stimulates aortic endothelial cells to proliferate

The effects of applied cyclic tensional deformation and relaxation on cultured bovine aortic endothelial cells were examined. Endothelial cells from passages 3 to 9 were seeded in flexible-bottomed plates and allowed to attach for 24 hours. Endothelial cells in the experimental group (n = 6 wells per time point) were placed in a vacuum-operated stress-providing instrument that exerted an average elongation of 10% at maximum downward deflection of the culture plate bottom. The stretched endothelial cells were subjected to repeating cycles of 10 seconds elongation and 10 seconds relaxation from days 1 through 7 in culture. Endothelial cells in the control group (n = 6 wells per time point) were subjected to similar incubation conditions as the experimental group but without tensional deformation. Tritiated thymidine was added to cells 24 hours before harvesting. On days 0, 1, 3, 5, and 7 cells were counted and analyzed for trichloroacetic acid-precipitable tritiated thymidine incorporation. The results showed that 3 cycles/min mechanical stretching stimulated deoxyribonucleic acid synthesis and endothelial cell division. We conclude that cyclic tensional deformation may stimulate endothelial cell proliferation. It is possible that naturally occurring cyclic mechanical deformation in vivo, such as the repetitive stretching and relaxation of aortic tissue by the heart, may invoke a particular pattern of synthesis and division in endothelial cells.     

Prostacyclin synthetic activity in cultured aortic endothelial cells undergoing cyclic mechanical deformation

The effect of mechanical stretching on prostacyclin (PGI2) synthesis was studied by growing bovine aortic endothelial cells on flexible-bottomed culture plates that could be deformed by vacuum. A stress unit was used to exert an elongation of 24% at maximum downward deflection of the culture plate bottom. The experimental group was subjected to cycles of 10 seconds of elongation, 10 seconds of relaxation for 1, 3, or 5 days. The control group was subjected to similar incubations but without cyclic stretch. Twenty-four hours before collection, the medium was replaced with new medium that was devoid of serum. On days 1, 3, and 5, the 24-hour culture medium was collected (basal state). Arachidonic acid (20 mumol/L) was then added to each culture and incubated for 5 minutes at 37 degrees C. The medium was then collected to assess prostacyclin synthetic activity (stimulated state). Media were assayed for PGI2 and thromboxane A2 by radioimmunoassays for 6-keto-PGF1 alpha and thromboxane B2, the respective stable hydrolysis product. The results indicate that cyclic stretching, while not altering the basal production of PGI2, increases PGI2 synthetic capacity in a time-dependent manner. These data suggest that it may be inappropriate to extrapolate the mechanisms of in vivo PGI2 release from studies of endothelial cells in stationary culture.     

Stimulation of adenylate cyclase activity in cultured endothelial cells subjected to cyclic stretch

While vascular endothelial cells are repeatedly stretched by the pulsatile nature of cardiac output, in vitro models traditionally used to study vascular biology involve static culture techniques. We have recently shown that pulsatile stretching of endothelial cells in culture will increase their rates of proliferation and regulate their secretion of macromolecules. The aim of this study was to determine whether membrane adenylate cyclase is involved in intracellular signalling during pulsatile stress. Bovine aortic endothelial cells were seeded on flexible-bottomed culture wells (3 x 10(5) cells/25 mm well) and allowed to attach for 48 hours. The culture wells were placed in a vacuum-operated stress providing instrument and subjected to 0.5 s of 24% strain, 0.5 s relaxation (60 cycles/min) for 0, 1, 3, 5, 7, 10 and 15 minutes (N = 24 wells/time point). Cells were homogenized and a crude membrane preparation (27,000 x g) was assayed for adenylate cyclase under basal and forskolin (100 microM) stimulated conditions. The results indicate that there is a time-dependent increase in both basal and stimulated adenylate cyclase with cyclic deformation and suggest that there may be a "stretch receptor" coupled to adenylate cyclase which can modulate endothelial cell function with hemodynamic changes.     

