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D Amiel

Publications and source records attributed to D Amiel.

At least 73 records · Page 4Linked to original sources
PubMed1995

Integrin display increases in the wounded rabbit medial collateral ligament but not the wounded anterior cruciate ligament.

The differential capacities of the anterior cruciate and medial collateral ligaments to heal may be related to differences in cellular function. This study tested the hypothesis that differential expression of integrins occurs in these ligaments after injury. The integrins are a family of cell surface receptors that mediate adhesion, migration, and other cellular functions critical to the healing of a wound. A similar complement and amount of the beta 1 subfamily of integrins are known to be present on the unperturbed anterior cruciate and medial collateral ligaments in humans and rabbits. A partial laceration was surgically created in these two ligaments in 12 anesthetized New Zealand White rabbits. Immunohistochemistry was performed on sections from the ligaments at 1, 3, 7, and 10 days after injury, using monoclonal antibodies directed against the integrin subunits beta 1, alpha 5, alpha 6, and alpha v. Between 3 and 7 days, the wounded medial collateral ligament demonstrated a striking increase in staining for the beta 1, alpha 5, and alpha v subunits on the fibroblasts, within the repair site, and on capillary endothelium. Increased staining was most marked for the beta 1 subunit and less marked for the alpha 5 and alpha v subunits. The alpha 6 subunit stained exclusively vascular structures within the healing medial collateral ligament. In marked contrast, the anterior cruciate ligament, which does not mount an effective repair response, demonstrated no comparable alteration of integrin expression from baseline levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Synergistic effect of growth factors on cell outgrowth from explants of rabbit anterior cruciate and medial collateral ligaments.

Cellular migration and proliferation are integral aspects of wound healing. An in vitro assay for cellular migration and proliferation using explants of rabbit anterior cruciate and medial collateral ligaments was developed previously. This study presents the effects of serum-free culture medium supplemented with basic fibroblast growth factor, bovine insulin, transforming growth factor-beta 1, and platelet-derived growth factor-B, added either individually or in combination, on cell outgrowth in explants of rabbit anterior cruciate and medial collateral ligaments. Outgrowth was assessed at 3 and 6 days by counting the number of cells surrounding the tissue explants. For explants of both ligaments, cell outgrowth was dependent on the presence of 10% fetal bovine serum or the combination of growth factors. Little outgrowth occurred in explants of either ligament in the presence of basic fibroblast growth factor, transforming growth factor-beta 1, or bovine insulin. Platelet-derived growth factor-B at concentrations of 20 and 100 ng/ml seemed to increase cell outgrowth from medial collateral ligament explants at 6 days. The outgrowth from the explants of both ligaments was much greater in the presence of all four growth factors than the sum of the outgrowth with the individual factors. This synergistic effect was as much as 10-fold and 20-fold for the anterior cruciate ligament explants at days 3 and 6, respectively, but no more than 3-fold for the medial collateral ligament explants at these times. Medial collateral ligament explants exhibited greater cell outgrowth than anterior cruciate ligament explants in 10% serum and in the presence of the four growth factors.

Animals↗
PubMed1995

Autogenous intrasynovial and extrasynovial tendon grafts: an experimental study of pro alpha 1(I) collagen mRNA expression in dogs.

On the basis of recent evidence that the healing processes of tendon grafts are donor-tissue specific, in situ hybridization, using a 372 bp cDNA fragment complementary to a portion of pro alpha 1(I) collagen mRNA, was utilized to compare the cellular responses to transplantation exhibited by autogenous intrasynovial and extrasynovial flexor tendon grafts. Intrasynovial and extrasynovial tendons from the hindpaw were transferred to synovial sheaths in the forepaw of 12 mongrel dogs (24 tendons) and treated with immediate controlled passive motion. The tendon grafts were harvested at 2, 4, and 6 weeks, and each was divided into a proximal, central (8 mm), and distal portion. Sections from the central portion were embedded in paraffin and subjected to in situ hybridization, autoradiography, and staining; levels of procollagen mRNA then were assessed by microscopic examination. The two types of tendon grafts exhibited different levels of pro alpha 1(I) collagen mRNA expression at all three time points. Intrasynovial tendon grafts displayed no areas of increased type-I procollagen mRNA at 2, 4, and 6 weeks. The extrasynovial tendon grafts displayed increased surface levels of type-I procollagen mRNA at 2 and 4 weeks; the levels decreased to background levels by 6 weeks. The high levels of procollagen mRNA exhibited by the extrasynovial grafts suggest increased collagen synthetic activity, indicative of a cellular response to injury, whereas the preservation of low levels of expression in the intrasynovial grafts may signify a less inflammatory cellular response.

