Rate Of Collagen Synthesis Research Articles

Effect of Extracellular pH on Matrix Synthesis by Chondrocytes in 3D Agarose Gel

In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3-6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7-day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH-dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.

Decreased response of osteoblasts obtained from aged Cohen diabetic sensitive rats to sex steroid hormones and 1,25(OH)2D3 in culture

Patients with diabetes mellitus are known to develop osteopenia and osteoporosis, apparently as a reduction in the process of bone formation. In order to evaluate whether bone-modulating hormones--estradiol, testosterone, and 1,25(OH)(2)D(3)--have different effects on osteoblasts derived from diabetic and from normal non-diabetic rats, we studied the specific effects of these hormones on the differentiation and function of cultured osteoblasts derived from 1-year-old Cohen diabetic rats. (The Cohen diabetic model consists of a diabetic-sensitive strain [CDs; diabetic] and a diabetic-resistant strain [CDr; normal]). The CDs and CDr male and female rats were fed on a regular diet (RD) or a high-sucrose low-copper diet (HSD; diabetogenic). On the HSD diet, only CD rats develop type 2 diabetes, while CDr do not. Bones were removed for primary osteoblast cultures, and osteoblastic responses to the bone-modulation hormones--estradiol, testosterone, and 1,25(OH)(2)D(3)--were studied. In male rats fed RD, primary cultures of osteoblasts without hormone addition to the culture medium showed that alkaline phosphatase (ALP) activity was similar in the Cohen diabetic rats (both CDr and CDs) to that of the original Sabra strain. However, collagen synthesis was reduced in the CDr and CDs compared to the Sabra strain. The addition of the hormones to the culture medium did not change ALP activity or collagen synthesis in the male-derived osteoblasts, but increased mineralization in all strains. In female rats (studied only in CDs and CDr animals) there were no differences between animals fed the RD. HSD increased the basal activity of ALP in the CDr but not in the CDs rats, and decreased the rate of collagen synthesis in both CDr and CDs (diabetic) animals. The addition of the bone-modulation hormones to the culture medium further increased ALP activity in the osteoblasts derived from the CDr animals, while decreasing ALP activity in the CDs. These hormones also decreased collagen synthesis in both strains and increased mineralization in all osteoblasts. In conclusion, the metabolic status (HSD and diabetes) in rats prior to culture affected the phenotype of cultured osteoblasts, decreasing their response to bone-modulation hormones. This decreased response, especially to estradiol, may be a major cause of the osteopenia observed in diabetes.

Tendon collagen synthesis at rest and after exercise in women

In general, there is a higher incidence of musculoskeletal injuries during physical activity in women than in men. We hypothesized that in women rates of tendon collagen synthesis would be lower than in men at rest and after exercise, especially in the later luteal phase when estrogen and progesterone concentrations are higher than the early follicular phase. We studied tendon collagen fractional synthesis rate (FSR) in 15 young, healthy female subjects in either the early follicular (n = 8) or the late luteal phase (n = 7) 72 h after an acute bout of one-legged exercise (60 min kicking at 67% workload maximum) (72 h) and compared the results with those previously obtained for men. Samples were taken from the patellar tendon in both the exercised and rested legs to determine collagen FSR by the incorporation of [15N]proline into tendon collagen hydroxyproline. There was no effect of menstrual phase on tendon collagen synthesis either at rest or after exercise. However, there was a significant difference between women and men at rest (women = 0.025 +/- 0.002%/h, men = 0.045 +/- 0.008%/h; P < 0.05) and 72 h after exercise (women = 0.027 +/- 0.005%/h; men = 0.058 +/- 0.008%/h). Furthermore, rest and 72-h tendon collagen synthesis were not different in women, whereas in men tendon collagen synthesis remained significantly elevated 72 h after exercise. It is concluded that both in the resting state and after exercise, tendon collagen FSR is lower in women than in men, which may contribute to a lower rate of tissue repair after exercise.