Combination of pre-operative radiotherapy and surgery suppresses local accumulation of collagen and TGF-beta1 in rats

BACKGROUND: The systemic and local tissue repair responses of radiation in combination with surgery are still unclear. We have studied the effect of fractionated pre-operative radiotherapy with or without subsequent laparotomy on collagen accumulation using a rodent model. MATERIALS AND METHODS: Thirty-two male Sprague-Dawley rats were divided into four groups (eight rats per group): 1) sham radiation and sham laparotomy (control); 2) sham radiation and laparotomy; 3) radiation and sham laparotomy; and 4) radiation followed by laparotomy. Expanded polytetrafluoroethylene (ePTFE) tubes were implanted subcutaneously in the abdominal wall in the radiotherapy field and on the back outside the radiotherapy field day 0. The abdomen (3 cm x 4 cm) was irradiated day 3 (10 Gy) and again day 7 (10 Gy). On day 10, implants were extirpated, laparotomy undertaken in groups 2 and 4 and new ePTFE tubes implanted subcutaneously. The second implants were extirpated on day 20. Implants were analyzed for hydroxyproline, total protein and transforming growth factor-beta1 (TGF-beta1) levels. RESULTS: On day 10, hydroxyproline (P < 0.05) and TGF-beta1 (P < 0.001) were lower in ePTFE tubes in irradiated compared with non-irradiated rats. On day 20, the abdominal ePTFE hydroxyproline remained low (P < 0.001) in animals subjected to laparotomy and pre-operative irradiation while hydroxyproline levels of rats subjected to irradiation only were similar to controls. The effects of radiation on hydroxyproline were confined to the irradiated abdominal area. There was a positive correlation between hydroxyproline and TGF-beta1 levels in the abdominal wall implant day 20 (r = 0.53, P < 0.005). CONCLUSION: A clinically relevant fractionated radiation scheme reduced subcutaneous collagen accumulation pre-operatively and profoundly within the radiation field post-operatively after laparotomy, possibly because of lowered TGF-beta1 levels.     

Integrative articular cartilage repair: dependence on developmental stage and collagen metabolism

OBJECTIVES: The objectives of this research were to determine whether the integrative repair of bovine cartilage explants was dependent on developmental stage, and whether observed differences in integration with developmental stage were related to deposition of newly synthesized collagen and lysyl oxidase-mediated collagen cross-linking. METHODS: Pairs of fetal, newborn calf, and adult bovine cartilage blocks were cultured in partial apposition for 2 weeks in medium supplemented with serum, ascorbate, and [3H]proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength in a single-lap shear configuration. Formation and stabilization of newly synthesized protein and collagen was investigated by determination of [3H]proline and [3H]hydroxyproline in tissue digests and guanidine extracts. RESULTS: Calf cartilage exhibited a relatively high integrative repair phenotype, achieving an adhesive strength that was three--four-fold that of adult or fetal specimens. The low and high integrative repair phenotypes appeared related in part to different levels of collagen biosynthesis, which was approximately four--five-fold higher in calf cartilage samples than in the adult. However, fetal cartilage also exhibited a high level of biosynthesis. The different integrative repair phenotypes were not associated with marked differences in the kinetics of chemical stabilization of newly synthesized collagen, as the proportion of incorporated [3H]proline and newly-formed [3H]hydroxyproline that was resistant to extraction by 4M guanidine-HCl following culture was similar for cartilage from all developmental stages. Integration of calf cartilage appeared to depend on lysyl oxidase-mediated collagen cross-link formation, since inclusion of beta-aminopropionitrile (BAPN) in the culture medium completely eliminated development of adhesive strength. BAPN treatment also increased the percentage of newly synthesized protein in the guanidine extracts from 10% to 36% of the total, and that of newly synthesized collagen from 2% to 20%, while having only slight inhibitory effects on overall protein and collagen biosynthesis. CONCLUSION: The finding that cartilage exhibits enhanced integrative repair at a certain developmental stage suggests that it may ultimately be possible to enhance repair when needed in clinical situations.     

