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Critical pathways help institutions in efficient and appropriate resource use to increase the quality of health care and minimize health care costs. However, many opportunities for pathway development and implementation are unexplored. This article delineates the development process for critical pathways and discusses the outcomes realized from use of the total joint pathway at the Medical College of Georgia, Augusta, GA.

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To overcome rapid diffusion and clearance from the implant site and to increase stability, recombinant transforming growth factor beta2 (TGF-beta2) was covalently bound to injectable bovine dermal fibrillar collagen (FC) and its activity compared to admixed TGF-beta2. Covalent binding was achieved in a two-step procedure: First, TGF-beta2 was reacted with the difunctional polyethylene glycol (PEG) linker, and then the PEG-attached TGF-beta2 (PEG-TGF-beta2) was bound to the fibrillar collagen (FC-PEG-TGF-beta2). Initial binding of TGF-beta2 to difunctional succinimidyl glutarate (D-SG-PEG) or succinimidyl propionate polyethylene glycol (D-SE-PEG) linkers was completed after reacting for 8 or 10 min as monitored by reverse-phase high-performance liquid chromatography. After reaction with injectable fibrillar collagen, extraction of unbound PEG-TGF-beta2 and Western blot analysis, using a TGF-beta specific antibody, demonstrated that at least 85% of the TGF-beta2 was bound to the fibrillar collagen. The activity of PEG-TGF-beta2 was fully stable in phosphate-buffered saline at 4 degrees C and 37 degrees C for at least up to 4 weeks. Unmodified TGF-beta2 mixed with fibrillar collagen was completely inactivated after 1 week of incubation, as measured by the mink lung epithelial cell (Mv1Lu) growth inhibition assay. Formulations of FC-PEG-TGF-beta2 containing 40 microg/ mL TGF-beta2 were implanted subcutaneously into rats and analyzed after days 7, 21, and 42. All TGF-beta2-containing formulations showed the TGF-beta typical fibroblastic response at the day 7 time point. Covalent binding of TGF-beta2 to collagen with both difunctional PEG crosslinkers resulted in a significantly stronger and longer-lasting TGF-beta2 response than that observed with admixed formulations of collagen and TGF-beta. The TGF-beta response with FC-PEG-TGF-beta2 lasted up to day 42 but was not seen after day 7 for TGF-beta2 admixed to FC. These findings clearly demonstrate that TGF-beta2 remains fully active after being covalently bound to collagen via difunctional PEG. In addition, covalent binding potentiates and prolongs in vivo TGF-beta responses and stabilizes the TGF-beta in vitro. Results suggest that this method of formulation could be useful to stabilize and deliver similar peptide growth factors or biologically active agents.

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When applied at low concentrations (< 10 micrograms/ml), staphylococcal alpha-toxin generates a small channel in keratinocyte and lymphocyte membranes that permits selective transmembrane flux of monovalent ions. Here we show that a moderate concentration (1-50 micrograms/ml) of alpha-toxin similarly produces a small pore in membranes of human fibroblasts. This process leads to rapid leakage of K+ and to a drop in cellular ATP to 10-20% of normal levels in 2 h. In the presence of medium supplemented with serum and at pH 7.4, the cells are able to recover from toxin attack, so that normal levels of K+ and ATP are reached after 6-8 h at 37 degrees C. The repair process is dependent on the presence of serum in the medium and is very sensitive towards pH. Decreases of pH in the medium to < or = 7.0 as well as increases to > or = 7.8 causes the repair mechanism to fail. The fate of cell-bound toxin molecules was investigated by using a radiolabelled tracer and by immunological detection of toxin exposed at the cell surface. The results indicated that 50-70% of the toxin was shed from cell membranes. However, there was no clear correlation between shedding and recovery, and shedding was also observed in cells that died at pH 7.8. Shedding was not decisive for repair, since cells that had recovered from toxin attack continued to carry 30-40% of initially bound toxin on their cell surface. Blockade of Na+/K(+)-ATPases with ouabain evoked similar kinetics of K(+)-depletion in control cells, compared with cells that had just recuperated from toxin attack and that still carried 30-40% alpha-toxin on their surface. We therefore tentatively concluded that repair of alpha-toxin lesions was due to closure of small pores, rather than from compensation of membrane leaks by up-regulation of Na+/K(+)-ATPase activity. We speculate that repair of small membrane lesions may extend to other agents that produce channels of similar nature in nucleated cells. Larger pores created by E. coli hemolysin or streptolysin O, both of which form larger functional transmembrane lesions, could not be repaired by fibroblasts.