Animals↗
PubMed1995

Effect of tissue culture storage on the in vivo survival of canine osteochondral allografts.

In vitro studies in our laboratory have shown that the biomechanical and biochemical characteristics of osteochondral grafts can be preserved for as long as 28 days under tissue culture conditions. This study represents an attempt to extend these results to an in vivo model. In adult mongrel dogs, either an autograft, a fresh allograft, or a stored allograft was placed in a standardized defect on the weight-bearing surface of the medial femoral condyle. The stored grafts were kept at 4 degrees C in tissue culture medium for 14 days prior to implantation. The animals were killed at 12 weeks. Cartilage from the contralateral knee served as a control. The modulus and permeability of the cartilage were assessed with confined compression creep tests. The collagen and glycosaminoglycan contents were measured, and the cartilage was analyzed histologically with hematoxylin and eosin and safranin O stains. Grossly, the cartilage appeared viable at harvest. The histologic results were similar in the treatment groups, with the same spectrum of mild degenerative changes being noted in each group. The glycosaminoglycan content was significantly less in the autograft group than in its control group and than in the fresh allograft group. The glycosaminoglycan content did not differ significantly between fresh and stored allografts. The collagen content, modulus, and permeability did not differ either between experimental and control groups or between graft types. Our results support the conclusion that osteochondral allografts can be stored for as many as 14 days without significantly affecting the results of the procedure.

Animals↗
PubMed1995

Increased expression of the beta 1, alpha 5, and alpha v integrin adhesion receptor subunits occurs coincident with remodeling of stress-deprived rabbit anterior cruciate and medial collateral ligaments.

The biomechanical, biochemical, and morphological properties of the anterior cruciate and medial collateral ligaments are dramatically altered in response to deprivation of normal physical forces and joint motion. Integrin adhesion receptors are known to play important roles in the tissue remodeling that occurs in the course of normal wound repair. We propose that integrins play a similar role in the remodeling of the extracellular matrix in stress-deprived periarticular ligaments. This study tests the hypothesis that altered expression of integrins on ligament fibroblasts accompanies this remodeling. The left knees of 15 New Zealand White rabbits were surgically immobilized in acute flexion and the right knees served as controls (no operation). The anterior cruciate and medial collateral ligaments were harvested at 1, 3, 5, 9, or 12 weeks after immobilization. Sections from the ligaments were immunostained with monoclonal antibodies specific for the integrin subunits beta 1, alpha 5, alpha 6, and alpha v, as well as with a negative control antibody. Fibroblasts within both the stress-deprived anterior cruciate and medial collateral ligaments demonstrated markedly increased staining for the beta 1, alpha 5, and alpha v subunits, as compared with the controls. The increased staining was greatest at 9 weeks in the anterior cruciate ligament and at 12 weeks in the medial collateral ligament. Western blot study of ligament proteins extracted with sodium dodecyl sulfate demonstrated an increased amount of beta 1 subunit protein in both ligaments from knees that were stress deprived for 9 and 12 weeks, as compared with the control ligaments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

In situ assessment of cell viability within biodegradable polylactic acid polymer matrices.

Efforts to expand treatment options for articular cartilage repair have increasingly focussed on the implantation of cell polymer constructs. Primary cells cultured from perichondrium, a chondrogenic tissue, were found to survive in vitro within a biodegradable porous polylactic acid matrix. The novel application of an in situ fluorescent double-stain protocol to cell polymer constructs was supported by increased 3H-thymidine uptake and the ability of cell seeded polylactic acid to form first passage explant cultures. This in situ viability staining technique allowed for rapid determination of cell viability and, in conjunction with confocal microscopy, assessment of cellular distribution within a biodegradable scaffold. Advantages of using this method over histological and electron microscopic analysis include in situ observation, absence of distortion in scaffold architecture due to polymer dissolution and disruption during processing, and obtaining a viability assessment within 30 min. Potential applications of this protocol as a screening tool for laboratory engineered tissues and in the evaluation of cellular injury in natural tissues are discussed.

