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Biomedical subjects

C B Frank

Publications and source records attributed to C B Frank.

At least 37 records · Page 2Linked to original sources
PubMed2000

In-vitro cyclic tensile loading of an immobilized and mobilized ligament autograft selectively inhibits mRNA levels for collagenase (MMP-1).

To test the hypothesis that loading conditions can be used to "engineer" ligament autograft behaviors, the effect of cyclic tension on the mRNA levels of matrix molecules and collagenase in in-vivo immobilized and mobilized 6-week rabbit medial collateral ligament (MCL) autografts was examined using an in-vitro system. Femur-[autograft MCL]-tibia complexes were subjected to a tensile stress of 4 MPa at 0.5 Hz for 1 min, followed by 14 min of rest. This 15-min testing cycle was repeated for 4 h. Semi-quantitative reverse transcrip-tase polymerase chain reaction (RT-PCR) was performed on RNA from mechanically treated MCL autografts, using rabbit-specific primer sets for types I and III collagen, biglycan, decorin, fibromodulin, lumican, versican, matrix metalloproteinase-1 (MMP-1, collagenase-1), MMP-13 (collagenase-3), and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, 4 h of culture of normal control MCLs led to increased mRNA levels for MMP-1 (P < 0.05), but there were no significant changes in MMP-13 mRNA levels. Total RNA levels in that normal MCL tissue were, however, decreased after culture (P < 0.05). In-vitro tensile loading of in-vivo mobilized autografts resulted in a significant increase in total RNA (185% of in-vitro non-loaded autografts). On the other hand, in-vitro tensile loading of in-vivo immobilized autografts resulted in no significant changes in total RNA levels compared with levels in non-loaded control grafts. MMP-1 mRNA levels in both the in-vivo mobilized (47% of non-loaded autograft) and in-vivo immobilized (38% of non-loaded autograft) MCL autografts were significantly lower than those in non-loaded control tissue following in-vitro tensile loading, but there were no significant changes in the mRNA levels for the seven other matrix molecules assessed. These results show that it is possible to selectively inhibit MMP-1 mRNA levels in autograft ligaments by supplying mechanical stimuli in vitro. The results also demonstrate that in-vivo immobilization leads to a decrease in the effects of subsequent in-vitro mechanical loading in such autografts with respect to total RNA levels. Collectively, these results demonstrate that both in-vivo and in-vitro loading have implications in the engineering of an ideal ligament graft.

Animals↗
PubMed2000

Complexity of determining cause and effect in vivo after antisense gene therapy.

Injuries to joint tissues are major clinical problems occurring with significant frequency and resulting in the formation of scar tissue or in some tissues with no healing at all. Such scar tissue has compromised biomechanical integrity, which leads to impaired function, increased risk of reinjury, induction of remodeling in other joint tissues and increases the risk of diseases such as ostheoarthritis. Development of new therapies, such as gene therapy, to enhance repair could have a significant impact on quality of life for patients. The well-characterized rabbit medial collateral ligament injury model was used to transiently modulate the expression of specific molecules during early stages of healing. The small matrix proteoglycan decorin, known to influence matrix assembly and to bind and growth factors, was targeted in vivo using decorin-specific antisense oligodeoxynucleotides and Hemagglutinating Virus of Japan-Liposome method. After 4 weeks of healing, scar tissue was assessed after antisense exposure by reverse transcription polymerase chain reaction, Western Blot analysis, light and transmission electron microscopy, and biomechanically for low and high load behavior. Ligament scar messenger ribonucleic acid and protein levels for decorin decreased and collagen fibril diameter size increased after antisense treatment. Creep and stress at failure improved after antisense treatment indicating a functional improvement in the scar tissue. However, messenger ribonucleic acid levels for multiple genes were affected by the decorin-specific antisense treatment and therefore all of the observed improvements in the scar tissue cannot be directly ascribed to depressing decorin levels.

Animals↗
PubMed2000

Adaptive time-frequency analysis of knee joint vibroarthrographic signals for noninvasive screening of articular cartilage pathology.