Collagen synthesis of articular cartilage explants in response to frequency of cyclic mechanical loading

Articular cartilage in vivo experiences the effects of both cell-regulatory proteins and mechanical forces. This study has addressed the hypothesis that the frequency of intermittently or continuously applied mechanical loads is a critical parameter in the regulation of chondrocyte collagen biosynthesis. Cyclic compressive pressure was applied intermittently to bovine articular cartilage explants by using a sinusoidal waveform of 0.1-1.0 Hz frequency with a peak stress of 0.5 MPa for a period of 5-20 s followed by a load-free period of 10-1,000 s. These loading protocols were repeated for a total duration of 6 days. In separate experiments, cyclic loading was continuously applied by using a sinusoidal waveform of 0.001-0.5 Hz frequency and a peak stress of 1.0 MPa for a period of 3 days. Unloaded cartilage discs of the same condyle were cultured in identically constructed loading chambers and served as controls. We report quantitative data showing that (1) no correlation exists between the relative rate of collagen synthesis expressed as the proportion of newly synthesized collagen among newly made proteins and either the frequency of intermittently or continuously applied loads or the overall time cartilage is actively loaded, and (2) individual protocols of intermittently applied loads can reduce the relative rate of collagen synthesis and increase the water content, whereas (3) continuously applied cyclic loads always suppress the relative rate of collagen synthesis compared with that of unloaded control specimens. The results provide further experimental evidence that collagen metabolism is difficult to manipulate by mechanical stimuli. This is physiologically important for the maintainance of the material properties of collagen in view of the heavy mechanical demands made upon it. Moreover, the unaltered or reduced collagen synthesis of cartilage explants might reflect more closely the metabolism of normal or early human osteoarthritic cartilage.

Eccentric rehabilitation exercise increases peritendinous type I collagen synthesis in humans with Achilles tendinosis

It has been shown that 12 weeks of eccentric heavy resistance training can reduce pain in runners suffering from chronic Achilles tendinosis, but the mechanism behind the effectiveness of this treatment is unknown. The present study investigates the local effect of an eccentric training regime on elite soccer players suffering from chronic Achilles tendinosis on the turnover of the peritendinous connective tissue. Twelve elite male soccer players, of whom six suffered from unilateral tendinosis and six were healthy controls, participated in this study. All participants performed 12 weeks of heavy-resistance eccentric training apart from their regular training and soccer activity. Before and after the training period the tissue concentration of indicators of collagen turnover was measured by the use of the microdialysis technique. After training, collagen synthesis was increased in the initially injured tendon (n=6; carboxyterminal propeptide of type I collagen (PICP): pre 3.9+/-2.5 microg/L to post 19.7+/-5.4 microg/L, P<0.05). The collagen synthesis was unchanged in healthy tendons in response to training (n=6; PICP: pre 8.3+/-5.2 microg/L to post 11.5+/-5.0 microg/L, P>0.05). Collagen degradation, measured as carboxyterminal telopeptide region of type I collagen (ICTP), was not affected by training neither in the injured nor in the healthy tendons. The clinical effect of the 12 weeks of eccentric training was determined by using a standardized loading procedure of the Achilles tendons showing a decrease in pain in all the chronic injured tendons (VAS before 44+/-9, after 13+/-9; P<0.05), and all subjects were back playing soccer following the eccentric training regime. The present study demonstrates that chronically injured Achilles tendons respond to 12 weeks of eccentric training by increasing collagen synthesis rate. In contrast, the collagen metabolism in healthy control tendons seems not to be affected by eccentric training. These findings could indicate a relation between collagen metabolism and recovery from injury in human tendons.

Examination of the rheumatoid hand and wrist

Rheumatoid disease affects almost all of the musculoskeletal system as well as some internal organs, such as the heart and lungs, pleura, eyes, lymph nodes and vessels. In the case of the upper extremity, we should know the time of onset and type of disease, as the extent and degree of tissue involvement varies among the different types of rheumatoid disease. Surgical treatment and postoperative management will vary significantly in patients suffering from sclerodermia to those of systemic lupus erythematosus. This can also be said about the type of medication used for treating the disease, as more recent medications may alter collagen synthesis and infection rates. Before examining the hand, shoulder and elbow function should also be assessed. Clinical examination of the hand joints is most important, as all deformities, including most tendon ruptures and tendon dislocations are secondary to joint involvement. We should carefully examine for the presence and intensity of joint synovitis, and the degree of joint deformity and record the range of active and passive joint mobility. Radiological examination should be done in all cases to determine the degree of joint cartilage destruction, joint subluxation, and even joint ankylosis which is some times difficult to asses on clinical examination due to a fixed joint deformity from extraarticular causes. CAT scan examination can be useful for wrist joint assessment. Magnetic resonance examinations may be required only in certain circumstances, i.e. to determine the presence of joint or tendon synovitis not clearly assessed by clinical exploration, and the location of a flexor tendon rupture.