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Transforming growth factor beta is a multifunctional protein with known actions on bone and bone cells. The ability of TGF-beta to stimulate osteogenic parameters in vitro and osteogenesis adjacent to injection sites in vivo is well established. The purpose of this study was to determine if systemic administration of recombinant TGF-beta 2 (rTGF-beta 2) could stimulate bone formation in rats of different ages. Juvenile (25-day-old) and adult (160-day-old) rats were treated daily for 5 days and 14 days, respectively, with rTGF-beta 2 given by subcutaneous injection. Bone formation was measured in cancellous bone of the lumbar vertebrae in juvenile rats and the femoral epiphysis in adult rats. Endochondral bone growth rates were measured in the distal femurs from both juvenile and adult rats using histomorphometric methods. Systemic administration of rTGF-beta 2 resulted in substantial increases in bone formation rates (both surface and volume referent) in both juvenile and adult rats. In the juvenile rats, rTGF-beta 2 increased the percent double labeled surface and the mineral appositional rate. In the adult rats, TGF-beta 2 treatment increased the double labeled surface and also endochondral (longitudinal) growth parameters without changing the number of osteoclasts or the number of osteoclast nuclei per cell. These results demonstrate that short-term systemic administration of rTGF-beta 2 substantially increases cancellous bone formation rate in rats.

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Six monoclonal antibodies (mAb) are described that bound to the bovine bone glycoprotein, osteoglycin. The protein osteoglycin was originally called Osteoinductive Factor (OIF). The antibodies were characterized with respect to their reaction patterns in Western blots, indirect immunoprecipitation, and binding epitope. The antibodies bound to one of two sequential determinants, either residues 62-76 or 95-105, in the C-terminal region of mature osteoglycin. One mAb, 2C11, was found to be useful for affinity purification of osteoglycin. Another mAb, 3B2, was able to immunohistochemically stain osteoblasts, osteocytes, chondrocytes, occasional osteoclasts and nail bed epithelial cells in rat neonatal forelimb. The mAbs will provide an essential tool for the further characterization of this unique glycoprotein.

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We studied the effects of highly purified bone morphogenetic protein 2 and 3 (BMP-2 and -3) on growth plate chondrocytes and osteoblastic cells in vitro and compared to TGF-beta. A mixture of BMP-2 and 3 (BMPs) strongly stimulated DNA synthesis of chondrocytes in the presence of fibroblast growth factor (FGF). BMPs induced rapid maturation of chondrocytes at a growing stage: BMPs transformed the cells into rounded cells and induced marked accumulation of cartilage matrix; TGF-beta slightly reduced matrix accumulation and changed cell morphology into spindle-like in the presence of FGF. Moreover, exposure of chondrocytes to BMPs resulted in a dramatic increase of the putative approximately 80 kD PTH receptors expressed on the cell surface. In multilayered chondrocytes at the calcifying stage, BMPs stimulated alkaline phosphatase (ALPase) activity but TGF-beta inhibited it. In osteoblastic MC3T3-E1 cells, BMPs were found to be the most potent stimulator of ALPase activity thus far described: ALPase in the cells treated with approximately 100 ng/ml of BMPs reached 5- to 20-fold over the basal, whereas TGF-beta inhibited expression of ALPase activity in these cells. The stimulatory action of BMPs overrode the inhibition of ALPase activity by TGF-beta when the cells were incubated with TGF-beta and BMPs. BMPs also upregulated expression of the approximately 80 kD PTH receptor on the cells. These results suggest that BMPs have unique biologic activities in vitro that lead to growth and phenotypic expression of cells playing a critical role in endochondral bone formation.

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We previously identified a novel glycoprotein in an osteoinductive fraction from bovine bone (Bentz et al.: Amino acid sequence of bovine osteoinductive factor. J. Biol. Chem. 265: 5024-5029, 1990). We now find that this fraction also contained small amounts of bone morphogenetic protein-2 and 3 (BMP-2 + 3) previously identified by others (see Wozney, J.M.: Bone morphogenetic proteins. Prog. Growth Factor Res. 1: 267-280, 1990). Separation of BMP-2 + 3 from the glycoprotein was achieved with a modified reversed phase-high pressure liquid chromatographic procedure. When assayed in the rat subcutis using a collagen-ceramic carrier, the osteoinductive activity was found in the subfraction containing BMP-2 + 3. This activity was potentiated and the ratio of cartilage to bone was increased by transforming growth factor-beta 2. The glycoprotein, originally called osteoinductive factor, has been renamed osteoglycin. In its precursor form, osteoglycin is a member of the leucine-rich family of proteins showing the characteristic 24-residue internal homology. Its biological function is unknown.