Vibroarthrographic (VAG) signals emitted by human knee joints are nonstationary and multicomponent in nature; time-frequency distributions (TFD's) provide powerful means to analyze such signals. The objective of this paper is to construct adaptive TFD's of VAG signals suitable for feature extraction. An adaptive TFD was constructed by minimum cross-entropy optimization of the TFD obtained by the matching pursuit decomposition algorithm. Parameters of VAG signals such as energy, energy spread, frequency, and frequency spread were extracted from their adaptive TFD's. The parameters carry information about the combined TF dynamics of the signals. The mean and standard deviation of the parameters were computed, and each VAG signal was represented by a set of just six features. Statistical pattern classification experiments based on logistic regression analysis of the parameters showed an overall normal/abnormal screening accuracy of 68.9% with 90 VAG signals (51 normals and 39 abnormals), and a higher accuracy of 77.5% with a database of 71 signals with 51 normals and 20 abnormals of a specific type of patellofemoral disorder. The proposed method of VAG signal analysis is independent of joint angle and clinical information, and shows good potential for noninvasive diagnosis and monitoring of patellofemoral disorders such as chondromalacia patella.

Algorithms↗
PubMed1999

The gross morphology of torn human anterior cruciate ligaments in unstable knees.

To evaluate the presence and incidence of reattachments of torn human anterior cruciate ligaments (ACL), we prospectively investigated 101 patients undergoing arthroscopic ACL reconstruction to study the intra-articular morphology of ACLs under circumstances in which functional healing had failed. Results showed that roughly 72% of these unstable knees had reattachment of the torn ACL to the posterior cruciate ligament (PCL). Eighteen percent had no signs of ACL reattachment but only 2% of previously torn ACLs were absent. These results suggest that even in chronic situations in which the knee remains functionally unstable, human ACLs rarely resorb. It also suggests that torn human ACLs commonly reattach in the knee, mainly to the PCL via a process that is consistent with scarring. While the function of these reattachments is clearly inadequate in people with unstable knees because of a combination of reattachment location, scar quantity, or quality, these results nonetheless show that the intra-articular environment in humans often maintains ACL stumps and it is not totally inhibitory to ACL reattachment via some biological process.

Adolescent↗
PubMed1999

Molecular biology and biomechanics of normal and healing ligaments--a review.

OBJECTIVE: In this review article, we discuss current data and concepts concerning the molecular biology and biomechanics of both normal and healing ligaments in a rabbit model. METHOD: Data is presented from light microscopy, transmission electron microscopy, molecular biology (RT-PCR), and biomechanical measurements (laxity, stress at failure, modulus, and static creep) or normal, pregnant and healing rabbit medial collateral ligaments. RESULTS: 'Flaws' in scar matrix, smaller-than-normal diameter collagen fibrils, and failure of collagen cross-link maturation may be particularly important deficiencies which appear to be related to ligament scar weakness and perhaps to scar creep. The mechanical behaviours of both normal and healing ligaments are altered by relative states of joint motion and normal ligaments are affected by systemic hormones (particularly during pregnancy). DISCUSSION: Molecular analysis of ligaments and ligament scars, combined with ongoing morphological and biomechanical studies of ligament structure and function, will ultimately reveal which factors can be manipulated clinically to optimize the restoration of normal ligament properties after ligament injuries. Further studies on the mechanisms of ligament healing, genetic markers of repair, and gender-specific differences in ligament repair responses are required.

Animals↗
PubMed1999

Collagenase degradation decreases collagen fibril diameters--an in vitro study of the rabbit medial collateral ligament.

Based on the similarity of fibril diameters in healing and grafted ligaments, it has been speculated that all small fibrils represent newly synthesized collagen. Alternatively, small fibrils in grafts could be due to enzymatic degradation of endogenous large fibrils. This study examined the effect of collagenase on collagen fibril diameters in normal NZW rabbit MCLs. Midsubstance MCL slivers were incubated in buffer for 72 or 144 h for comparison with slivers incubated in buffer containing 4 units/ml bacterial collagenase. The samples were examined under TEM for fibril diameter analysis. Mean fibril diameters of 3-day and 6-day collagenase-treated MCLs were significantly reduced, resembling 40-week scar values. These results suggest that collagenase treatment can alter collagen fibril diameter and shape in normal rabbit MCL, thus it is possible that despite their similarity to ligament scars, that at least some small fibrils in ligament grafts may be enzymatically reduced endogenous fibrils.

