Biomedical subjects

D Amiel

Publications and source records attributed to D Amiel.

At least 37 records · Page 2Linked to original sources

PubMed2000

Cartilage repair with autogenic perichondrium cell and polylactic acid grafts.

The repair of articular cartilage injuries remains a challenge, with many of the current therapeutic strategies based on the grafting or recruitment of chondrogenic tissues or cells. This 1-year study compared the repair of a 3.7-mm diameter by 3-mm deep osteochondral defect in the medial femoral condyle of 24 New Zealand White rabbits; the defect was obtained using an autogenic perichondrium cell polylactic acid composite graft with a contralateral control in which the osteochondral defect remained empty. To elucidate the effect of host immune responses on the repair process after perichondrium cell transplantation, the results of the autogenic perichondrium cell polylactic acid graft group were compared with those obtained in the authors' previous 1-year study of allogenic perichondrium cell polylactic acid composite grafts implanted in a similar model. One year after surgery, the repair site underwent gross inspection and histologic, histomorphometric, biochemical, and biomechanical analyses. The autogenic perichondrium cell polylactic acid graft group (92%) and the control group in which the osteochondral defect remained empty (88%) resulted in a high percentage of grossly acceptable repairs. The autogenic grafts appeared to augment the intrinsic healing capacity of the animals (as compared with the animals in the No Implant Group). The autogenic perichondrium cell polylactic and grafts improved the histologic appearance and percentage of Type II collagen of the cartilaginous repair tissue. Compared with allogenic grafts, the autogenic grafts had better reconstitution of the subchondral bone. However, the results of this experimental model suggest a suboptimal concentration of glycosaminoglycans in the neocartilage matrix, a depressed surface of the repair tissue, a histologic appearance that was not equivalent to that of normal articular cartilage, and reduced biomechanical properties for the repair tissue. The future application of growth factors to this model may yield a treatment that can be applied in the clinical arena.

Animals↗

PubMed2000

Nonviral in vivo gene therapy for tissue engineering of articular cartilage and tendon repair.

Heretofore, nonviral methods have been used primarily for in vitro transfection of cultured cell lines. These methods were substantially less efficient when compared with the use of viruses, particularly when used in vivo. Herein a three-step, highly efficient method of nonviral gene delivery is presented. Using this method, genes have been delivered successfully into tissues of orthopaedic importance with high-efficiency by nonviral means. Transforming growth factor-beta 1, parathyroid hormone related protein, and a marker gene were transfected into primary perichondrium and cartilage cells with efficiencies in excess of 70%. They overexpressed their cognate gene products showing efficacy of expression in a rabbit model of osteochondral defect repair. Using the same method, a marker gene was delivered into a canine model for intrasynovial flexor tendon injury and repair. This was achieved by direct gene delivery during surgery. An estimated 5 additional minutes were required during surgery to complete the transfection steps. High efficiency gene delivery was achieved in the flexor tendons, tendon sheaths, tendon pulleys, surrounding tissues, and skin. The efficiency of transfection approached 100% in the exposed superficial tissue layers and transfected cells were found several layers below the exposed tissue surfaces. The data show the potential of direct nonviral gene therapy in orthopaedics for ex vivo and in vivo applications.

Animals↗

PubMed2000

The tetrabasic KKKK(147-150) motif determines intracrine regulatory effects of PthrP 1-173 on chondrocyte PPi metabolism and matrix synthesis.

Expression of PTHrP is a major regulator of growth cartilage development and also becomes robust in osteoarthritic cartilage. We further defined how PTHrP 1-173, which we observed to be the preferentially expressed PTHrP isoform in normal and osteoarthritic cartilage, functions in chondrocytes. We transfected both immortalized human juvenile costal chondrocytes (TC28 cells) and rabbit articular chondrocytes with wild-type PTHrP 1-173 and mutants of putative PTHrP 1-173 endoproteolytic processing sites. Wild-type PTHrP 1-173 inhibited collagen synthesis and decreased extracellular PPi (which critically regulates hydroxyapatite deposition) by 50-80% in both chondrocytic cell types. In contrast, PTHrP 1-173 mutated at the PTHrP 147-150 motif KKKK (but not the other site-directed mutants) and increased both extracellular PPi and collagen synthesis by >50%. Synthetic PTHrP 140-173 mutated at amino acids 147-150 and also increased extracellular PPi, and wild-type 140-173 decreased extracellular PPi in permeabilized cells. The 147-nuclear localization of PTHrP. We conclude that the tetrabasic 147-150 motif functions to determine how PTHrP 1-173 regulates collagen synthesis and levels of extracellular PPi by an intracrine mechanism in chondrocytes, and it may prove useful as a therapeutic target for regulation of mineralization.