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cDNA clones encoding osteoinductive factor (OIF) have been isolated from a bovine osteoblast library. Sequence analysis of these clones indicated that the 105-amino-acid OIF is synthesized as a larger 299-amino-acid precursor, the carboxyl terminus of which is cleaved to yield the mature protein. Northern blot analysis of bovine osteoblast mRNA revealed two OIF-specific transcripts of 1.9 and 2.4 kb. The polymerase chain reaction was used to obtain clones coding for human OIF from the osteosarcoma cell line, MG-63. The human OIF cDNA encodes a precursor of 298 amino acids that exhibits 94% identity to the bovine protein. Northern blot analysis of various cell lines and tissues indicated that expression of OIF transcripts is limited and may be restricted to cells of bone lineage.

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The complete amino acid sequence of bovine osteoinductive factor (OIF) was determined by automated Edman degradation of S-pyridylethylated bovine OIF and selected fragments. Cleavage with endoproteinase Lys-C, endoproteinase Glu-C, or endoproteinase Asp-N established all fragments in an unambiguous sequence. Bovine OIF contains 105 residues with a calculated molecular weight of 12,055. It is a single chain polypeptide containing two intramolecularly linked cysteines at residues 62 and 95. Two asparagine-linked glycosylation sites at positions 52 and 65 were found by comparing sequence data and peptide profiles of native and deglycosylated OIF fragments. The amino acid sequence of OIF has no homology to other reported proteins.

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A unique protein that promotes ectopic osteoinduction in the rat has been isolated and characterized. Osteoinductive factor (OIF) was extracted from the organic matrix of bovine bone with 4 M guanidine HCl and purified by gel filtration, ion-exchange chromatography, affinity chromatography, and reversed phase high performance liquid chromatography. OIF is a glycoprotein with an apparent molecular mass of 22-28 kDa based on sodium dodecyl sulfate gel electrophoresis. Enzymatic or chemical deglycosylation of OIF reduces its mass to about 12 kDa with apparent loss of activity. OIF activity in the model used is substantially increased by addition of transforming growth factor (TGF)-beta 1 or TGF-beta 2, suggesting an important role for TGF-beta 1 and -2 in bone regeneration and repair. The N-terminal sequence of OIF has no homology to other reported proteins.

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In a multi-generation trial with 120 or 96 hens of the breed White Leghorn per group the effect of diets containing 7.5% or 15.0% of methanol-grown dried bacterial cells was examined. The hens were kept in conventional three-stage battery. In two of the three experiments the feed intake was decreased if it was used methanol-grown bacterial cells as a protein source. The period of intensive laying (50% egg production) started later and the egg production has been decreased by using the bacterial cells. For the traits egg weight and mortality no relation to the nutrition could be found. The feeding of methanol-grown bacterial cells showed in two incubatory trials no negative effects on reproductive ability of the hens. The cock's sperm quality was not influenced in two experiments. In one trial the sperm volume was reduced and the sperm concentration was raised if diets contained methanol-grown dried bacterial cells. Indications of toxic influences of the methanol-grown bacterial cells were not found.

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Subcutaneous (S.C.) implantation of allogeneic demineralized bone matrix in rats results in endochondral bone formation. In contrast, implants of bovine demineralized bone matrix in rat S.C. tissue show inconsistent cartilage and bone formation, presumably due to an intense inflammatory reaction at the implant site. To overcome this response, a partially purified bone inducing extract was prepared from bovine bone by a series of steps that included demineralization, guanidine/HCl extraction, gel filtration, and cation exchange chromatography. To develop a carrier, the inactive guanidine/HCl-extracted matrix was then trypsinized to remove the inflammatory and immunogenic components, thus yielding a predominantly collagenous matrix. Bovine composites were prepared by combining different amounts of the bone inducing extract with a carrier that consisted of the trypsinized bone matrix and purified soluble bovine dermal collagen. Subcutaneous implantation of the composite preparation resulted in dose-dependent endochondral bone formation in rats. The inductive activity and the low-level inflammatory response were comparable to allogeneic implants.

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Problems of drug supply by veterinary doctors are discussed and explained. It has to be pointed out that the cancellation of drug substances needed for drugs and veterinary drugs, respectively, can lead to a sensible gap in the veterinary drug assortment, which is often difficult to compensate. Proposals to solve this problem are offered. In addition a growing demand on the treatment of small animals and pets, which are kept in increasing numbers, has to be considered. An adequate training and further education of pharmaceutical staff would be recommendable to meet the demands of an effective drug supply as well as a sufficient ability to give proper advice to proprietors of animals at the pharmacist's. This is already realised in Czechoslovakia in an exemplary way.

