Microplate Assay for the Measurement of Hydroxyproline in Acid-Hydrolyzed Tissue Samples

Brown, Sharon J.; Worsfold, M.; Sharp, Christopher

doi:10.2144/01301bm06

Cited by 72 publications

(48 citation statements)

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“…Concentrations at or above 20% have been shown to result in decreased cell viability , Khattak et al, 2005. Secondly, studies have shown (Brunet-Maheu et al, 2009) the importance of frequent media replenishment. We replenished the culture media every third day but this may not have been frequent enough to maintain viability.…”

Section: Discussionmentioning

confidence: 99%

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The composition of hydrogels for cartilage tissue engineering can influence glycosaminoglycan profile

Wang

Hughes²,

Cartmell³

et al. 2010

eCM

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The injectable and hydrophilic nature of hydrogels makes them suitable candidates for cartilage tissue engineering. To date, a wide range of hydrogels have been proposed for articular cartilage regeneration but few studies have quantitatively compared chondrocyte behaviour and extracellular matrix (ECM) synthesis within the hydrogels. Herein we have examined the nature of ECM synthesis by chondrocytes seeded into four hydrogels formed by either temperature change, self-assembly or chemical crosslinking. Bovine articular cartilage chondrocytes were cultured for 14 days in Extracel®, Pluronic F127 blended with Type II collagen, Puramatrix® and Matrixhyal®. The discriminatory and sensitive technique of fluorophoreassisted carbohydrate electrophoresis (FACE) was used to determine the fine detail of the glycosaminoglycans (GAG); hyaluronan and chondroitin sulphate. FACE analysis for chondroitin sulphate and hyaluronan profiles in Puramatrix® closely matched that of native cartilage. For each hydrogel, DNA content, viability and morphology were assessed. Total collagen and total sulphated GAG production were measured and normalised to DNA content. Significant differences were found in total collagen synthesis. By day 14, Extracel® and Puramatrix® had significantly more total collagen than Matrixhyal® (1.77±0.26 μg and 1.97±0.26 μg vs. 0.60±0.26 μg; p<0.05). sGAG synthesis occurred in all hydrogels but a significantly higher amount of sGAG was retained within Extracel® at days 7 and 14 (p<0.05). In summary, we have shown that the biochemical and biophysical characteristics of each hydrogel directly or indirectly influenced ECM formation. A detailed understanding of the ECM in the development of engineered constructs is an important step in monitoring the success of cartilage regeneration strategies.

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Section: Discussionmentioning

confidence: 99%

“…Total collagen production was determined at 0, 5, 7 and 14 days using the hydroxyproline assay as described by Brown et al (2001). Total collagen was not detectable until after 3 days in culture (unpublished observations).…”

Section: Determination Of Total Collagen Productionmentioning

confidence: 99%

The composition of hydrogels for cartilage tissue engineering can influence glycosaminoglycan profile

Wang

Hughes²,

Cartmell³

et al. 2010

eCM

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“…Collagen content of wound edge tissue was assessed by determining the amount of hydroxyproline (23). Frozen wound tissue was pulverized under liquid nitrogen and then hydrolyzed in 1 ml of 5 N HCl overnight at 110°C.…”

Section: Collagen Contentmentioning

confidence: 99%

Oxidant-induced Vascular Endothelial Growth Factor Expression in Human Keratinocytes and Cutaneous Wound Healing

Sen

Khanna

Babior

et al. 2002

Journal of Biological Chemistry

291

218

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Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair. We observed that H 2 O 2 potently induces vascular endothelial growth factor (VEGF) expression in human keratinocytes. Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp ؊194 to ؊50 of the VEGF promoter is responsible for the H 2 O 2 response. It was established that at M concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible factor (HIF)-1 and dependent on Sp1 activation. To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention. Rac1 gene transfer accelerated wound contraction and closure. Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human keratinocytes. Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole. Taken together, the results of this study indicate that wound healing is subject to redox control.