Animals↗

PubMed2000

Effect of hyaluronan on chondrocyte apoptosis and nitric oxide production in experimentally induced osteoarthritis.

OBJECTIVE: Nitric oxide (NO) plays an important role in cartilage degeneration, and NO donors induce chondrocyte apoptosis. This study evaluated the effect of intraarticular injections of hyaluronan (HA) on chondrocyte apoptosis and NO production using an experimental osteoarthritis (OA) model. METHODS: Thirty-six New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT), and were divided into 3 groups. Four weeks after ACLT, the HA group started intraarticular HA injections once a week for 5 weeks; the vehicle group started to receive the carrier of HA; the no injection group received no treatment. All ACLT knees were harvested at Week 9 and evaluated for OA severity. Culture supernatants of the cartilage were analyzed for nitrite concentration. Cartilage sections were analyzed by TUNEL for apoptosis and by immunohistochemistry for nitrotyrosine. RESULTS: OA in the HA group was less severe than the other 2 groups. The number of apoptotic chondrocytes was significantly lower in the HA group. HA injection did not influence NO production in cartilage. CONCLUSION: HA protects against chondrocyte apoptosis during the development of OA, while it may not have definite effects on NO production in the joints. These inhibitory effects of HA on chondrocyte apoptosis may play a role in its mechanism of action in chondroprotection.

Animals↗

PubMed2000

Transforming growth factor beta one (TGF-beta 1) enhancement of the chondrocytic phenotype in aged perichondrial cells: an in vitro study.

BACKGROUND: Perichondrium is recognized as a tissue with chondrogenic potential yielding cells which can be used for osteochondral repair. Factors which influence the proliferative ability and chondrocytic phenotype of such cells include age and presence of specific growth factors, i.e. TGF-beta 1. The present in vitro study assessed proliferation and markers of chondrocytic phenotype in cells extracted from the rib perichondrium of four- to five-year-old aged rabbits, and assessed the effects of exogenously added TGF-beta 1 on those cells. METHODS: Assays included 3H-thymidine incorporation (cell proliferation), 35S-sulfate incorporation (proteoglycan synthesis) and quantitative RT-PCR for determination of type II collagen gene expression. RESULTS: The results demonstrated that addition of TGF-beta 1 to the culture media stimulated thymidine incorporation and proteoglycan synthesis up to four- and five-fold, respectively, in aged perichondrium-derived cells. Moreover, the exogenous addition of TGF-beta 1 to the culture media resulted in an upregulation of transcriptional expression of the type II collagen gene. CONCLUSIONS: In summary, the present study has demonstrated that exogenously added TGF-beta 1 can stimulate proliferation and chondrocytic phenotype in aged perichondrium-derived cells in vitro.

Age Factors↗

PubMed1999

Nitric oxide production and apoptosis in cells of the meniscus during experimental osteoarthritis.

OBJECTIVE: To examine the pathologic changes in meniscus tissue during experimental osteoarthritis (OA) and to determine the relationship between nitric oxide (NO) synthesis, apoptosis, and meniscus degradation. METHODS: OA was induced in rabbits by anterior cruciate ligament (ACL) transection. Knees were harvested after 9 weeks and assessed for OA severity. Menisci were subjected to histologic, immunohistochemical, and electron microscopic analyses for the presence of nitrotyrosine and apoptosis. Menisci were also cultured for analysis of NO production. RESULTS: All menisci from joints with ACL transection demonstrated degenerative changes. A high number of apoptotic cells was present in the medial part of menisci, which contains chondrocytic cells. Menisci from nonoperated contralateral knees contained only small numbers of cells in apoptosis. Conditioned media from meniscus cultures contained similarly elevated levels of nitrite as cartilage cultures from the same arthritic knees. Nitrotyrosine immunoreactivity, an indicator of in vivo NO production, was prominent in menisci from knees with ACL transection. In addition, menisci from normal knees produced high levels of NO in response to in vitro stimulation with interleukin-1beta or lipopolysaccharide. CONCLUSION: These observations suggest that pathologic changes in menisci are a regular feature of experimentally induced OA and are associated with NO production and meniscus cell apoptosis.

