Date of Award

Spring 1993

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Program/Concentration

Biomedical Sciences

Committee Director

Lloyd Wolfinbarger, Jr.

Committee Member

Christopher J. Osgood

Committee Member

Mark S. Elliot

Committee Member

Mickey Castle

Abstract

The purpose of this research was to study the effects of cryopreservation on the proteoglycan (PG) and mineral content of aorta conduit tissue. Proteoglycans from fresh and cryopreserved porcine aorta conduit tissues were isolated by extraction with 4 M guanidine (Gdn) - hydrochloride (HCl) for 48 hours. Quantitative analysis of extracted proteoglycans revealed that the content of proteoglycan material from cryopreserved tissue, measured as uronate and protein per unit weight of wet tissue, was similar to that from fresh tissue. Gel permeation column chromatography studies suggested that proteoglycans present in three CsCl fractions from cryopreserved tissues had molecular weights similar to proteoglycans present in similar fractions from fresh tissue.

Cryopreserved and fresh tissues were extracted using Gdn-HCl followed by sequential digestion of the tissues with collagenase, elastase, and papain. Glycosaminoglycans (GAGs) of the PGs were isolated and quantitated. Gdn-HCl extracted about 61% and 62% of the total GAG (proteoglycan) material from cryopreserved and fresh tissues, respectively. Collagenase solubilized proteoglycans from Gdn-HCl-extracted tissue represented 20% and 13%, respectively, of the total GAGs present in cryopreserved and fresh tissue. Subsequent elastase hydrolysis of collagenase digested tissue released about 11% of total GAGs from cryopreserved tissue and 16% from fresh tissue. The remaining 8%, from cryopreserved tissues, and 9%, from fresh tissue, of total GAGs were obtained through use of a final papain hydrolysis.

The histologic status of the fresh and cryopreserved porcine aortic conduit did not differ markedly. The normal tissue architecture was not affected markedly by the cryopreservation procedure as neither alteration of elastic structure, fibrous proteins nor alteration of nuclear distribution or smooth muscle cell morphology was detected.

Quantitative tissue mineral studies revealed that the mean calcium content of the cryopreserved aorta conduit tissue was higher than that of the fresh tissue. The mean phosphorus content was 703 +/- 35 ug/g wet tissue from cryopreserved tissue and 720 +/- 26 ug/g wet tissue from fresh tissue.

The studies indicate that there is no significant alteration in the content, molecular size, or distribution of PGs in properly cryopreserved tissue. However, the total calcium level appears to be increased in cryopreserved tissue

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/p3xg-hv56

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