Animals↗
PubMed1998

Localization and characterization of porcine patellar tendon xenograft antigens in a rabbit model of medial collateral ligament replacement.

BACKGROUND: Ligament injuries of the knee are common and, if severe, can predispose to joint pain, instability, reinjury, and, ultimately, osteoarthritis. Xenograft replacement of ligaments could have potential; however, a limited understanding of the immunology of ligament xenograft rejection has inhibited their use. The purpose of this study was to characterize the antigenic elements of a fresh porcine tendon xenograft in a rabbit model and to provide a better understanding of what would need to be done to either block or extract these antigenic elements. METHODS: Three experimental situations were evaluated in a pig to rabbit ligament transplantation model: subcutaneous implantation of fresh porcine patellar tendon (PPT), implantation of fresh PPT into a medial collateral ligament midsubstance gap, and replacement of the entire medial collateral ligament complex with either fresh or guanidinium hydrochloride-extracted PPT. Preimmune and immune sera were collected from rabbits and used to localize antigenic targets in PPT, meniscus, and cartilage with indirect immunofluorescence techniques. The reactivities of the same rabbit sera towards tissue extracts of PPT, meniscus, and cartilage by Western immunoblot analyses were used to characterize the antigenic components. RESULTS: Indirect immunofluorescence with preimmune rabbit sera on PPT showed staining of tendon fibroblasts. Immune sera from rabbits transplanted with xenografts stained regions of the extracellular matrix of PPT. Fresh PPT induced antibodies that consistently recognized six extracellular matrix components with molecular masses of >200 kDa, 180 kDa, 135 kDa, 108 kDa, 63 kDa, and 59 kDa. CONCLUSIONS: Our results suggest that naturally occurring rabbit anti-pig antibodies of the IgG isotype recognize immunogenic components on tendon fibroblasts, whereas induced rabbit anti-pig antibodies recognize a specific subset of six extracellular matrix components of PPT. PPT xenografts appeared to induce a similar humoral immune response irrespective of graft location. Finally, our results indicate that selective extraction of PPT xenograft components before implantation altered the induced rabbit anti-pig antibody response; however, such extraction did not change the ultimate fate of the transplant tissue.

Animals↗
PubMed1998

Postmortem stability of total RNA isolated from rabbit ligament, tendon and cartilage.

The stability of RNA, particularly mRNA, in tissues is under complex regulation. Most studies to date have focused on very cellular tissues and not connective tissues such as ligaments, tendons and cartilage. As the availability of such tissues for transplantation or research purposes is frequently delayed following death, it is important to determine whether RNA stability in such tissues is influenced by time postmortem. To approach this question, skeletally mature NZW rabbits were used to investigate RNA integrity over time in dense, hypocellular connective tissues and in several hypercellular organ tissues such as brain, kidney, liver and lung. Samples were analyzed at varying intervals postmortem with respect to rRNA integrity by agarose gel electrophoresis and ethidium bromide staining and mRNA integrity by Northern blot analysis and RT-PCR. No degradation of rRNA or loss in integrity of mRNA for genes of low and high copy number was observed up to 96 h postmortem. These findings confirm that it is likely appropriate to use properly stored postmortem dense connective tissues for molecular biological investigations.

Animals↗
PubMed1998

Comparison of mRNA levels for matrix molecules in normal and disrupted human anterior cruciate ligaments using reverse transcription-polymerase chain reaction.