Ageing and human bone collagen synthesis

A loss of bone mineral density and collagen concentration occurs with ageing, but possible deficits in human bone collagen synthesis have not previously been examined. We studied 4 healthy elderly men (age 62 ± 7 y) and 2 healthy postmenopausal elderly women (55,62y) in the post absorptive state and compared the results with those from our previously published data for young healthy men. Subjects fasted overnight, were given a flooding dose of 1-13C proline over 3 min with an iliac crest biopsy being taken under local anesthesia and mild sedation 120 min later. There were no differences in the rate of immature bone collagen synthesis between the elderly and young men (NaCl fraction: 0.57±0.18 v 0.57±0.10 %.h−1; HOAc fraction: 0.23±0.10 v 0.24±0.05 %.h−1 respectively), but the rate of mature bone collagen synthesis was less in the elderly than the young men (pepsin fraction: 0.04±0.01 v 0.07±0.01 %.h−1, P< 0.002; hot water fraction: 0.04±0.01 v 0.06±0.01 %.h−1, P<0.002, respectively). In elderly women the pattern of rates of collagen synthesis was different to that in men with lower rates of immature collagen synthesis than in the young men (NaCl: 0.36 %.h−1; HOAc: 0.17 %.h−1) and higher rates of mature collagen synthesis (pepsin: 0.08 %.h−1; hot water: 0.08 %.h−1). Ageing in men does not appear to effect osteoblasts ability to synthesis collagen but extracellular processing into fibrillar collagen appears to be defective, whereas in women procollagen synthesis may be decreased. Supported by The Wellcome Trust, Diabetes UK, UK MRC, BBSRC.

Effect of orally administrated amino acids on skin collagen synthesis in UV irradiated mice

Abnormality of skin collagen metabolism is one of main causes for ultraviolet (UV) induced skin ageing. It has been shown that single UV irradiation decreases procollagen mRNA expression and increases matrix metalloproteinases mRNA expression in dermis, and that chronic UV irradiation decreases collagen amount and induces wrinkle formation. Amino acid is known to regulate protein metabolism, in general. Therefore, we investigated effects of UV irradiation and orally administrated various amino acids on skin collagen synthesis rate. Hr-1 hairless mice (male, 8weeks old) were irradiated with UVB (66 J/cm2) twice at one day interval, and then fasted for 16hr. Fractional synthesis rate (FSR) of skin tropocollagen was measured by flooding dose method using stable isotope L-[ring-2H5]Phenylalanine. We confirmed that dermal tropocollagen FSR was decreased by UVB irradiation. Subsequently, FSR of dermal tropocollagen was measured at 30 min after oral administration of amino acid (1g/kg) in UVB irradiated mice. Neither branched-chain amino acids (BCAA), Arginine (Arg), Glutamine (Gln), nor Glutamate, but amino acids mixtures, BCAA+Arg+Gln and BCAA+Gln increased dermal tropocollagen synthesis. It is suggested that the combination of BCAA and glutamine is key for recovery of dermal collagen synthesis impaired by UV irradiation.

Topical application of epidermal growth factor accelerates wound healing by myofibroblast proliferation and collagen synthesis in rat

Recombinant human epidermal growth factor (rhEGF) stimulates the proliferation and migration of epithelial cells in human cell culture systems and animal models of partial-thickness skin wounds. This study investigated the effect of a topical rhEGF ointment on the rate of wound healing and skin re-epithelialization in a rat full thickness wound model, and verified whether or not the rhEGF treatment affected both myofibroblast proliferation and collagen synthesis in the dermis. When rhEGF (10 µg/g ointment) was applied topically twice a day for 14 days, there was significantly enhanced wound closure from the 5th to the 12th day compared with the control (ointment base treatment) group. A histological examination at the postoperative 7th day revealed that the rhEGF treatment increased the number of proliferating nuclear antigen immunoreactive cells in the epidermis layer. In addition, the immunoreactive area of alpha-smooth muscle actin and the expression of prolyl 4-hydroxylase were significantly higher than those of the control group. Overall, a topical treatment of rhEGF ointment promotes wound healing by increasing the rate of epidermal proliferation and accelerating the level of wound contraction related to myofibroblast proliferation and collagen deposition.

