Delia Ertl

Biochemical differences between dystrophic calcification of cross-linked collagen implants and mineralization during bone induction

SummaryEctopic calcification of diseased tissues or around prosthetic implants can lead to serious disability. Therefore, calcification of implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen was compared with mineralization induced by demineralized bone matrix (DBM). Whereas implants of DBM accumulated large amounts of calcium and a bone-specific γ-carboxyglutamic acid protein (BGP or osteocalcin) following implantation in both young and older rats, implants of cross-linked pericardium calcified with only traces of BGP. Glutaraldehyde-cross-linked DBM failed to calcify after implantation in 8-month-old rats for 2–16 weeks. Implants of cross-linked type I collagen exhibited small calcific deposits 2 weeks postimplantation but calcium content eventually dropped to levels equal to those of soft tissues as the implants were resorbed. The calcium content of DBM implanted in 1- and 8-month-old rats reached comparable levels after 4 weeks, but the BGP content was approximately twice as high in the younger animals than in the older ones. Glutaraldehyde-cross-linked implants of DBM, tendon, and cartilage calcified significantly in young but not in old animals. This form of dystrophic calcification was associated with only trace amounts of BGP. Alkaline phosphatase activity was high in implants of DBM and undetectable in implants of cross-linked collagenous tissues. These results show that implants of glutaraldehyde-cross-linked collagenous tissues and reconstituted collagen calcify to different extents depending upon their origin and the age of the host, and that the mechanism of dystrophic calcification differs significantly from the process of mineralization associated with bone induction as reflected by alkaline phosphatase activity and BGP accumulation.

Mechanism of Crosslinking of Proteins by Glutaraldehyde IV: In Vitro and In Vivo Stability of a Crosslinked Collagen Matrix

The use of native or reconstituted collagen as a bioprothesis for tissue augmentation requires the introduction of exogenous synthetic crosslinks. The degree of crosslinking determines the rate of resorption or replacement of the implanted materials by the host. Since biophysical and chemical methods to quantify these crosslinks have in general been difficult to evaluate, we have developed in vitro enzymatic approaches which enable us to correlate the degree of crosslinking with the rates of enzymatic degradation. When the number of stable crosslinks formed is large it is essential to partially unfold the collagen fibrils by heating or by exposure to denaturing agents to enhance their susceptibility to hydrolysis. In the present study we demonstrate that increasing the number of reactive amino groups on collagen by coupling 1,6-diaminohexane to carboxyl groups using a water soluble carbodiimide can significantly enhance the number of crosslinks introduced by glutaraldehyde. We also show that the enzymatic method developed correlates well with the biodegradation of radiolabeled crosslinked collagenous tissues implanted subcutaneously in rats.

New evidence and new hope concerning endothelial seeding of vascular grafts.

Endothelial cell (EC) seeding of prosthetic bypass grafts has been promoted as a method of improving graft patency. However, an efficient and reliable method of seeding vascular prostheses with ECs is lacking due to inefficient harvesting of ECs and poor attachment and proliferation of cells on the prosthetic surfaces. To investigate the effect of a commonly used prosthetic surface on EC attachment and proliferation, we measured the attachment and proliferation of ECs on polytetrafluoroethylene (PTFE) grafts uncoated or coated with gelatin, laminin, fibronectin, collagen type I and/or III, or RGD (arginine-glycine-aspartate)-containing peptide. EC attachment and proliferation were both significantly decreased on the untreated PTFE graft surface. Conversely, coating of PTFE with fibronectin, RGD, laminin, or gelatin significantly (p less than 0.05) improved the attachment of ECs, with the most striking increases occurring with laminin and gelatin. Similarly, all matrix components in this study improved EC proliferation compared with untreated PTFE, with RGD and gelatin producing the most significant improvement. PTFE adversely effects EC attachment and proliferation. These properties can be improved by treating PTFE graft surfaces with extracellular matrix components in relatively low concentrations. Future investigations are needed to determine whether there are combinations and concentrations of matrix components that will optimize these cellular functions on vascular prostheses.