Animals↗
PubMed1995

Intrinsic properties of ACL and MCL cells and their responses to growth factors.

Different intrinsic properties of the constituent cells of the anterior cruciate ligament (ACL) and medial collateral ligament (MCL) have been proposed to be one of the factors in the differential repair mechanisms. We have found that the outgrowth of cells from rabbit ACL explants was slower than from MCL explants after 10 d. Growth curves of ACL and MCL cultures at both Passage numbers 2 and 6 showed a slower rate of proliferation of ACL cells than MCL cells (P < 0.005). The proliferative response of rabbit ACL and MCL cells to b-FGF and TGF-beta was also investigated. Both b-FGF and TGF-beta had no significant effect on cell proliferation of ACL and MCL cultures after 48 h. However, TGF-beta did have an inhibitory effect on thymidine incorporation, especially at concentrations greater than 1 ng.ml-1, while b-FGF stimulated thymidine incorporation in ACL and MCL in a dose-dependent manner. Differences in the cell morphology of the ACL and MCL cells grown in culture were seen also. Cells from Passages 3-6 demonstrated these differences more prominently, and phalloidin staining for actin showed that ACL cells appeared to have more intracellular actin fibers.

Animals↗
PubMed1994

Preventing postlaminectomy adhesion. A new experimental model.

STUDY DESIGN: The efficacy of various interpositional membranes for prevention of extradural adhesion was investigated by a new animal model that quantified the biomechanical effect of scar formation. Twenty-one dogs were treated with autologous free fat graft, hyaluronic acid or no interpositional membrane after undergoing two-level laminotomy, nerve root exploration, and disk injury. An additional 11 dogs that did not undergo spine surgeries served as control animals. OBJECTIVES: Inter-animal variability in inherent propensity to form scar was first measured before any spine surgery. Twelve weeks after spine surgery, the lumbosacral spine of each dog was harvested en bloc for biomechanical testing of extradural adhesion ultimate load. Scar stiffness coefficient was also calculated. SUMMARY OF BACKGROUND DATA: Adhesion ultimate load was significantly less in the nonoperative control group when compared with the fat graft and no interpositional membrane group, but not when compared with the hyaluronic acid group. A beneficial effect of hyaluronic acid in lowering adhesion ultimate load was demonstrated, although a statistically significant difference from the fat graft and no interpositional membrane groups was not reached. No difference in scar stiffness coefficient was found between the four groups. METHODS: A new experimental model allowing objective biomechanical quantification of the effect of postoperative scar was described. Ultimate load of adhesions to both nerve roots and dura was measured. A biochemical assay that determined collagen content was also used to assess inter-animal propensity to form scar after a standardized surgical insult. Results were compared with other relevant studies. RESULTS: Findings suggest a beneficial effect of hyaluronic acid in decreasing the biomechanical strength of extradural adhesions following laminotomy, nerve root exploration, and disk injury when compared with use of fat graft or no interpositional membrane. These results support other recent investigations that study the use of hyaluronic acid treatment in a laminectomy model. The adverse consequence of intraoperative epidural bleeding was also demonstrated. CONCLUSIONS: The new experimental model described in the current study was reproducible and permitted objective quantification of the effect of postoperative adhesion rather than measuring its mere presence. A beneficial effect of hyaluronic acid treatment and a lack of such beneficial effect of free fat graft interpositional membrane was suggested. The importance of avoiding active epidural bleeding was also evident.

Adipose Tissue↗
PubMed1994

Integrin expression by human articular chondrocytes.

