Fred W. Keeley

Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion: a follow-up report at one year

An autogenous graft of tibial periosteum was sutured (with its cambium layer facing into the joint) to the base of a five by ten-millimeter full-thickness defect in the patellar groove of each of forty-five adolescent rabbits. The rabbits were randomly treated postoperatively by either four weeks of immobilization in a cast, intermittent active motion in a cage, or two weeks of continuous passive motion. One year postoperatively, the regenerated tissue from each rabbit was analyzed macroscopically, histologically, histochemically, and biochemically. Gross degenerative changes were seen in 57 per cent of the rabbits that had been immobilized in a cast, in 73 per cent of the rabbits that had been allowed intermittent active motion, and in 22 per cent of the rabbits that had been subjected to continuous passive motion (p less than 0.05). Out of a possible score of 7.0 points for the nature of the regenerated tissue, the scores for the three groups were: immobilization in a cast, 4.1 points; intermittent active motion, 4.0 points; and continuous passive motion, 5.9 points (p greater than 0.05). Out of a possible perfect combined score of 10.0 points for the structural characteristics of the regenerated tissue, the cast-immobilization group scored 3.8 points; the intermittent active-motion group, 2.5 points; and the continuous passive-motion group, 6.4 points (p less than 0.001). The total scores for freedom from cellular changes of degeneration, a perfect score being 5.0 points, were: immobilization in a cast, 2.4 points; intermittent active motion, 2.3 points; and continuous passive motion, 3.9 points (p less than 0.01). Degenerative changes in the adjacent cartilage, which were noted in 42 and 46 per cent of the knees in the immobilization and intermittent active-motion groups, respectively, were not found in the knees that had been subjected to continuous passive motion (p less than 0.05). The total indices, which were derived by combining the scores for all categories (maximum, 24.0 points), revealed that the index for the continuous passive-motion group was significantly better than the index for either of the other two groups: immobilization in a cast, 12.9 points; intermittent active motion, 11.2 points; and continuous passive motion, 19.2 points (p less than 0.0005).(ABSTRACT TRUNCATED AT 400 WORDS)

The chondrogenic potential of free autogenous periosteal grafts for biological resurfacing of major full-thickness defects in joint surfaces under the influence of continuous passive motion. An experimental investigation in the rabbit.

A rectangular graft of autogenous tibial periosteum was sutured (with its cambium layer facing into the joint) onto the base of a five by ten-millimeter full-thickness defect in the patellar groove of each of 143 adolescent and adult rabbits. The rabbits were managed postoperatively by either immobilization, intermittent active motion, continuous passive motion for two weeks, or continuous passive motion for four weeks. When the animals were killed four weeks postoperatively, the contour of the patellar groove had been restored in all of the rabbits in the group that had had four weeks of continuous passive motion, and the newly formed tissue in all of the defects in this group had the gross, histological, and histochemical appearance of smooth, intact hyaline articular cartilage. Histologically, the nature of the tissue that had formed, as well as its surface regularity, structural integrity, and bonding to the adjacent cartilage, were significantly better in the group that had had four weeks of continuous passive motion than in any of the other groups. The results were significantly worse when the orientation of the periosteal graft was reversed (that is, when it had been sutured into the defect with the cambium layer of the graft facing the subchondral bone rather than into the joint) or when no periosteal graft was used. Biochemical analyses revealed that, in the group that had had four weeks of continuous passive motion, the total hexosamine content, the levels of chondroitin sulphate and keratan sulphate, and the ratio of galactosamine to glucosamine were all comparable with the values for normal articular cartilage. In contrast, in the groups that were treated by immobilization, intermittent active motion, or two weeks of continuous passive motion, as well as in the adult rabbits, the content of the first three of these substances was significantly less than normal. In the groups that were treated by immobilization, intermittent active motion, or two weeks of continuous passive motion, 32 to 47 per cent of the total collagen was type II, while in the group that had had four weeks of continuous passive motion, 93 per cent of the total collagen was type II. These results demonstrate that, under the influence of continuous passive motion, free autogenous periosteal grafts can repair a large full-thickness defect in a joint surface by producing tissue that resembles articular cartilage grossly, histologically, and biochemically, and that contains predominantly type-II collagen.