Animals↗

PubMed1999

PT-12, a putative ras-activated proliferation-dependent gene, is expressed in patellar tendon and not in anterior cruciate ligament.

We describe a gene (PT-12) that is expressed in the patellar tendon and not in the anterior cruciate ligament. We used a recently developed polymerase chain reaction-based subtractive cDNA analysis to discover genes that are overexpressed in the patellar tendon but not expressed in the anterior cruciate ligament; the long-term objective was to find genes that are central to the self-repair of the patellar tendon, in contrast with the inability of the anterior cruciate ligament to launch a repair response following injury. PT-12 is a homologue of human S2 or mouse LLRep3 ribosomal genes, which are known to be overexpressed in highly proliferating cells. This study opens a new vista to the development of techniques and reagents to study the differences between two periarticular tissues (i.e., the patellar tendon and anterior cruciate ligament) that differ primarily in their ability to self-repair.

Animals↗

PubMed1999

Effects of increased in vivo excursion on digital range of motion and tendon strength following flexor tendon repair.

Postoperative rehabilitation is an important factor in determining functional outcome following intrasynovial flexor tendon repair. We hypothesized that a rehabilitation protocol that produced increased in vivo excursion would lead to increased digital range of motion and tendon strength and decreased adhesion formation in a canine model. Ninety-six flexor digitorum profundus tendons from 48 dogs were cut transversely and repaired by a multistrand suture technique. Postoperative rehabilitation was performed daily with a low excursion-low force (1.7-mm average excursion; < 10 N force) or a high excursion-low force (3.6 mm excursion; < 10 N force) protocol. After death of the dogs at 10, 21, or 42 days, specimens were evaluated for digital range of motion, tensile mechanical properties, elongation of the repair site, and adhesion formation. Our data indicate that the range of motion of digits whose tendons were at low or high excursion was similar to that of controls. Increased in vivo tendon excursion due to synergistic wrist motion did not significantly affect ex vivo flexion of the distal and proximal interphalangeal joints or tendon displacement (p > 0.05). Similarly, tensile properties (ultimate load, repair site rigidity, and repair site strain at 20 N and at failure) and length of the gap at the repair site were not significantly affected by increased excursion (p > 0.05). Severity of adhesion formation was reduced slightly by increased excursion (p = 0.04). Our findings indicate that 1.7 mm of tendon excursion is sufficient to prevent adhesion formation following sharp transection of the canine flexor tendon and that additional excursion provides little added benefit.

Animals↗

PubMed1999

Integrative cartilage repair: inhibition by beta-aminopropionitrile.

The effects of beta-aminopropionitrile, a known inhibitor of lysyl oxidase, on the extractability of newly synthesized collagen and integrative cartilage repair were determined in explant cultures of adult bovine articular cartilage. Dose-escalation studies indicated that treatment of cartilage explants for 6 days with beta-aminopropionitrile caused a dose-dependent inhibition of proteoglycan synthesis ([35S]sulfate incorporation) with a 50% inhibition at 2.2 mM. However, 0.25 mM beta-aminopropionitrile had no detectable effect on proteoglycan synthesis and was thus used for subsequent experiments. Treatment of cartilage with beta-aminopropionitrile for 14 days increased the extractability of newly synthesized collagen with 4 M guanidine-HCl while having little effect on proteoglycan synthesis, proteoglycan deposition, collagen synthesis (formation of [3H]hydroxyproline after labeling with [3H]proline), collagen deposition, or cartilage cellularity (DNA content). In untreated cultures, the percentage of radiolabeled collagen ([3H]hydroxyproline) that was extractable after 1 day of radiolabeling, 6 days of radiolabeling, or 6 days of label and 6 days of chase decreased from 81 to 25 and 9%, respectively. In beta-aminopropionitrile-treated cultures, the extractability was relatively higher (96, 62, and 47%, respectively). Treatment with beta-aminopropionitrile after radiolabeling with [14C]lysine also significantly inhibited the formation of the reducible crosslink [14C]dihydroxylysinonorleucine without affecting the overall deposition in cartilage of [14C]lysine and [14C]hydroxylysine. In functional repair studies, treatment with beta-aminopropionitrile caused an almost complete inhibition of integration between pairs of cartilage explants maintained in apposition for 2 weeks. These results indicate that beta-aminopropionitrile blocks the formation of collagen crosslinks in cartilage explants and suggest that such crosslinks are critical to integrative cartilage repair.