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“…The other 54 explants were used for hydroxyproline (HYP) measurement. 18 Data obtained from postcyclic loaded explants were normalized by the precyclic loaded values and subsequently analyzed with two-way repeated measures ANOVA and StudentNeuman-Keuls post hoc tests to determine differences between groups (SigmaStat v.2.0, SPSS Inc., Chicago, IL). PG and HYP content was normalized by tissue wet weight and reported in mg PG and mg HYP per mg wet weight, respectively.…”

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confidence: 99%

High levels of glucosamine–chondroitin sulfate can alter the cyclic preload and acute overload responses of chondral explants

Wei

Haut

2009

Journal Orthopaedic Research

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A recent study by our laboratory showed that 14 days of low intensity, intermittent cyclic preloading of chondral explants elevated the concentration of proteoglycans (PGs) to cause a mechanical stiffening of the explants prior to an acute overload and limit the extent of tissue damage. Longer term loading to 21 days resulted in tissue degradation prior to the acute traumatic event and excessive damage from an acute overload. Previous studies by others showed that bathing chondral explants in a supplement of glucosaminechondroitin sulfate (glcN-CS) upregulated the synthesis of tissue PGs, particularly in stressed tissue, and the supplement served as an antiinflammatory agent. Our current hypothesis was that the supplementation of culture media with a high concentration of glcN-CS would upregulate the production of tissue PG and limit or mitigate long-term degradation of chondral explants under cyclic preloading and limit tissue damage in an acute overload. We showed that, in the presence of supplement, cyclic preloading significantly increased tissue PG content and matrix modulus by about 65 and 300%, respectively, at 21 days, resulting in a reduction of matrix damage and cell death following an acute overload. These data show a biological action of high concentrations of this supplement and its effect on the mechanical properties in this in vitro model. In vitro studies of impact loading 1,2 on cartilage show cell death and matrix damage. In contrast, in vitro and in vivo studies showed positive aspects of low intensity, intermittent compressive loading, such as increased biosynthesis of proteoglycans (PGs). [3][4][5] Changes in PG and collagen contents of cartilage significantly alter the mechanical properties of this tissue. 6 Thus, it is reasonable to assume that cyclic preloading of cartilage can alter its mechanical properties and help determine the extent of matrix damage and cell death that will occur due to the blunt force overloading of a joint, such as during an acute knee ligament rupture. 7 Recent studies in our laboratory show that low intensity, intermittent cyclic preloading of chondral explants prior to an acute overload yields an increase in the synthesis of tissue PGs, resulting in a less severe traumatic insult for an acute overload. 8 Between 14 and 21 days of cyclic preloading, the explants experience a significant PG loss that leads to a dramatic loss in mechanical properties. Acute overloading of the explanted tissue then yields matrix damage and cell death significantly greater than nonpreloaded controls. A recent study by others shows a significant loss of tissue PG and cell death with production of matrix metalloproteinase (MMP-3) adjacent to damaged collagen fibers under cyclic compression at 0.5 Hz for intensities of 1 and 5 MPa over 24 h. 9 Similarly, under 0.5 MPa of 1 Hz cyclic loading, a significant increase in the MMP-2 and -9 synthesis was found after 3 h. 10 The combination of glucosamine-chondroitin sulfate (glcN-CS) was shown in in vitro studies to function as a ''...

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Microplate Assay for the Measurement of Hydroxyproline in Acid-Hydrolyzed Tissue Samples

Cited by 72 publications

References 0 publications

The composition of hydrogels for cartilage tissue engineering can influence glycosaminoglycan profile

The composition of hydrogels for cartilage tissue engineering can influence glycosaminoglycan profile

Oxidant-induced Vascular Endothelial Growth Factor Expression in Human Keratinocytes and Cutaneous Wound Healing

High levels of glucosamine–chondroitin sulfate can alter the cyclic preload and acute overload responses of chondral explants

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