Alcohol-induced bone degradation and its early detection in the alcohol-fed castrated rats

The objective of this study was to examine alcohol-induced changes of bone in hormone-deficient males using the developed method. In the process of bone resorption, type I collagen crosslinking molecules, pyridinoline (PYD), are released into the circulation and cleared by the kidneys. (2)H(2)O as a tracer has been applied to measure the synthesis rates of slow-turnover proteins and successfully applied to bone collagen synthesis in our hormone deficiency rats. This study demonstrated for the first time, the early changes of the femur bone degradation in hormone-deficient male individuals, more influenced by alcohol through histopathological study, serum PYD assay, and (2)H(2)O labeling. We also observed that serum PYD was a sensitive pathological marker of bone degradation in castrated osteoporosis males and the unique features of (2)H(2)O labeling to measure the bone turnover collagen synthesis rates were excellent markers of bone degradation and aging.

Collagen biosynthesis in normal and hypertrophic scars and keloid as a function of the duration of the scar.

The rates of collagen biosynthesis and the tissue concentrations of collagen in normal scar, hypertrophic scar and keloid were determined as a function of the duration of the lesions. The rate of collagen synthesis in normal scar was approximately constant between 6 months and 20 years after the initial wounding, but in both hypertrophic scar and keloid the rate was initially approximately twice that in normal scar, and 2-3 years after wounding it fell to approximately the same level as in normal scar. The tissue concentration of collagen appeared to be relatively constant in normal scar with respect to the time elapsed after wounding, but in both types of abnormal scar it was initially somewhat lower than normal scar and then rose to values higher than in normal scar after about 2-3 years. Since similar results were obtained for both types of abnormal scar in terms of the rates of collagen biosynthesis with respect to the time elapsed after wounding and also in terms of the tissue concentrations of collagen, this may indicate that the events in the pathogenesis of both abnormal scar types are similar. The results also raise the possibility that the formation of both types of abnormal scar may occur in two phases: an initial phase characterized by abnormally high rates of collagen biosynthesis and a later phase characterized by essentially normal rates of collagen biosynthesis.

Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise

We hypothesized that an acute bout of strenuous, non-damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups (n = 8 and 6) of healthy young men were studied over 72 h after 1 h of one-legged kicking exercise at 67% of maximum workload (W(max)). To label tissue proteins in muscle and tendon primed, constant infusions of [1-(13)C]leucine or [1-(13)C]valine and flooding doses of [(15)N] or [(13)C]proline were given intravenously, with estimation of labelling in target proteins by gas chromatography-mass spectrometry. Patellar tendon and quadriceps biopsies were taken in exercised and rested legs at 6, 24, 42 or 48 and 72 h after exercise. The fractional synthetic rates of all proteins were elevated at 6 h and rose rapidly to peak at 24 h post exercise (tendon collagen (0.077% h(-1)), muscle collagen (0.054% h(-1)), myofibrillar protein (0.121% h(-1)), and sarcoplasmic protein (0.134% h(-1))). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF-I and IGF binding proteins (IGFBP-3 and IGFBP-4) or procollagen type I N-terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise in human tendon and muscle. The similar time course of changes of protein synthetic rates in different cell types supports the idea of coordinated musculotendinous adaptation.

Collagen biosynthesis of mechanically loaded articular cartilage explants

The purpose of this study was to investigate systematically the effect of load amplitudes, frequencies and load durations of intermittently applied mechanical pressure on the biosynthesis of collagen and non-collagenous proteins (NCP) as well as on the water content of cultured bovine articular cartilage explants. Cyclic compressive pressure was applied using a sinusoidal waveform of 0.5 Hz frequency with a peak stress of 0.1, 0.5 or 1.0 MPa for a period of 10s followed by a load-free period of 10, 100 or 1000s. These intermittent loading protocols were repeated for a total duration of 1, 3 or 6 days. During the final 18 h of experiments, the incorporation of [(3)H]-proline into collagen and NCP, the content of water as well as the deformation of loaded explants were determined. Intermittently applied, cyclic mechanical loading of articular cartilage explants consistently reduced the relative rate of collagen synthesis compared to load-free conditions. This reduced proportion of newly synthesized collagen among newly made proteins was independent of the mechanical stimuli applied. The release of newly synthesized collagen and NCP from loaded explants into the nutrient media was unaffected by any of the loading protocols applied. In addition, quantitative data are provided showing that only high amplitudes of loads and frequencies enhanced the water content of the explants. Previous studies reporting that osteoarthritic cartilage in vivo can synthesize elevated amounts of collagen imply that the loading protocols chosen were inadequate for simulating in vitro osteoarthritic-like alterations of collagen synthesis. In our experiments the collagen biosynthesis of chondrocytes was only minor responsive to alterations in mechanical stimuli, applied over a wide range. Thus, our results imply that the synthesis of these structural macromolecules is under the strict control of normal chondrocytes enabling them to maintain the shape of this physical demanded tissue.

