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Abstract

Objective

To compare chondrocyte proliferation and metabolism in three-dimensional fibrin cultures formed from polymerized autogenous fibrinogen with that of commercially manufactured fractionated fibrinogen.

Animals

Fibrinogen and chondrocytes for in vitro experimentation derived from 2 horses, ages 12 and 14 months, donated for reasons unrelated to skeletal or hematologic abnormalities.

Procedure

Fibrinogen was isolated from whole blood, using plasma cryoprecipitation and centrifugation, and fractionated fibrinogen was purchased. Each was mixed with 10 × 106 chondrocytes/0.5 ml of fibrinogen, and was polymerized by addition of 0.5 ml of calcium-activated thrombin. Thirty 1-ml fibrin-chondrocyte disks were formed from each fibrinogen source and cultured for 0 (n = 6), 7 (n = 12), or 14 (n = 12) days. Chondrocyte metabolism and cell proliferation in each fibrin type were objectively assessed by assays for total proteoglycan content, [35S]proteoglycan accumulation, proteoglycan monomer size, and total DNA. Cell morphology and cartilage-specific cell function was evaluated by routine histologic, alcian blue histochemical, type-II collagen immunohistochemical, and type-II collagen in situ hybridization methods.

Results

Histologic examination indicated better retention of chondrocyte morphology in autogenous composites. Autogenous fibrinogen also stimulated greater chondrocyte proliferation (DNA content increased 1.4-fold on day 14) and supported higher proteoglycan accumulation (increased 1.4-fold on day 14), compared with commercial, fractionated fibrinogen. Abundant intracellular type-II procollagen mRNA was detected in autogenous fibrin cultures by in situ hybridization, and translation was confirmed by extensive pericellular type-II collagen accumulation.

Conclusions

Autogenous fibrinogen has an inherent capacity to maintain chondrocyte phenotypic metabolism that is reduced or absent in commercially prepared fibrinogen. Enhanced, differentiated cell function may be useful for in vivo applications, but represents an added variable that may confound in vitro experiments, and should be considered when designing studies of chondrocyte function. (Am J Vet Res 1998;59:514–520)

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in American Journal of Veterinary Research

. Stata, version 10.0, StataCorp, College Station, Tex. References 1. Eufinger H Rasche C Lehmbrock J , et al . Performance of functionally graded implants of polylactides and calcium phosphate/calcium carbonate in an ovine model for

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in American Journal of Veterinary Research

.3 ± 0 Calcium (mg/dL) 8.0–10.4 7.8 ± 0.6 10.0 ± 0.4 Phosphorus (mg/dL) 3.6–7.3 7.3 ± 1.3 5.7 ± 0.8 Potassium (mg/dL) 4.1–5.5 5.7 ± 0.5 5.2 ± 0.4 Urinalysis was performed for 1 male and 2 female ferrets

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in American Journal of Veterinary Research

, gastroprokinetic agents, antihistamines, opioids, anticholinergics, tricyclic antidepressants, calcium channel blockers, progesterone compounds, corticosteroids, synthetic hormones or other hormone treatments, antacids, and β-adrenergic receptor agonists or

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in American Journal of Veterinary Research

more pronounced in bone because of the high calcium content 37 and subsequent absorption of x-rays. 38 Attempts to optimize corrections for beam hardening exist in clinical applications 37 and in industrial metrological applications. 38

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in American Journal of Veterinary Research

.8, total alkalinity of 150 to 200 ppm, and calcium hardness of 60 to 100 ppm. Water temperature was maintained at 32°C. The tank was filled to the level of the greater trochanter, as described elsewhere. 24 The first session in the UWTM was also used for

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in American Journal of Veterinary Research

through deposition of calcium salts. 24 Compromised epithelium was apparent on the basis of subserosal dissection by infusate with increasing amounts of infused solution. Damage to capillaries in the lamina propria may have contributed to increased

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in American Journal of Veterinary Research

calcium, phosphorus, sodium, and potassium concentrations; Hct; and total leukocyte and platelet counts. Thromboelastography —Thromboelastography was performed on citrated plasma samples with diluted recombinant human tissue factor h as an activator

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in American Journal of Veterinary Research

. Polyglactin 910 is a multifilament suture material composed of a copolymer of lactic acid and glycolic acids that are coated with calcium stearate and a second copolymer of glycolide and lactide. Absorption of polyglactin 910 is through hydrolysis, and

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in American Journal of Veterinary Research

aminotransferase, alkaline phosphatase, γ-glutamyl transferase, amylase, and lipase activities and measurement of glucose, BUN, creatinine, cholesterol, total bilirubin, albumin, total protein, fructosamine, bile acids, calcium, magnesium, phosphate, sodium, and

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in American Journal of Veterinary Research