Activation of serum complement inhibits collagen synthesis in fetal rat bone in organ culture

Summary Activation of rabbit serum complement caused a marked reduction in collagen synthesis but a much smaller change in noncollagen protein synthesis in fetal rat calvaria maintained in organ culture. In the periosteum of the fetal rat calvarium, both collagen and noncollagen protein synthesis were reduced, whereas in the central bone, presumably enriched in osteoblasts, only collagen synthesis was inhibited. This large decrease in bone collagen synthesis could not be attributed to enhanced degradation of newly synthesized collagen or its release into the culture medium. Activation of complement also stimulated the production of PGE in fetal rat calvaria. Antagonists of prostaglandin cyclooxygenase decreased prostaglandin synthesis but did not restore collagen synthesis in complement-treated bones, suggesting that complement decreases osteoblast collagen synthesis by a mechanism largely independent of prostaglandin production.     

Activation of serum complement inhibits collagen synthesis in fetal rat bone in organ culture

Activation of rabbit serum complement caused a marked reduction in collagen synthesis but a much smaller change in noncollagen protein synthesis in fetal rat calvaria maintained in organ culture. In the periosteum of the fetal rat calvarium, both collagen and noncollagen protein synthesis were reduced, whereas in the central bone, presumably enriched in osteoblasts, only collagen synthesis was inhibited. This large decrease in bone collagen synthesis could not be attributed to enhanced degradation of newly synthesized collagen or its release into the culture medium. Activation of complement also stimulated the production of PGE in fetal rat calvaria. Antagonists of prostaglandin cyclooxygenase decreased prostaglandin synthesis but did not restore collagen synthesis in complement-treated bones, suggesting that complement decreases osteoblast collagen synthesis by a mechanism largely independent of prostaglandin production.     

Effects of deflazacort on aspects of bone formation in cultures of intact calvariae and osteoblast-enriched cells.

Deflazacort, a synthetic glucocorticoid reported to have bone-sparing properties in vivo, and cortisol were compared for their effects on bone formation in vitro. Deflazacort and cortisol were studied for their effects on DNA and collagen synthesis in cultures of intact fetal rat calvariae and of osteoblast-enriched (Ob) cells from 21- to 22-day-old fetal rat parietal bone. Both steroids were also examined for their effects on skeletal insulin-like growth factor (IGF) I production, which is decreased by cortisol and appears relevant to its mode of action. After 24 h of culture, deflazacort and cortisol had limited effects on the parameters studied, although cortisol at 100 nM decreased [3H]proline incorporation into collagen in intact calvariae. In contrast, after 72 h deflazacort and cortisol at 1-100 nM inhibited the incorporation of [3H]thymidine into DNA and at 100 nM decreased the incorporation of [3H]proline into collagen and noncollagen protein in intact calvariae. Deflazacort and cortisol at 10-1000 nM decreased calvarial collagen degradation to a similar extent. Both steroids had a similar activity, and at 100 nM for 72 h they decreased IGF-I production by calvariae; however, cortisol at 10 nM was somewhat more effective than deflazacort in decreasing IGF-I levels. Deflazacort and cortisol had analogous effects in Ob cell cultures. After 24 h of treatment, deflazacort at 100-1000 nM and cortisol at 10-1000 nM decreased the labeling of DNA, and both steroids at 100-1000 nM caused a similar decrease in [3H]proline incorporation into collagen in Ob cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate

We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.     

Biology of fetal wound healing: collagen biosynthesis during dermal repair.