Midsubstance samples of anterior cruciate ligaments from seven normal human cadaver knees (16-50 years old) were harvested and compared with midsubstance pieces of scarred anterior cruciate ligaments from 30 patients (15-40 years old). RNA was isolated from each ligament, and the expression of type-I collagen, type-III collagen, biglycan, decorin, lumican, and tissue inhibitor of metalloproteinase-1 was evaluated by quantitative reverse transcription-polymerase chain reaction with use of beta-actin as the housekeeping gene. Data for injured ligaments were further compared statistically as a function of time after injury to better define patterns of cellular expression over time. Our hypothesis was that injured ligaments would show minimal cellular activity and decreasing activity over time. The results revealed that both normal and injured anterior cruciate ligaments contain cells that express mRNA for all molecules studied. However, cells in injured ligaments express much higher, but still proportional, quantities of message for type-I collagen and type-III collagen (p < 0.000001) and higher quantities of biglycan (p < 0.02) and tissue inhibitor of metalloproteinase-1 (p < 0.0003) than do cells in normal anterior cruciate ligaments. These levels remained elevated for longer than 1 year after injury. Linear regression analysis showed biglycan expression correlated with time from injury (r2 = -0.69; p = 0.007). These results collectively demonstrate that injured human anterior cruciate ligaments contain cells that express scar-like molecules and that the injured ligaments are likely continuing to remodel matrix over time. Furthermore, they suggest that human anterior cruciate ligaments have not failed to heal due to the failure of scar formation per se. The quality and quantity of this scar remain questionable; however, the possibility of its enhancement as a healing strategy for human anterior cruciate ligaments cannot be dismissed.

Adolescent↗
PubMed1998

Method to assess in vivo knee stability longitudinally in an animal model of ligament injury.

The purpose of this study was to develop a method to prospectively quantify passive knee stability in an animal model of joint injury over time. Knee stability is defined here as the amount of translation or rotation of the tibia relative to the femur for a given application of force or moment, respectively. Five animals that had undergone transection of the anterior cruciate ligament and three control animals that had undergone a sham operation were anaesthetized and positioned in a stereotaxic frame. Motion of the tibia relative to the femur was quantified with use of reflective markers secured to modified bone pins and a three-dimensional motion analysis system. External forces and moments in the transverse plane of the tibia were measured with use of force transducers based on a strain-gauge design. Longitudinal measurements of knee stability were made before either sham surgery (control animals) or transection of the ligament (experimental animals), immediately after surgery, and at 2 and 4 months after transection. The results showed that the animals tolerated the procedures well and that systematic measurements could be obtained. The method described here has the practical advantage over cross-sectional experimental designs in that the number of subjects can be decreased while maintaining statistical power and has the further conceptual advantage that individual changes can be accounted for over time.

Animals↗
PubMed1998

Longitudinal measurement of tibial motion relative to the femur during passive displacements in the cat before and after anterior cruciate ligament transection.

Passive anterior-posterior displacement and medial-lateral rotation of the tibia on the femur in the feline knee were assessed before transection of the anterior cruciate ligament, immediately after transection, and 2 and 4 months after transection. Four anaesthetized experimental and three sham-operated control animals were positioned in a stereotaxic frame. Motions of the tibia relative to the femur were measured with use of 60-Hz video motion analysis, while a strain-gauged system allowed measurement of forces and moments applied to the tibia. Displacement at 15 N of anterior force and 30 degrees of knee flexion increased by an average of 6 mm following transection, and stiffness decreased by an average of 6 N/mm. At 2 and 4 months following transection, there were statistically significant reductions in this abnormal displacement. Stiffness during anterior displacement of the tibia at 30 degrees increased significantly from immediately after transection to 4 months. At 90 degrees, mean anterior displacement decreased from 5.1 mm immediately after transection to 2.9 mm at 4 months. Medial rotation at 30 degrees of knee flexion was significantly decreased from a mean of 16.5 degrees after transection to a mean of 10.7 degrees at 4 months. Changes in medial rotation at 90 degrees, lateral rotation at 90 degrees, and lateral rotation at 30 degrees were not statistically significant. These results indicate a significant change in secondary constraints to tibial motion in response to knee instability.

Animals↗
PubMed1998

Identification of sex hormone receptors in human and rabbit ligaments of the knee by reverse transcription-polymerase chain reaction: evidence that receptors are present in tissue from both male and female subjects.