Collagen synthesis in human musculoskeletal tissues and skin

We have developed a direct method for the measurement of human musculoskeletal collagen synthesis on the basis of the incorporation of stable isotope-labeled proline or leucine into protein and have used it to measure the rate of synthesis of collagen in tendon, ligament, muscle, and skin. In postabsorptive, healthy young men (28 +/- 6 yr) synthetic rates for tendon, ligament, muscle, and skin collagen were 0.046 +/- 0.005, 0.040 +/- 0.006, 0.016 +/- 0.002, and 0.037 +/- 0.003%/h, respectively (means +/- SD). In postabsorptive, healthy elderly men (70 +/- 6 yr) the rate of skeletal muscle collagen synthesis is greater than in the young (0.023 +/- 0.002%/h, P < 0.05 vs. young). The rates of synthesis of tendon and ligament collagen are similar to those of mixed skeletal muscle protein in the postabsorptive state, whereas the rate for muscle collagen synthesis is much lower in both young and elderly men. After nutrient provision, collagen synthesis was unaltered in tendon and skeletal muscle, remaining at postabsorptive values (young: tendon, 0.045 +/- 0.008%/h; muscle, 0.016 +/- 0.003%/h; elderly: muscle, 0.024 +/- 0.003%/h). These results demonstrate that the rate of human musculoskeletal tissue collagen synthesis can be directly and robustly measured using stable isotope methodology.

Human Bone Collagen Synthesis Is a Rapid, Nutritionally Modulated Process

We developed a direct assay of human bone collagen synthesis using [13C] or [15N] proline and applied it to determine the effects of feeding in young healthy men. Surprisingly, postabsorptive bone collagen synthesis is not sluggish, being approximately 0.07%/h more rapid than that of muscle protein, and capable of being stimulated within 4 h of intravenous feeding by 66 +/- 13%. All current methods for estimation of bone collagen turnover are indirect, depending on the assay of collagen "markers." Our aim was to develop a direct method for human bone collagen synthesis to be used to study its physiology and pathology, and specifically, in the first instance, the effect of feeding. We applied, over 2 h, flooding doses of [13C] and [15N] proline to label iliac crest bone collagen in eight young healthy men. The rate of collagen synthesis was determined as the rate of labeling of collagen hydroxyproline (assayed by gas chromatography-combustion-isotope ratio mass spectrometry in collagen extracted by differential solubility) compared with plasma proline labeling (assayed by gas chromatography-mass spectrometry). We also determined (in a second group of eight young healthy men) the effect of intravenous nutrition (glucose, lipid emulsion, and amino acids (in the ratio of 55%:30%:15% energy, respectively). Free bone proline labeling was 92 +/- 6% of that of plasma proline, supporting the flooding dose assumption. Human iliac crest bone collagen is heterogeneous, with NaCl-EDTA, 0.5 M acetic acid, pepsin-acetic acid, and hot water-extractable pools being responsible for approximately 1%, 3%, 8%, and 81% of content, respectively. The synthetic rates were 0.58 +/- 0.1, 0.24 +/- 0.05, 0.07 +/- 0.02, and 0.06 +/- 0.01%/h, respectively, giving an average rate of approximately 0.066%/h. [13C] and [15N] proline gave identical results. Intravenous nutrition caused the disappearance of proline label from the procollagen pool and its increased appearance in the less extractable pools, suggesting nutritional stimulation of collagen processing. The results show (1) that iliac crest bone collagen synthesis is faster than generally assumed and of the same order as muscle protein turnover and (2) that feeding increases synthesis by approximately 66%. Given its ability to detect physiologically meaningful responses, the method should provide a new approach to studying the regulation of bone collagen turnover.

Anterior Cruciate Ligament Tear Prevention in the Female Athlete

Anterior Cruciate Ligament Tear Prevention in the Female Athlete

Effects of a Long-Term Dietary Xylitol Supplementation on Collagen Content and Fluorescence of the Skin in Aged Rats