The rapid restoration of tissue integrity and breaking strength in healing fetal wounds is mainly a function of fetal wound collagen. In this study, the fetal and adult tissue responses to injury were characterized in terms of changes in collagen biosynthesis. Linear wounds and unwounded skin were incubated with radioactive proline, and collagen synthesis was measured as isotope incorporation into collagenase-sensitive protein. Likewise, noncollagen protein synthesis was represented by isotope incorporation into collagenase-resistant protein. Adult wounds demonstrated a preferential stimulation of collagen as compared with noncollagen protein synthesis after wounding. In contrast, both collagen and noncollagen protein synthesis were significantly elevated in the fetus during the first 5 days postwounding. Despite marked increases in fetal wound collagen synthesis above both unwounded fetal skin and adult wound levels, fetal wounds exhibited no evidence of excessive collagen deposition or scar formation after wounding. These findings suggest that the fetal response to tissue injury is a function of the distinctive qualities of fetal fibroblasts associated with the extracellular wound matrix and may involve rapid collagen turnover and degradation.     

Transdermal nicotine patch enhances type I collagen synthesis in abstinent smokers

Cigarette smokers deposit less collagen, expressed as hydroxyproline, in granulation tissue than nonsmokers. We studied the effect of abstinence from smoking and transdermal nicotine patches on deposition of hydroxyproline, proline, type I procollagen, and total proteins. Fifty-four healthy smokers were studied during 10 days of smoking and again from days 10 to 20 following smoking cessation. After the first 10 days of abstinence they were randomized to double-blind treatment with transdermal nicotine patches of 25 mg/day or placebo for a period of 10 days. During this period and during smoking, an expanded polytetrafluoroethylene tube was implanted into the subcutis. Following removal of the implant, total amino acids and peptides were extracted. Hydroxyproline and proline were analyzed by high-pressure liquid chromatography, type I procollagen was analyzed by enzyme-linked immunoassay, and total proteins were determined colorimetrically. In the 39 subjects who complied with the study protocol, abstinence from smoking did not affect the deposition of hydroxyproline, proline, type I procollagen, or total protein in the implants. During abstinence, the type I procollagen level increased by 18% in the transdermal nicotine patches group and decreased by 10% in the placebo group (p<0.05). We conclude that 20 days of abstinence from smoking does not affect collagen deposition in granulation tissue. However, in abstinent smokers, transdermal nicotine patches appears to increase type I collagen synthesis.     

Alterations in aortic endothelial cell morphology and cytoskeletal protein synthesis during cyclic tensional deformation

Previous studies have shown that bovine aortic endothelial cells (ECs) in culture respond to repetitive tensional deformation with an increase in deoxyribonucleic acid synthesis and cell proliferation. This study was designed to determine whether cyclic tensional deformation of ECs in vitro induces different morphologic or protein synthetic responses. ECs from passages 6 through 9 were seeded in 35 mm2 well silicone rubber plates at 2 x 10(5) cells/well and allowed to attach for 24 hours. The experimental group was placed in a vacuum-operated stress-providing device that exerted an elongation of 24% at maximum downward deflection of the culture plate bottom and was subjected to repetitive cycles of 10 seconds of 24% maximum elongation and 10 seconds of relaxation for 5 days. The control group was subjected to similar incubation conditions but without stretch. 35S-methionine (500 muCi/well) was added to the plates 24 hours before harvesting, and two-dimensional gels of the harvested lysates (isoelectric focusing with pH 3 to 10 ampholytes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12.5% gels) were performed and the labeled proteins visualized by autoradiography. The data indicate that there is a differential synthesis of proteins, with synthesis of some proteins decreased or ablated whereas other proteins were increased in response to cyclic mechanical tension. The actin filament organization was evaluated after staining with rhodamine phalloidin, a fluorescent F-actin probe. The ECs subjected to tension had a more polygonal shape and demonstrated pseudopods and actin stress fibers, whereas ECs cultured under static conditions were more rounded and did not express actin stress cables.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of vascularization on avian flexor tendon repair. A biochemical study