Gender-related factors have been attributed to observed differences in the rate of injury to ligaments (e.g., anterior cruciate ligament) between male and female subjects. These differences may be a result of unique regulatory mechanisms within the tissue in response to the sex hormones estrogen and progesterone. These hormones, when bound to specific intracellular receptors (estrogen receptor and progesterone receptor, respectively), modulate gene expression within hormone-responsive tissue. The purpose of this study was to evaluate the expression of the estrogen and progesterone receptors in ligament tissue from male and female rabbits and humans by the sensitive molecular technique of reverse transcription-polymerase chain reaction. Total RNA was extracted from human anterior cruciate ligament tissue and from medial cruciate ligament, anterior cruciate ligament, patellar tendon, and synovium tissue of the New Zealand White rabbit by the newly developed TRIspin method. The total RNA was reverse transcribed and analyzed by polymerase chain reaction to assess the expression of estrogen and progesterone receptors. Our results demonstrate that estrogen and progesterone receptor transcripts are expressed in ligament tissue of male and female rabbits and humans and that alterations in receptor expression occur in ligaments during pregnancy. In the human samples, only a small percentage of the estrogen receptor appeared to be a nonfunctional mRNA splice variant, and the predominant form contained the estrogen-binding domain.

Alternative Splicing↗
PubMed1998

Immobilization increases the vulnerability of rabbit medial collateral ligament autografts to creep.

Rehabilitation after soft-tissue autograft reconstructions is controversial because there is indirect evidence that some grafts fail by creeping over time. The vulnerability of soft-tissue grafts to creep over healing time and the effects of the load environment during healing on this vulnerability have never been studied specifically. We hypothesized that immobilization would decrease the magnitude of the vulnerability of ligament grafts to creep. Thirty-nine skeletally mature New Zealand White rabbits underwent a standardized medial collateral ligament autograft procedure to the right hindlimb, and 19 of the rabbits also had the limb rigidly pinned into flexion. Subgroups were killed at 3 or 8 weeks, and all isolated tibia/medial collateral ligament/femur complexes were tested for creep at 4.1 MPa under a standardized protocol. Eight normal medial collateral ligament controls were tested similarly. Results showed that all grafts were quantitatively more susceptible to cyclic and static creep than were normal medial collateral ligament controls (p < 0.05). By 3 weeks of healing, immobilization significantly increased the magnitude of the vulnerability of the grafts to cyclic, static, and total creep (all: p < 0.05). Furthermore, the grafts had more unrecovered creep strain than did the controls following a 20-minute recovery period. Contrary to our hypothesis, immobilization resulted in increased vulnerability of these ligament autografts to creep even with this relatively nonprovocative test of short duration and low stress. We postulate that following immobilization, this increase in the magnitude of susceptibility of the grafts to creep will result in functionally significant elongation of the graft if it is exposed to higher loads and over longer periods of time in vivo.

Animals↗
PubMed1998

Altered levels of extracellular matrix molecule mRNA in healing rabbit ligaments.

RT-PCR methods were used to amplify, semi-quantify, clone and sequence cDNA fragments specific for rabbit extracellular matrix molecules biglycan, collagen I, collagen III, decorin, lumican, versican, fibromodulin, and also glyceraldehyde-3-phosphate dehydrogenase (G3PDH), using RNA isolated from rabbit ligaments. Sequence analysis of two independent clones of PCR products was used to verify the identity of the cDNA. Semi-quantitative RT-PCR was used to study mRNA levels for these matrix molecules in normal and healing rabbit ligament at three, six, and fourteen weeks post-injury. The yield of RNA from the ligament scar was increased at three and six weeks post-injury, but it had returned to near normal levels by fourteen weeks. On a microgram RNA basis, it was demonstrated that biglycan, collagen I, collagen III and lumican mRNA levels are significantly elevated, versican mRNA levels significantly depressed, and decorin and fibromodulin mRNA levels showed no significant change in response to tissue injury in the ligament during the course of healing. These findings suggest that differential regulation of mRNA levels for these extracellular matrix molecules occurs during ligament healing.

Animals↗
PubMed1998

A comparison of in vivo gene delivery methods for antisense therapy in ligament healing.