In Vivo Collagen Turnover Following Experimental Balloon Angioplasty Injury and the Role of Matrix Metalloproteinases

Extracellular matrix formation is the major component of the restenosis lesion that develops after balloon angioplasty. Although ex vivo studies have shown that the synthesis of collagen is stimulated early after balloon angioplasty, there is a delay in accumulation in the vessel wall. The objectives of this study were to assess collagen turnover and its possible regulation by matrix metalloproteinases (MMPs) in a double-injury iliac artery rabbit model of restenosis. Rabbits were killed at four time points (immediately and at 1, 4, and 12 weeks) after balloon angioplasty. In vivo collagen synthesis and collagen degradation were measured after a 24-hour incubation with [14C]proline. Arterial extracts were also run on gelatin zymograms to determine MMP (gelatinase) activity. Collagen turnover studies were repeated in a group of 1-week postangioplasty rabbits that were treated with daily subcutaneous injections of either a nonspecific MMP inhibitor, GM6001 (100 mg/kg per day), or placebo. Collagen synthesis and degradation showed similar temporal profiles, with significant increases in the balloon-injured iliac arteries compared with control nondilated contralateral iliac arteries immediately after angioplasty and at 1 and 4 weeks. Peak collagen synthesis and degradation occurred at 1 week and were increased (approximately four and three times control values, respectively). Gelatin zymography was consistent with the biochemical data by showing an increase of a 72-kD gelatinase (MMP-2) in the balloon-injured side immediately after the second injury, peaking at 1 week, and still detectable at 4 and 12 weeks (although at lower levels). In balloon-injured arteries, the MMP inhibitor reduced both collagen synthesis and degradation. Overall, at 1 week after balloon angioplasty, GM6001 resulted in a 33% reduction in collagen content in balloon-injured arteries compared with placebo (750 +/- 143 to 500 +/- 78 micrograms hydroxyproline per segment, P < .004), which was associated with a nonsignificant 25% reduction in intimal area. Our data suggest that degradation of newly synthesized collagen is an important mechanism regulating collagen accumulation and that MMPs have an integral role in collagen turnover after balloon angioplasty.

Self-aggregation characteristics of recombinantly expressed human elastin polypeptides.

Elastin is an extracellular matrix protein found in tissues requiring extensibility and elastic recoil. Monomeric elastin has the ability to aggregate into fibrillar structures in vitro, and has been suggested to participate in the organization of its own assembly into a polymeric matrix in vivo. Although hydrophobic sequences in elastin have been suggested to be involved in this process of self-organization, the contributions of specific hydrophobic and crosslinking domains to the propensity of elastin to self-assemble have received less attention. We have used a series of defined, recombinant human elastin polypeptides to investigate the factors contributing to elastin self-assembly. In general, coacervation temperature of these polypeptides, used as a measure of their propensity to self-assemble, was influenced both by salt concentration and polypeptide concentration. In addition, hydrophobic domains appeared to be essential for the ability of these polypeptides to self-assemble. However, neither overall molecular mass, number of hydrophobic domains nor general hydropathy of the polypeptides provided a complete explanation for differences in coacervation temperature, suggesting that the specific nature of the sequences of these hydrophobic domains are an important determinant of the ability of elastin polypeptides to self-assemble.

Impaired elastin fiber assembly related to reduced 67-kD elastin-binding protein in fetal lamb ductus arteriosus and in cultured aortic smooth muscle cells treated with chondroitin sulfate.

In the fetal ductus arteriosus (DA) disruption in the assembly of elastin fibers is associated with intimal thickening and we previously reported that fetal lamb DA smooth muscle cells incubated with endothelial conditioned medium produce two-fold more chondroitin sulfate (CS) compared with aorta (Ao) cells (Boudreau, N., and M. Rabinovitch. 1991. Lab. Invest. 64:187-199). We hypothesized that CS or dermatan sulfate (DS), both N-acetylgalactosamine glycosaminoglycans (GAGs), may be similar to free galactosugars in causing release of the 67-kD elastin binding protein (EBP) from the smooth muscle cell surfaces and impaired elastin fiber assembly. Using immunohistochemistry, immunoelectron microscopy, and western immunoblot we demonstrated a reduction in the 67-kD EBP in fetal lamb DA smooth muscle in tissue and in cultured cells. Also, reduced EBP was observed in fetal lamb and neonatal rat Ao smooth muscle cells incubated with N-acetylgalactosamine GAGs, CS, and DS, but not with N-acetylglucosamine containing GAGs, heparan sulfate (HS), or hyaluronan. Reduction in EBP was related to shedding from cell surfaces into the conditioned medium. This was associated with impaired elastin fiber assembly in cultured cells, assessed both morphologically and by a relative increase in tropoelastin and decrease in desmosines. The EBP extracted from smooth muscle cell membranes binds to an elastin affinity gel and can be eluted from it with CS but not with HS. Moreover, the amount of EBP extractable from smooth muscle cell membranes correlated with the morphologic assessment. We propose that increased CS or DS, may impair assembly of newly synthesized elastin in the media of the ductus arteriosus associated with the development of intimal thickening.

Tropoelastin interacts with cell-surface glycosaminoglycans via its COOH-terminal domain.