Although it has been demonstrated in experimental animals that vascular perfusion functions less effectively than diffusion as a nutrient pathway to the uninjured flexor tendon, it is recognized that the nutritional requirements of an injured flexor tendon may be different. The present study examines several biochemical parameters of avian flexor tendon repair, during a six-week period, in the presence of an intact vinculum longum and with the vinculum longum ligated. Formation of collagen/noncollagen protein, glycosaminoglycan synthesis, and tissue levels of both DNA and hexosamine were quantified. The absence of the vincular blood supply did not affect the levels of total DNA and hexosamine, did not alter the pattern of either protein or glycosaminoglycan formation in the early healing phase, and did not impair the overall synthesis of collagen and noncollagen protein throughout the six-week study period. However, the pattern of collagen and noncollagen protein synthesis differed in lacerated tendons during the period of repair; noncollagen protein synthesis peaked at ten days, while collagen synthesis increased uniformly throughout the 42-day period of study. There was a notable decrease in total matrix hydroxyproline (collagen) during repair, which continued throughout the entire first three weeks after laceration. Thus, the actual percentage of collagen synthesis decreased during the first three weeks of repair and began to return to its normal uninjured level by the sixth week.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of hydroxyproline by high performance liquid chromatography and its application to collagen turnover studies in bone cultures

Summary We describe a high performance liquid chromatography (HPLC) technique for separating and quantitating hydroxyproline in calvarial cultures. Using a reverse-phase Nova-Pak C₁₈ column and a 140 mM sodium acetate, 0.05% triethylamine (TEA), 6% acetonitrile solvent system, we obtained a complete separation of hydroxyproline. Recovery of added standards ranged from 89 to 103% and intraassay variability was <8%. [³H]hydroxyproline measurements were used to examine changes in collagen turnover in rat calvariae labeled with [³H]proline and “chased” in the presence of 10 mM unlabeled proline. The addition of parathyroid hormone (PTH) during a 24–48 hour “chase” period increased the release of acid-soluble [³H]hydroxyproline into the culture medium, indicating an increase of fully degraded collagen. This method offers a sensitive and reproducible technique for monitoring changes in bone matrix degradation and in studying agents that modify this process.     

Comparison of the effects of a potent synthetic analog of bovine parathyroid hormone with native bPTH-(1-84) and synthetic bPTH-(1-34) on bone resorption and collagen synthesis.

An analog of bobine PTH [nle-8, nle-18, tyr-34 bPTH-(1-34) amide, (PTH-Ana)] which is a potent stimulator of renal adenylate cyclase has been compared with the native hormone bPTH-(1-84) and the biologically active amino terminal portion, bPTH-(1-34), for its effects on bone resorption and bone collagen synthesis in organ culture. All three compounds stimulated the release of previously incorporated 45Ca from cultured fetal rat long bone shafts with similar dose-response curves at 10(-9) to 3 X 10(-8) M. All three compounds inhibited bone collagen synthesis as measured by incorporation of proline into collagenase digestible protein, whereas incorporation into noncollagen protein was not inhibited. The effects were dose related and decreases in percent collagen synthesis were significant at 10(-9) M. Thus PTH-Ana appears to have the same effects on bone resorption and collagen synthesis as bPTH-(1-84) and (1-34) and is likely to be a valid probe for investigating PTH receptors in bone as well as in kidney.     