Ectopic bone formation is enhanced in senescent animals implanted with embryonic cells

Ectopic bone formation induced by the subcutaneous implantation of demineralized bone matrix (DBM) is very significantly reduced in older Fischer 344 rats. Cells originating from calvaria of 20-day-old embryo donors were introduced into cylinders of DBM sealed at the ends with a Millipore filter or collagen sponges prior to subcutaneous implantation. Cells within the chambers had access to vascular channels that could penetrate through the interstices of the DBM. After four weeks of implantation in 26-month-old rats, the cylinders were full of bone. This bone was assessed by histologic techniques, by calcium and bone gamma-carboxyglutamic acid (gla) protein (BGP) concentrations, and by alkaline phosphatase activity. Cylinders to which no cells were added produced no bone. Bone marrow cells enclosed in similar cylinders or injected weekly at the implantation site also enhanced new bone formation but to a much lesser extent. Embryonic muscle cells formed large amounts of cartilage and less bone. Fibroblasts were inactive in this system. Prior treatment of the DBM with trypsin inhibited the myoblast response but not that of calvaria cells.

A Modified rhTGF-β1 and rhBMP-2 Are Effective in Initiating a Chondro-Osseous Differentiation Pathway in Bone Marrow Cells Cultured In Vitro

Rat bone marrow cells were cultured in vitro in a collagen-gel medium at 0.5% fetal bovine serum concentration for 10 days in the presence of recombinant human transforming growth factor-beta-1, genetically engineered to contain a collagen binding domain (rhTGF- g 1-F2), or a commercial rhTGF- g 1. To compare the effects of TGF- g s with other growth factors in which the osteogenic capacity has been widely documented, a recombinant human bone morphogenetic protein (rhBMP-2) was evaluated. Once serum conditions compatible with growth were re-established, the selected cells were cultured for 6 more days in the presence of the growth factor. In the last 2 days, dexamethasone (dex) and g -glycerophosphate ( g -GP) were added to promote osteogenesis. After this 16-day period, cells were placed into diffusion chambers or demineralized bone matrix (DBM) implants, and implanted subdermally on the backs of rats for 28 days. Biochemical, histological, and immunohistochemistry analysis provided evidence of cartilage (commercial rhTGF- g 1-treated cells), osteoid (rhTGF- g 1-F2-treated cells), and bone tissues (rhBMP-2 treated cells), inside the diffusion chambers, whereas bone, cartilage, and osteoid were observed inside the DBM implants under any of the three growth factors effect. Our study advances the technology capable of selecting a cell population from bone marrow that, in the presence of rhTGF- g 1 or rhBMP-2 in vitro, achieves chondro-osteogenic potential in vitro and in vivo.

Distribution of griseofulvin in the rat: comparison of the oral and topical route of administration.

Abstract— Effective penetration of griseofulvin across the dermal barrier has been achieved using an anhydrous solvent system of benzyl alcohol (10%), acetone (40%), and isopropanol (50%). There were quantitative differences in the relative accumulation of griseofulvin in skin compared with internal organs, when the topical and oral routes of administration were compared. The topical route enhanced localized concentrations of griseofulvin at the site of application, and these persisted for several days. After daily topical application a steady state was reached at day 3, when the diffusion across the skin barrier and epidermal loss seemed to equal the total amount applied to the skin surface. The application of griseofulvin topically, required a much smaller amount of drug to achieve similar integumentary levels compared with the amount required orally.

DYSTROPHIC CALCIFICATION AND MINERALIZATION DURING BONE INDUCTION: BIOCHEMICAL DIFFERENCES

The calcification of implants of glutaraldehyde-crosslinked collagenous tissues and collagen was studied in young and old rats and compared to bone induction by non-crosslinked osteogenically active demineralized bone matrix (DBM). Glutaraldehyde-crosslinked implants of DBM, tendon, and cartilage calcified in young but not in old animals and accumulated only trace amounts of BGP (Bone Gla protein, osteocalcin). Alkaline phosphatase activity was high in implants of DBM and undetectable in crosslinked implants. To try and understand why bone formation is so significantly reduced in older Fischer-344 rats, we developed a system which consists of cylinders of DBM sealed at the ends with a Millipore filter. Cells originating from 20-day-old embryo donors were introduced into the chambers prior to subcutaneous implantation. After 4 weeks of implantation in 26-month-old rats, the cylinders containing embryonic calvaria or muscle calls were found to be full of bone and/or cartilage.