Background: Dietary xylitol has been shown to increase the amounts of newly synthesized collagen, and to decrease fluorescence of the collagenase-soluble fraction in the skin of both healthy and diabetic rats. As in diabetic rats, a decreased rate of collagen synthesis and increased collagen fluorescence has also been detected in the skin of aged rats. We hypothesize that dietary xylitol supplementation may protect against these changes during aging. Objective: The purpose of the present study was to investigate whether a long-term dietary supplementation can protect against the decrease in the amounts of newly synthesized collagen, and against the increase in fluorescence in the collagenase-soluble fraction in the skin of aged rats. Methods:Twenty-four male Sprague-Dawley rats were used in the study. After weaning, the rats were divided into 2 groups of 12 animals. The rats in the control group were fed a basal RM1 diet, while the rats in the experimental group were fed the same diet supplemented with 10% xylitol. After 20 months, the rats were killed and pieces of skin from their dorsal areas were excised. The thickness of the samples was measured with a micrometer screw gauge. The collagen contents of rat skin were measured as hydroxyproline, and glycosylation as fluorescent intensity of collagen. Statistical significances of the differences between the groups were determined using the unpaired t test. Results: No general side effects were detected in the rats during the experimental period. The skin of the xylitol-fed rats was a little thicker than that of the control rats. The hydroxyproline content of the acid-soluble fraction was significantly greater in the xylitol group as compared to the controls. However, there were no significant differences in the hydroxyproline content of the collagenase-soluble fraction between the groups. The fluorescence of the collagenase-soluble fraction was significantly smaller in the xylitol-fed aged rats than in the aged rats fed the basal diet. Conclusions: The results of this study indicate that xylitol caused an increase in the amount of newly synthesized collagen and a decrease in collagen fluorescence in the skin of aged rats.

Protein synthesis rates in human muscles: neither anatomical location nor fibre‐type composition are major determinants

In many animals the rate of protein synthesis is higher in slow-twitch, oxidative than fast-twitch, glycolytic muscles. To discover if muscles in the human body also show such differences, we measured [13C]leucine incorporation into proteins of anatomically distinct muscles of markedly different fibre-type composition (vastus lateralis, triceps, soleus) after an overnight fast and during infusion of a mixed amino acid solution (75 mg amino acids kg(-1) h(-1)) in nine healthy, young men. Type-1 fibres contributed 83 +/- 4% (mean +/-s.e.m.) of total fibres in soleus, 59 +/- 3% in vastus lateralis and 22 +/- 2% in triceps. The basal myofibrillar and sarcoplasmic protein fractional synthetic rates (FSR, % h(-1)) were 0.034 +/- 0.001 and 0.064 +/- 0.001 (soleus), 0.031 +/- 0.001 and 0.060 +/- 0.001 (vastus), and 0.027 +/- 0.001 and 0.055 +/- 0.001 (triceps). During amino acid infusion, myofibrillar protein FSR increased to 3-fold, and sarcoplasmic to 2-fold basal values (P < 0.001). The differences between muscles, although significant statistically (triceps versus soleus and vastus lateralis, P < 0.05), were within approximately 15%, biologically probably insignificant. The rates of collagen synthesis were not affected by amino acid infusion and varied by < 5% between muscles and experimental conditions.

Cyclic hydrostatic compression stimulates chondroinduction of C3H/10T1/2 cells

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/10T1/2 murine embryonic fibroblasts by recombinant human bone morphogenetic protein-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied 10 min on/10 min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/10T1/2 cell line in a duration-dependent manner.

Transport and intracellular accumulation of vitamin C in endothelial cells: relevance to collagen synthesis

Endothelial cells preserve vascular integrity in part by synthesizing type IV collagen for the basement membrane of blood vessels. Vitamin C, which at physiologic pH is largely the ascorbate mono-anion, both protects these cells from oxidant stress and is required for collagen synthesis. Therefore, cultured endothelial cells were used to correlate intracellular concentrations of ascorbate with its uptake and ability to stimulate collagen release into the culture medium. The kinetics and inhibitor specificity of ascorbate transport into EA.hy926 endothelial cells were similar to those observed in other cell types, indicative of a specific high affinity transport process. Further, transport of the vitamin generated intracellular ascorbate concentrations that were 80–100-fold higher than concentrations in the medium following overnight culture, and transport inhibition with sulfinpyrazone and phloretin partially prevented such ascorbate accumulation. On the other hand, low millimolar intracellular concentrations of ascorbate impaired its transport measured after overnight culture. Synthesis and release of type IV collagen into the culture medium was markedly stimulated by ascorbate in a time-dependent manner, and was saturable with increasing medium concentrations of the vitamin. Optimal rates of collagen synthesis required intracellular concentrations of the vitamin up to 2 mM. Since such concentrations can only be generated by the ascorbate transporter, these results show the necessity of transport for this crucial function of the vitamin in endothelium.