OBJECTIVE: To perform a comprehensive analysis of the integrin forms expressed by normal human articular chondrocytes. METHODS: Cartilage sections and collagenase-released chondrocytes were probed with a comprehensive panel of integrin isoform-specific monoclonal antibodies (MAb), using in situ immunohistochemistry techniques, indirect immunofluorescence and flow cytometry, and immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Chondrocytes in cartilage sections reacted with MAb specific for the alpha 5, alpha v, and beta 1 integrin subunits and the alpha v beta 3 and alpha v beta 5 heterodimers. They also reacted with a polyclonal antibody specific for the intracytoplasmic portion of the alpha 1 subunit. MAb specific for the alpha v subunit reacted more strongly with chondrocytes near the articular surface than with those in deeper layers of cartilage, and the alpha v beta 3-specific MAb reacted exclusively with chondrocytes within the most superficial 30 microns of cartilage. Flow cytometric analysis and SDS-PAGE analysis of immunoprecipitates prepared from extracts of cell-surface radioiodinated chondrocytes confirmed the above observations, and additionally revealed the presence of the alpha 3 beta 1 integrin. CONCLUSION: Normal human articular chondrocytes prominently display substantial quantities of the alpha 1 beta 1, alpha 5 beta 1, and alpha v beta 5 integrin heterodimers, as well as lesser quantities of the alpha 3 beta 1 and alpha v beta 3 heterodimers. The alpha v subunit-containing integrins are detected more readily on the more superficial chondrocytes than on chondrocytes deep within cartilage. These observations provide the basis for analysis of the role of chondrocyte integrins in cartilage homeostasis and in the pathogenesis of joint diseases.

Adult↗
PubMed1994

Cellular survival and proliferation in autogenous flexor tendon grafts.

In order to investigate fibroblast survival and proliferation in autogenous flexor tendon grafts, hindlimb intrasynovial and extrasynovial donor tendons were placed within the synovial sheaths of the medial and lateral forepaw digits of 21 dogs (42 tendons) and treated with controlled early passive motion. Intravital histologic evaluations with confocal microscopy and biochemical determinations of total DNA content and DNA synthesis were carried out at 10 days, 3 weeks, and 6 weeks. Intravital staining of the extrasynovial tendon grafts demonstrated variable degrees of cellular necrosis at the earliest intervals followed by cellular repopulation with fibroblasts and neovascularization from surface vessels. In contrast, intrasynovial tendon grafts were populated predominantly by viable cells at each interval, with occasional patches of cell necrosis and fibroblast ingrowth. Total DNA content and DNA synthesis values in the intrasynovial donor tendons were significantly lower than those seen in the extrasynovial tendon grafts at each interval. Extrasynovial tendons appear to act as scaffolds, undergoing extensive cellular death followed by a rapid repair response. Findings that intrasynovial tendon fibroblasts survive the tendon grafting process suggest that the nutritional supplies and metabolic requirements of intrasynovial and extrasynovial donor tendons differ largely.

Animals↗
PubMed1994

An in vitro assay of anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cell migration.

Explants from rabbit anterior cruciate ligaments (ACL) and medial collateral ligaments (MCL) were utilized to compare the relative rates that fibroblasts migrate onto glass and plastic culture surfaces in vitro. During the first two weeks in culture, a monolayer of cells appeared on the periphery of all the ACL and MCL explants. From 45 to 134 hrs in culture, the mean total MCL cell count per explant was 6-12 times greater than that for the ACL on the plastic culture dishes, and this difference was even greater for the cells attached to the glass cover slides over the explants. These differences were significant at the p < .005 level. The rates of cell proliferation were quite similar for primary cultures of ACL and MCL grown in the same medium as that used for the migration assay. The large difference in cell number at early times of culture is thus due to the more rapid MCL cell migration out of the explants, and not to a difference in the rate of cell proliferation. These data support the hypothesis that differences in cell migration rate play a role in the greater healing capacity of the MCL as compared with the ACL. The assay described in this work may then be useful in assessing factors that promote wound healing.

Animals↗
PubMed1994

The effects of immobilization on the maturation of the anterior cruciate ligament of the rabbit knee.

