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


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.


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.


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.


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)

Free access
in American Journal of Veterinary Research


Cartilage resurfacing by chondrocyte transplantation, using porous collagen matrices as a vehicle to secure the cells in cartilage defects, has been used experimentally in animals. This in vitro study evaluated the temporal morphologic features and proteoglycan synthesis of chondrocyte-laden collagen matrices. Forty-two porous collagen disks were implanted with a minimum of 6 × 106 viable chondrocytes, covered by a polymerized collagen gel layer, and 6 disks were harvested after 0, 3, 7, 10, 14, 18, or 22 days of incubation in supplemented Ham's F12 medium at 37 C and 5% CO2. Histologic and histo-chemical evaluation of formalin-fixed segments of the cultured disks indicated that the chondrocytes proliferated in the implant, producing small groups and linear segments of cells by day 14. The collagen framework remained intact over the course of the study with thick areas attributable to depositions of matrix material after day 10. Alcian blue-stained matrix was evident in the pericellular region of chondrocytes in sections of disks harvested on days 14, 18, and 22.

Glycosaminoglycan (GAG) assay by dimethyl-methylene blue dye binding after papain digestion of the disk segments revealed negligible amounts of GAG at day 0. Significant (P ≤ 0.0001) increase in total GAG content was observed by day 3 (0.329 M-μg of disk) and further increases were observed until a plateau in GAG quantity was seen on day 14. Mean peak GAG content was 0.553 ± 0.062 μg/mg. Secondary treatment of the papain-digested implants with keratanase and chondroitinase ABC yielded similar trends in chondroitin sulfate (CS) and keratan sulfate (KS) concentrations. The CS content significantly (P = 0.0002) increased for the first 14 days of incubation, then a plateau was observed for the remainder of the study. Peak CS content was 0354 ± 0.037 μg/mg. Concentration of KS reached a plateau earlier than did CS content, with peak amount of 0.193 ± 0.027 μg/mg on day 10. Fluctuations in K5 content were not significant until an increase on day 22.

Chondrocytes actively populated the collagen implants, increasing in number and synthesizing matrix GAG epitopes over the 22 days of incubation. These results indicate that chondrocyte-laden porous collagen matrices may be suitable cartilage analogue materials and the optimal metabolic time for transfer to cartilage defects is 10 to 14 days.

Free access
in American Journal of Veterinary Research