Aminopropionitrile↗

PubMed1999

Wrist and digital joint motion produce unique flexor tendon force and excursion in the canine forelimb.

The force and excursion within the canine digital flexor tendons were measured during passive joint manipulations that simulate those used during rehabilitation after flexor tendon repair and during active muscle contraction, simulating the active rehabilitation protocol. Tendon force was measured using a small buckle placed upon the tendon while excursion was measured using a suture marker and video analysis method. Passive finger motion imposed with the wrist flexed resulted in dramatically lower tendon force (approximately 5 N) compared to passive motion imposed with the wrist extended (approximately 17 N). Lower excursions were seen at the level of the proximal interphalangeal joint with the wrist flexed (approximately 1.5 mm) while high excursion was observed when the wrist was extended or when synergistic finger and wrist motion were imposed (approximately 3.5 mm). Bivariate discriminant analysis of both force and excursion data revealed a natural clustering of the data into three general mechanical paradigms. With the wrist extended and with either one finger or four fingers manipulated, tendons experienced high loads of approximately 1500 g and high excursions of approximately 3.5 mm. In contrast, the same manipulations performed with the wrist flexed resulted in low tendon forces (4-8 N) and low tendon excursions of approximately 1.5 mm. Synergistic wrist and finger manipulation provided the third paradigm where tendon force was relatively low (approximately 4 N) but excursion was as high as those seen in the groups which were manipulated with the wrist extended. Active muscle contraction produced a modest tendon excursion (approximately 1 mm) and high or low tendon force with the wrist extended or flexed, respectively. These data provide the basis for experimentally testable hypotheses with regard to the factors that most significantly affect functional recovery after digital flexor tendon injury and define the normal mechanical operating characteristics of these tendons.

Animals↗

PubMed1999

The effects of hyaluronan on matrix metalloproteinase-3 (MMP-3), interleukin-1beta(IL-1beta), and tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression during the development of osteoarthritis.

OBJECTIVE: To assess the influence of intra-articular injection of hyaluronan (HA) on expression of matrix metalloproteinase-3 (MMP-3), interleukin-1beta(IL-1beta), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in cartilage and synovium during the process of osteoarthritis (OA). DESIGN: Eighteen mature New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT) and were divided into two groups. The first group (HA injection group) received 0.3 ml of intra-articular HA injections into the ACLT knees 4 weeks after transection, once a week for 5 weeks as per clinical treatment presently utilized. The animals in the second group (no injection group) were not injected after ACLT. At death, 9 weeks following surgery, synovium and cartilage were harvested and total RNA was extracted. Gene expressions of MMP-3, IL-1beta and TIMP-1 were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) for each subgroup created according to morphological grade of OA. RESULTS: The extent and grade of cartilage damage in the HA injection group was less severe than in the no injection group. In synovium, expression of MMP-3 and IL-1beta mRNA was suppressed in the mild grades of OA in the HA injection group. HA treatment had either no effect on MMP-3 expression in cartilage at all grades of OA or on enhanced MMP-3 and IL-1beta expression in synovium at a progressed grade. No effect of HA treatment on TIMP-1 expression was observed in either cartilage or synovium. CONCLUSIONS: These results suggest that one of the mechanism of therapeutic effect of HA is down-regulation of MMP-3 and IL-1beta in synovium during early development of OA.

Animals↗

PubMed1999

Articular cartilage transplantation. Clinical results in the knee.

Between December 1983 and August 1991, 55 consecutive patients (55 knees) who underwent articular cartilage transplantation to their damaged knees were enrolled in the study. Average followup was 75 months (range, 11-147 months). Eight-two percent were younger than 45 years of age. Patients were evaluated through an 18-point scale, with 6 points each allocated to pain, range of motion, and function. An excellent knee was pain free, had full range of motion, and permitted unlimited activity. A good knee allowed full time employment and moderate activity. Eleven of 15 (73%) allografts transplanted 10 or more years ago were still good or excellent at the time of last followup. Overall, 45 of 55 (76%) knees that received the transplants were rated good or excellent. Specifically, 36 of 43 (84%) patients with unipolar transplants regained normal use of their resurfaced knee. The results after bipolar resurfacing were less encouraging, with only six of 12 (50%) knees rated good or excellent. The described technique of osteochondral shell allograft resurfacing of the knee capitalize on the different healing potentials of bone and cartilage by transplanting the viable articular cartilage organ in its entirety along with just enough of the underlying bone to allow for graft incorporation through creeping substitution. The results support the use of fresh osteochondral shell allograft transplantation for the treatment of large, full thickness articular cartilage defects to the medial or lateral femoral condyles and to the patella.