Immobilization-induced alterations occurred in young anterior cruciate ligament (ACL) samples, including the loss of the rounded appearance of the cells. The mature ACL was minimally altered by immobilization at the light microscopy level. In the immobilized young ACL the fibroblasts became elongated and there was loss of the normal pericellular matrix. The immobilized mature ACL differed from controls primarily in the intracellular composition, as there was significantly more rough endoplasmic reticulum (RER) present. Collagen concentrations were reduced only in young immobilized ACL, while no differences were observed in the mature ACL. The collagen synthesis rate in the mature ACL increased with immobilization, although no significant change was observed in the young ACL. The increase in the rate of synthesis of the stress deprived ACL in the mature animals reflected an increase in collagen turnover rather than an increase in accumulation of collagen.

Aging↗
PubMed1994

Long-term fate and effects of exercise on sternal cartilage autografts used for repair of large osteochondral defects in horses.

Bilateral osteochondral defects (10 mm2 x 3 mm deep) were created on the distal articular surface of the radial carpal bone of ten, 2- to 3-year-old horses. One defect of each horse was repaired, using a sternal cartilage autograft (treated), and the other was left untreated (control). The horses were exercised on a high-speed treadmill at incrementally increased speed and duration over the course of 12 months. Horses were evaluated arthroscopically at 6 to 7 weeks, and clinical examinations were conducted weekly at exercise. Twelve months after surgery, carpuses of each horse were radiographed and clinically examined prior to euthanasia. A gross pathologic evaluation of each joint was conducted, and samples were collected for histologic, histochemical, histomorphometric, and biochemical evaluation. Radiographically, the grafted joints had more extensive evidence of arthropathy, and clinically, 8 of the 10 horses were more lame in the grafted limb. On the basis of histomorphometry, the repair tissue of the grafted defects contained a greater median percentage of hyaline cartilage (45%) than that of control defects (4.5%), and the control defects contained a greater percentage of fibrocartilage (82%) than did grafted defects (28.5%). A greater median percentage of repair tissue stained with safranin-O in the grafted defects (24.5%) than in the control defects (3.5%). On gross pathologic and histologic evaluation, repair tissue of the control defects had better continuity and was more firmly attached to the subchondral bone than was repair tissue of the grafted defects. Repair tissue of the grafted defects had extensive fissure and flap formation. Histologically, subchondral bone reactivity and fibroplasia was extensive in grafted joints. Repair tissue of grafted defects had a greater percentage of type II collagen (mean +/- SEM, 83.5 +/- 2.95%) than did controls (mean, 79.4 +/- 3.87%) that was not statistically significant. Hexosamine content was significantly higher (P < 0.05) in repair tissue of the grafted defect (mean, 28.9 +/- 3.00 mg/g of dry weight) vs control (mean, 20.6 +/- 1.85 mg/g of dry weight). On the basis of this experimental model, sternal cartilage autografts cannot be recommended at this time for repair of osteochondral defects in athletic horses.

Animals↗
PubMed1993

Development of a novel spontaneous metastasis model of human osteosarcoma transplanted orthotopically into bone of athymic mice.

There is a pressing need for in vivo models in which potential antitumor agents can be tested for their ability to inhibit the growth and metastatic spread of human sarcomas. A recent advance in this regard has been the development of a v-Ki-ras-oncogene-transformed human osteosarcoma cell line (KRIB) that efficiently colonizes the lungs of athymic nude mice when cells (1 x 10(5)) are administered by i.v. injection. In the present study, we have utilized this cell line to develop a spontaneous metastasis model in which a small number of tumor cells are injected into the tibial bones of athymic mice. When as few as 1000 KRIB cells are orthotopically implanted into the tibial bones of nude mice, bone tumors, which are radiographically and histologically similar to primary human osteosarcoma, develop within 4 weeks. Furthermore, as in the human disease, cells from these primary tumors subsequently seed the animals' lungs, resulting in reproducible and quantifiable pulmonary metastasis evident both upon gross inspection of the lungs and histologically 6 weeks after tumor inoculation. Surgical amputation of the tumor inoculation site up to 2 weeks after tumor injection prevents pulmonary metastasis, indicating that substantial local (tibial) growth and invasion of the primary tumor for at least 2 weeks is required for subsequent metastasis. Implantation of s.c. 5000 KRIB cells fails to produce local or metastatic tumors. We anticipate that this model will prove to be a powerful tool with which to study the mechanisms of human osteosarcoma growth and pulmonary metastasis, and to assess the efficacy of promising therapeutic agents.