Using a biochemical and cell biological approach, we have identified a cell interaction site at the carboxyl terminus of tropoelastin. Cell interactions with the COOH-terminal sequence are not through the elastin-binding protein (EBP67) because neither VGVAPG-like peptides nor galactoside sugars altered adhesion. Our results also show that cell adhesion to tropoelastin is not promoted by integrins. Through the use of mutant Chinese hamster ovary cell lines defective in glycosaminoglycan biosynthesis, as well as competition studies and enzymatic removal of specific cell-surface glycosaminoglycans, the tropoelastin-binding moieties on the cell surface were identified as heparan and chondroitin sulfate-containing glycosaminoglycans, with heparan sulfate being greatly preferred. Heparin affinity chromatography combined with cell adhesion assays identified the last 17 amino acids as the sequence element at the carboxyl terminus of tropoelastin responsible for the adhesive activity.

Molecular assembly and mechanical properties of the extracellular matrix: A fibrous protein perspective

The extracellular matrix is an integral and dynamic component of all tissues. Macromolecular compositions and structural architectures of the matrix are tissue-specific and typically are strongly influenced by the magnitude and direction of biomechanical forces experienced as part of normal tissue function. Fibrous extracellular networks of collagen and elastin provide the dominant response to tissue mechanical forces. These matrix proteins enable tissues to withstand high tensile and repetitive stresses without plastic deformation or rupture. Here we provide an overview of the hierarchical molecular and supramolecular assembly of collagens and elastic fibers, and review their capacity for mechanical behavior in response to force. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

The endogenous vascular elastase that governs development and progression of monocrotaline-induced pulmonary hypertension in rats is a novel enzyme related to the serine proteinase adipsin

We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats. We now report the isolation and characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat pulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA from rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtained encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this competitor of elastase from the PA extract and the elastolytic activity increased 100-fold. The enzyme was purified using an antibody that recognizes NH2-terminal sequences of serine proteinases and the eluate was further purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was resolved as an active enzyme on an elastin substrate gel. Immunogold labeling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin originally found in adipose tissue.

Coacervation of tropoelastin

The coacervation of tropoelastin represents the first major stage of elastic fiber assembly. The process has been modeled in vitro by numerous studies, initially with mixtures of solubilized elastin, and subsequently with synthetic elastin peptides that represent hydrophobic repeat units, isolated hydrophobic domains, segments of alternating hydrophobic and cross-linking domains, or the full-length monomer. Tropoelastin coacervation in vitro is characterized by two stages: an initial phase separation, which involves a reversible inverse temperature transition of monomer to n-mer; and maturation, which is defined by the irreversible coalescence of coacervates into large species with fibrillar structures. Coacervation is an intrinsic ability of tropoelastin. It is primarily influenced by the number, sequence, and contextual arrangement of hydrophobic domains, although hydrophilic sequences can also affect the behavior of the hydrophobic domains and thus affect coacervation. External conditions including ionic strength, pH, and temperature also directly influence the propensity of tropoelastin to self-associate. Coacervation is an endothermic, entropically-driven process driven by the cooperative interactions of hydrophobic domains following destabilization of the clathrate-like water shielding these regions. The formation of such assemblies is believed to follow a helical nucleation model of polymerization. Coacervation is closely associated with conformational transitions of the monomer, such as increased β-structures in hydrophobic domains and α-helices in cross-linking domains. Tropoelastin coacervation in vivo is thought to mainly involve the central hydrophobic domains. In addition, cell-surface glycosaminoglycans and microfibrillar proteins may regulate the process. Coacervation is essential for progression to downstream elastogenic stages, and impairment of the process can result in elastin haploinsufficiency disorders such as supravalvular aortic stenosis.

Decreased Elastin Synthesis in Normal Development and in Long-term Aortic Organ and Cell Cultures Is Related to Rapid and Selective Destabilization of mRNA for Elastin

We have previously shown that aortic organ cultures from 1- to 3-day-old chickens initially mimic the high levels of elastin production seen in vivo. However, more prolonged incubation of these tissues results in decreased synthesis of elastin. In the present study, we demonstrate that decreased production of elastin in these aortic organ cultures is selective for elastin compared with collagen and is correlated with decreased steady state levels of mRNA for elastin. These decreases in steady state levels of elastin mRNA are due at least in part to a rapid and selective destabilization of mRNA for elastin, the half-life of which falls from approximately 25 hours in fresh aortic tissues to approximately 15 hours after incubation for only 8 hours. Destabilization of elastin mRNA can be prevented by incubation in the presence of blockers of DNA transcription (5,6-dichlorobenzimidazole riboside and actinomycin D) and mRNA translation (cycloheximide). Furthermore, the half-life of aortic elastin mRNA decreases from approximately 25 hours in the 1-day-old chicken to approximately 7 hours in the 8-week-old chicken, demonstrating that destabilization of mRNA is an important contributing factor in the decline in production of aortic elastin taking place during normal postnatal growth.