Adolescent↗

PubMed1999

Collagen fibril diameter distributions in rabbit anterior cruciate and medial collateral ligaments: changes with maturation.

This study presents morphometric analyses of the collagen fibril diameters of rabbit anterior cruciate and medial collateral knee ligaments of New Zealand White rabbits (young, age two months and adult, age thirty-six to forty months). Measurements were made from transmission electron micrographs of transverse ligament sections of approximately 50,000x magnification. Statistically significant differences in the mean fibril diameters were found between the anterior cruciate and medial collateral ligaments of the thirty-six to forty month old animals (.069 +/- .005, .092 +/- .016 mm, p < .1); however, no statistical significance was found for differences between these ligaments in two month old animals (.077 +/- .006, .082 +/- .009, p > .1). These data support the idea that known differences in fibril distributions of adult rabbit anterior cruciate and medial collateral ligaments develop with maturation, and may reflect both the cellular environment in which the fibrocytes of these ligaments are subject to, as well as the developmental genetic program of these cell populations.

Animals↗

PubMed1998

Chondrocyte apoptosis and nitric oxide production during experimentally induced osteoarthritis.

OBJECTIVE: Chondrocytes produce nitric oxide (NO) and undergo apoptosis in response to exogenous NO. This study sought to examine the relationship between NO synthesis, chondrocyte apoptosis, and the development of cartilage degradation during experimental osteoarthritis (OA). METHODS: OA was induced in rabbits by anterior cruciate ligament transection (ACLT). Knees were harvested after 4 weeks and assessed for OA severity and chondrocyte apoptosis. Conditioned media from cultured cartilage explants were analyzed for nitrite content. Cartilage sections were analyzed by immunohistochemistry for the presence of nitrotyrosine. RESULTS: All ACLT knees demonstrated osteoarthritic changes. Conditioned media from ACLT cartilage organ cultures contained higher levels of nitrite as compared with cartilage samples from the nonoperated side or from rabbits that had not received ACLT. Cultures of specific areas of cartilage from ACLT knees showed high levels of NO production in the medial femoral and medial tibial cartilage. Approximately 28.7% of chondrocytes isolated from ACLT cartilage and 6.7% of chondrocytes from cartilage of the nonoperated side underwent apoptosis. In situ staining demonstrated apoptotic cells in the superficial and middle zones of ACLT cartilage. A high number of apoptotic cells was present at the pannus-cartilage junction. In control cartilage, the superficial zone contained a small number of cells in apoptosis. The prevalence of apoptotic cells was significantly correlated with the levels of nitrite production and OA grade. CONCLUSION: These observations suggest that, during the early phases of OA, NO production may lead to chondrocyte apoptosis, and that both events contribute to the pathogenesis of cartilage degradation. Inhibitors of NO synthesis and chondrocyte apoptosis may therefore be of therapeutic value after cartilage injury and in patients with OA.

Animals↗

PubMed1998

Age-related changes in the expression of cadherin-11, the mesenchyme specific calcium-dependent cell adhesion molecule.

Cadherin-11 is a calcium-dependent cell adhesion molecule that is expressed in cells of the mesenchymal lineage during embryonic development. In this study we show, for the first time, that cadherin-11 gene is expressed in the bone marrow and bone cells obtained from rabbits of various age groups. Furthermore, a quantitative measurement of gene expression revealed that cadherin-11 was expressed in young rabbits (6 week-old: open epiphysis) at a level of 6.7 x 10(5) +/- 0.7 x 10(5) molecules; in mature rabbits (8-10 month-old: closed epiphysis) at 11 x 10(5) +/- 0.9 x 10(5) molecules; and in aged rabbits (4-5 year-old) at a level of 1.2 x 10(5) +/- 0.2 x 10(5) molecules/microg total RNA. The relative level of cadherin-11 gene expression in mature rabbit marrow was found to be approximately 50% greater than in young rabbits. However, aged animals showed a reduction in cadherin-11 specific gene expression of greater than 900% as compared with mature animals. Age-related changes in bone remodeling/turnover lead to reduced bone density and high fracture risk, and since cadherins play a crucial role in tissue morphogenesis, this marked decrease may represent an index of the aging process in bone.