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Cartilage-inducing factors-A (CIF-A) and -B (CIF-B), purified from bovine bone on the basis of their ability to induce the cartilage phenotype in vitro, are proteins with molecular weights of 26,000 composed of two apparently identical disulfide-linked chains. CIF-A is apparently identical to TGF-beta from human platelets (Seyedin S. M., Thompson, A. Y., Bentz, H., Rosen, D. M., McPherson, J. M., Conti, A., Siegel, N. R., Galluppi, G. R., and Piez, K. A. (1986) J. Biol. Chem. 261, 5693-5695). We have now found that, like CIF-A and TGF-beta, CIF-B induces anchorage-independent proliferation of NRK-49F cells when these cells are simultaneously treated with epidermal growth factor. Furthermore, CIF-B competes with CIF-A for the same cell membrane receptors in NRK-49F cells. Partial amino acid sequencing reveals that CIF-B is a distinct molecule with extensive homology to CIF-A/TGF-beta. These results show that CIF-B and TGF-beta are structurally and functionally similar molecules, but differ more from each other than does TGF-beta from different species.

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Two apparently homologous proteins, designated CIF-A and CIF-B, were previously isolated from bovine bone on the basis of their cartilage-inducing activity in culture. CIF-A has been shown to probably be identical to transforming growth factor beta (TGF-beta). To address the question of tissue localization, antibodies to CIF-A were produced using a synthetic polypeptide identical to N-terminal residues 1-30. The antibodies were immunoreactive with bovine CIF-A and human TGF-beta, did not recognize CIF-B, and did not recognize other molecular weight species in crude bovine bone extracts. The antibodies were used to immunohistochemically localize CIF-A/TGF-beta in fetal bovine bone and other tissues. There was abundant staining of osteocytes throughout cancellous and cortical bone as well as chondrocytes within the articular cartilage, although growth plate-associated chondrocytes were not labeled. In addition, immunoreactive cells were detected in bone marrow (megakaryocytes and some mononuclear cells), fetal liver (hematopoietic stems cells), and the thymus (Hassall's corpuscle and some medullary thymocytes). In the kidney, the antibodies labeled a population of epithelial cells lining the calyces. Tissues which did not have detectable amounts of CIF-A/TGF-beta included the thyroid, adrenal, salivary gland, and aorta. Results presented here suggest that the factor may function in vivo as a general development and repair factor and may play a significant role in the differentiation of many cell types including chondrocytes, osteocytes, T-lymphocytes, and red blood cells.

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Comparison of the sequence of the N-terminal 30 amino acids of cartilage-inducing factor-A (CIF-A) from bovine demineralized bone with the corresponding sequence of human transforming growth factor-beta (TGF-beta) revealed 100% identity. Furthermore, CIF-A stimulated normal rat kidney fibroblasts to become anchorage-independent and form colonies in soft agar (in the presence of epidermal growth factor) in a manner similar to TGF-beta. Similarly, TGF-beta from human platelets induced rat muscle mesenchymal cells to differentiate and synthesize cartilage-specific macromolecules in a manner equivalent to CIF-A. These data show that CIF-A and TGF-beta are closely related or identical molecules and that these factors may be involved in cell differentiation including cartilage formation as the first step in endochondral bone formation.

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We recently reported the partial characterization of a new human collagen termed Type VII. This molecule is distinctive among the collagen family in that it contains three identical subunit alpha chains within a triple helical domain 424 nm in length. The molecule contains three identical alpha chains which are genetically distinct from other known collagens. Previous studies indicate that a portion of the limited pepsin-solubilized molecules appears to exist as antiparallel dimers associated by disulfide bonds. In this report, we demonstrate that the major tissue form of Type VII collagen is a dimer, associated by disulfide bonds through a 60-nm overlap of the aminoterminal triple helical ends. Intermolecular disulfide bonds occur only within this overlap region. Interchain disulfide bonds exist in the carboxyl terminal 7% of the molecule and may exist within the overlap region as well. Disulfide bond-stabilized aggregates larger than dimers are not seen.

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In a 3-generation experiment with a total of 2520 hens and 210 cocks of the species 'White Leghorn' kept in cages, the compatibility of 5.0%, 7.5% and 15% 'fermosin' torula yeast in the mixed feed ration was tested under long-term toxicologic aspects. The parameters investigated and relevant for the toxicological statement, with high probability, did not show a negative influence of the test ration. Thus, a good compatibility of the tested yeast product 'fermosin' for laying hens can be stated.

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In a multi-generation experiment with 120 hens of the species White Leghorn per group kept in cages, the influence of 5%, 7.5% and 15% 'fermosinR' torula yeast in the ration of mixed feed on various performance parameters in the 364-day laying period was investigated under long-term toxicologic aspects. In all probability there is no indication of toxic influences. The compatibility of the biomass can be considered good provided the basic nutrition-physiologic demands are met.

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