Morphologic alteration of cultured arterial smooth muscle cells by cyclic stretching

Cyclic stretching of smooth muscle cells in culture resulted in a two- to fivefold increase in protein and collagen synthesis. The same in vitro system was utilized to relate changes in smooth muscle cell morphology to mechanical stress. Smooth muscle cells, grown in culture from rabbit aorta explants, were transferred to purified elastic membranes derived from bovine aorta. The membranes were either subjected to stretching and relaxation 52 times per minute or stretched and held stationary for 8, 48, or 56 hr. Profiles of rough endoplasmic reticulum (RER) were counted and myofilament content estimated from electron micrographs of 100 cells for each experiment. Cells from cyclically stretched preparations were compared with stationary cells derived from the same subculture. Myofilaments were largely replaced by RER in cyclically stretched cells and there was a reciprocal relationship between RER and myofilament content in individual cells. In cells from stationary preparations, myofilament content also diminished with time but RER profiles were few. At 56 hr, RER profiles numbered 16.7 +/- 1.7 in stretched cells compared with 3.6 +/- 1.3 in stationary cells (P less than 0.05). Cyclically stretched cells formed numerous intercellular contacts and showed little evidence of cytoplasmic degradation while stationary cells showed few contacts and contained numerous cytosomes and lamellar bodies. The results suggest that cyclic stretching resulted in the formation of RER or the preservation of myofilaments and that immobility resulted in the disappearance of myofilaments and cytoplasmic degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the Nd:YAG laser on DNA synthesis and collagen production in human skin fibroblast cultures

Human skin fibroblasts were subjected to treatment with a Neodymium:YAG laser at 1060 nm with varying levels of energy determined by a reproducible method of dosimetry. DNA synthesis in the cells was measured by the incorporation of [3H]thymidine, and collagen production was monitored by the synthesis of nondialyzable [3H]hydroxyproline after incubation of cells with [3H]proline. Using energy levels equal to 1.7 X 10(3) J/cm2, a significant reduction in DNA synthesis was noted, while the cells remained viable as tested by the trypan blue exclusion test. With energy levels higher or equal to 2.3 X 10(3) J/cm2, the suppression of DNA synthesis was accompanied by cell nonviability. The collagen production, when measured immediately following the treatment with 1.7 X 10(3) J/cm2, was markedly reduced, and similar effects were observed with higher energy levels. However, when the cells were tested for collagen production at 20 hours following laser treatment, there was a significant decrease in collagen production at energy levels as low as 1.1 X 10(3) J/cm2, a dose that did not affect DNA synthesis or cell viability. Thus, the results indicate that the Nd:YAG laser can selectively suppress collagen production without affecting cell proliferation. These observations suggest that laser treatment could potentially be used to reduce collagen deposition in conditions such as keloids and hypertrophic scars.     

中间纤维对兔关节软骨细胞基质合成代谢的影响

目的 观察细胞骨架中间纤维破坏对培养的兔关节软骨细胞基质合成代谢的影响.方法 1个月龄新西兰白兔,无菌条件下切取双膝关节软骨,酶解法分离软骨细胞,接种于6孔板培养待细胞贴壁后弃去培养液并随机分为2组.对照组用DMEM/F12正常培养,中间纤维破坏组(简称IF破坏组)利用含5 mmoL/L丙烯酰胺的DMEM/F12培养,使细胞骨架中间纤维破坏.分别在换液后第4天和第7天以阿尔新蓝法检测2组细胞上清液中糖胺多糖(GAG)含量,同时检测上清液中羟脯氨酸含量以评估2组细胞胶原的合成量.结果 两组软骨细胞在换液后第4天和第7天时上清液中GAG含量差异无统计学意义(P>0.05);但2组上清液中GAG含量均随培养时间的延长而升高(P<0.05).IF破坏组上清液中羟脯氨酸含量在换液后第4天时与对照组差异无统计学意义(P>0.05),而第7天时明显低于对照组(P<0.05);对照组第7天上清液中羟脯氨酸含量较第4天有增高趋势,但统计学检验差异无统计学意义(P>0.05),而IF破坏组第7天上清液中羟脯氨酸含量与第4天相同.结论 软骨细胞GAG的合成具有时间累积效应,破坏中间纤维对软骨细胞GAG的合成没有影响,而破坏中间纤维可降低离体培养的关节软骨细胞羟脯氨酸的合成,表明中间纤维在软骨细胞胶原的合成中有着重要作用.