To determine the most efficient in vivo delivery method of oligonucleotides for antisense therapy in ligament healing, fluorescence-labelled phosphorothioate oligodeoxynuleotides (ODN) were introduced into 12 rabbit ligament scars 2 weeks after injury using haemagglutinating virus of Japan (Sendai virus: HVJ)-conjugated liposomes. We compared the efficiency of cellular uptake of fluorescence as a percentage of all cells in each scar using three delivery procedures: (1) direct free-hand injection into the ligament scar using a conventional syringe; (2) systematic direct sca injection using a repeating 10 microliters dispenser and a square mesh grid system; and (3) injection into the feeding (femoral) artery. Results showed that there was a significant difference in fluorescence uptake by scar cells on day 1 after injection between the three delivery methods: (1) direct free-hand, 9.7 +/- 7.6% (average +/- s.d.); (2) systematic direct, 58.4 +/- 15.9%; and (3) intra-arterial, 0.2 +/- 0.1%. Systematic direct injection was most efficient and it resulted in 25.9 +/- 13.0% of scar cells being labeled at 7 days after transfection. We then introduced antisense ODN for the rabbit proteoglycan, decorin, into ligament scars with this delivery method and confirmed a significant inhibition of decorin mRNA expression in antisense-treated scar tissues in vivo both at 2 days (42.3 +/- 14.7% of sense control +/- s.d.; P < 0.0025) and 3 weeks (60.5 +/- 28.2% of sense control +/- s.d.; P < 0.024) after treatment, compared with sense ODN-treated scars. Decorin was significantly suppressed also at protein level in antisense-treated scars at 4 weeks (66.6 +/- 35.7% of sense control +/- s.d.; P < 0.045) after treatment. These results demonstrate that in vivo transfection efficiency in ligament scars is 'delivery system dependent' and that introduction of antisense ODN for the small proteoglycan, decorin, with this delivery method can lead to significant suppression of its expression over 3 weeks both at mRNA and protein levels. Thus, an effective model for the potential manipulation of scar composition and quality in ligament healing has been established.

Animals↗
PubMed1998

Repetitive activity alters perfusion of proximal interphalangeal joints of the human hand.

OBJECTIVE: To examine whether competitive volleyball players show any difference in perfusion of their proximal interphalangeal (PIP) joints compared with a healthy group of subjects. Also to assess the viability of a dual wavelength laser Doppler imager (LDI) in making these measurements. SETTING: Physiology laboratory. PARTICIPANTS: Ten active volleyball players who had experienced repetitive finger joint injury and 12 age- and sex-matched normal control subjects. MAIN OUTCOME MEASURES: Using a modified LDI incorporating a near- infrared (850 nm) laser as well as a standard red (633 nm) laser, scans were performed over the dorsum of the hands of the volleyball players and the control group. RESULTS: Higher perfusion values were obtained with the 850-nm laser than with the red 633-nm laser. When referenced to adjacent skin blood flow, perfusion over PIP joints of volleyball players was found to be significantly higher than that in control subjects (p=0.00012; n=10-12). CONCLUSIONS: The higher perfusion values obtained using the 850-nm laser suggest that the longer wavelength laser is measuring perfusion in a greater volume of tissue, which could include subcutaneous structures. Volleyball players have significantly higher perfusion over the PIP joints, which is unlikely to be due to differences in skin perfusion over the two regions but is more likely to be related to hyperemia of the underlying PIP joints. The reason for increased PIP perfusion is not clear; it may represent ongoing tissue inflammation due to repeated injury, or it could be an adaptive response to the stresses placed on these joints by this type of repetitive activity. CLINICAL RELEVANCE: Near-infrared laser Doppler imaging has the potential to provide a noninvasive clinical assessment of finger joint injuries.

Adult↗
PubMed1998

Gender and neurogenic variables in tendon biology and repetitive motion disorders.

The incidence of repetitive motion disorders is increasing. Numerous studies have indicated that the incidence in females exceeds that in males. Some of the evidence regarding gender related factors in tendon biology is discussed and new data related to the regulation of gene expression in an animal model of tendon overuse, the determination of sex hormone receptors in tendons, and the influence of pregnancy associated factors on gene expression in four different tendons is provided. Furthermore, because neurogenic mechanisms may contribute to inflammatory conditions, new evidence is provided that supports the concept that neurotransmitters can influence expression of genes that could participate in such inflammation. By increasing our understanding of the regulation of tendon cellular and molecular biology, new approaches to preventing disease development and treatment of existing disease may evolve.

Animals↗