Age Factors↗

PubMed1998

Architectural remodeling in deep frozen meniscal allografts after total meniscectomy.

This study assesses the biological characteristics of cell repopulation and matrix remodeling in deep frozen meniscal allografts after total meniscectomy and allograft transplantation. Thirty-two mature New Zealand White rabbits were operated on, and the allografts were assessed at 12 and 26 weeks postsurgery. Before transplantation, fresh medial menisci were placed into liquid nitrogen to kill all intrinsic cells. Frozen meniscal allografts showed collagen remodeling coincident with revascularization and cellular repopulation. Biochemical characterization showed active collagen remodeling of the allografts at 12 and 26 weeks. In situ hybridization showed that expression of types I and III procollagen mRNAs were increased at 12 weeks, especially near the region of the synovial capsule. At 26 weeks after transplantation, the pattern of collagen-type expression progressed toward normal; that is, type I procollagen mRNA expression was prominent, whereas only a small amount of type III procollagen mRNA was observed. These data indicate the adaptation of the repopulating cells from the host to the frozen allograft at 26 weeks after transplantation.

Animals↗

PubMed1998

Long-term effects of hyaluronan on experimental osteoarthritis in the rabbit knee.

OBJECTIVE: Long-term assessment of the effect of hyaluronan (HA) on the articular cartilage and synovium in an animal knee joint during the development of osteoarthritis (OA). DESIGN: Sixty mature New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT) and were divided into two groups. Group 1 (SA) received intra-articular injections of 0.3 ml hyaluronan (HA) (i.e., ARTZ, MW: 8 x 10(5)) beginning 4 weeks after ACLT, once a week for 5 weeks. Group 2 (SV) received injections of the vehicle (phosphate buffered saline) in the same fashion as with the SA group. The contralateral nonoperated knee served as control. All animals were killed 21 weeks after surgery and their knee joints evaluated by gross morphologic, histologic, histomorphometric and biochemical analyses. RESULTS: Gross morphological inspection indicated that the femoral condyles from the knees injected with vehicle suffered more severe cartilage damage than cartilage from the knees injected with HA. Furthermore, two out of three histomorphometric parameters measured in the HA-treated cartilage (i.e., cartilage thickness and cartilage area which were not statistically different than control) provided evidence showing a protective effect of HA on the femoral condyles following ACLT. Biochemical analysis showed articular cartilage remaining on the femoral condyles following ACLT to have similar characteristics to contralateral controls. However, DNA concentration in the synovium from the ACLT knees of the vehicle-treated animals was greater than contralateral control, while this parameter was not statistically different than contralateral control in the HA treated animals. CONCLUSIONS: These results demonstrate a protective effect of HA on preservation of the articulating surface of the femoral condyle following ACLT up to 21 weeks post-surgery.

Animals↗

PubMed1998

Integrin expression is upregulated during early healing in a canine intrasynovial flexor tendon repair and controlled passive motion model.

To explore crucial early molecular events involved in contact healing of the intrasynovial flexor tendon, integrin expression was evaluated at the transcriptional and post-transcriptional levels during the first two weeks following injury, repair and controlled passive motion in a canine model. Specifically, immunohistochemical and reverse transcription polymerase chain reaction (RT-PCR) techniques were employed to evaluate expression of the fibronectin, vitronectin and endothelial cell binding integrin receptor subunits alpha5, alphav and alpha6, along with the common beta1 subunit. The two techniques revealed increasing expression of the four subunits over the two week post-repair period. Immunohistochemistry revealed that beta1 and alpha5 expression was concentrated in the epitenon layer near the repair site and interiorly within the wound area, while alpha6 was associated with capillary-forming endothelial cells near the wound. RT-PCR and quantitation by NIH image analysis demonstrated peak messenger RNA expression of beta1 and alpha5 at ten days post-repair and peak expression of alpha6 and alphav at 15 days. The results in this study correlate well with previous results demonstrating increased fibronectin deposition and angiogenesis during the same time period in a similar injury/repair model.

Animals↗