Animals↗
PubMed1993

The ligamentization process: a 4 year case study following ACL reconstruction with a semitendinosis graft.

This study evaluates the histological and biochemical changes that occurred in a semitendinosis autograft 4 years after intraarticular placement as an anterior cruciate ligament (ACL) substitute in a human patient. The graft was harvested during total knee replacement. Comparison to harvested ACL and hamstring tendon was made. Changes in collagen crimp pattern, cell type, glycosaminoglycan composition, and collagen crosslinking were present between the ACL autograft and the hamstring tendon. The appearance and biochemical properties of the semitendinosis autograft and the native ACL were similar 4 years following intraarticular placement. These observed phenomena could be attributed to functional adaptation, supporting the concept of ligamentization.

Adaptation, Physiological↗
PubMed1992

Characterization of the intrinsic properties of the anterior cruciate and medial collateral ligament cells: an in vitro cell culture study.

The poor healing abilities of the anterior cruciate ligament (ACL) in contrast to those of the medial collateral ligament (MCL) are well known. Different intrinsic properties of the constituent cells of these ligaments have been proposed to be one of the factors in the differential repair mechanisms. To examine this hypothesis, we have established primary cell lines of ACL and MCL from the tissue explants of approximately similar dimensions and have studied their behavior in vitro. The outgrowth of cells from ACL explants was slower than from MCL explants, as shown by the size of the surrounding clusters of cells. Both ACL and MCL cultures exhibited typical fibroblastic morphology. No significant differences were observed in either attachment or growth of cells from the attached explants derived from various segments of ACL and MCL. Growth curves of ACL and MCL cultures at both passage numbers 2 and 6 showed a slower rate of proliferation of ACL cells than MCL cells (p less than 0.005). DNA synthesis measured in terms of [3H]thymidine incorporation (CPM/10(3) cells) of both log phase (ACL = 607.5 +/- 5.4 vs. MCL = 1356.4 +/- 11.3) and confluent (ACL = 83.0 +/- 3.6 vs. MCL = 189.8 +/- 5.4) cultures, supports the conclusion that differential proliferation rates of these cells exist in culture. FITC-phalloidin staining (for actin) of later passage cultures (P3-P5) showed a spread-out appearance of ACL cells and an elongated appearance of MCL cells. Relatively more stress fibers were seen within ACL cells. SDS-PAGE and Western blot analysis of cellular proteins revealed higher actin (43 kDa) content in ACL cells than in MCL cells. In vitro wound closure assay was performed by creating a uniform wound of 0.6 mm width in the confluent layer of ACL and MCL cultures. By 48 h postwounding, cell-free zones created in ACL cultures were occupied partially by single cells in a nonconfluent fashion. In contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. Results presented in this report demonstrate a lower proliferation and migration potential of ACL cells in comparison with MCL cells. These differences in intrinsic properties of ACL and MCL cells that were observed in vitro might contribute to the differential healing potentials of these ligaments in vivo.

Actins↗
PubMed1992

Immunohistochemical localization of beta 1-integrins in anterior cruciate and medial collateral ligaments of human and rabbit.

The integrins are a family of adhesion-mediating cell surface receptors that play critical roles in cell-extracellular matrix interactions and have been shown to be important in the healing response in several tissues. We have studied integrin expression in normal human and rabbit anterior cruciate (ACL) and medial collateral (MCL) ligaments of the knee as a preamble to studies of beta 1-integrin expression in healing ligaments. Histologic sections of human and rabbit ACL and MCL were probed for integrin expression utilizing integrin-specific monoclonal antibodies (mAbs) followed by immunoperoxidase detection. Staining of human specimens with mAbs revealed the presence of beta 1-, alpha 1-, and alpha 5-integrin chains on the tissue fibroblasts of both ACL and MCL, while staining of rabbit specimens with rabbit integrin-reactive monoclonals revealed the presence of beta 1- and alpha 5-integrin on these ligaments. Equivalent amounts of the integrins studied were present on normal ACL and MCL. We conclude that the rabbit is an appropriate model for analyzing the expression and functional role of integrins in ligament wound healing.

Animals↗