Characterization of the collagen synthesized by cultured cartilage cells.

Cartilage cells from embryonic chick cartilage were grown in primary cultures. The cell layer was sequentially extracted with neutral saline, mercaptoethylamine and pepsin which revealed that these cells produced salt-soluble and salt-insoluble collagen. The alpha1- to alpha2-chain ratio was determined for the collagen extracted from the cultured cells and was found to be 13 to 1. Further analysis of the molecule was carried out by CNBr cleavage of the salt-extracted collagen and separation of resulting peptides by ion-exchange chromatography. It was shown that the cultured cartilage cells synthesize collagen of the type (alpha1[II])3.

Characterization and synthesis of macromolecules by adult collateral ligament.

Bovine collateral ligament was found to have a water content of 67.5 +/- 2.5%, the tissue was highly collagenous containing 100.3 +/- 15.1 micrograms hydroxyproline/mg dry weight. Type I collagen was the major collagen present with small amounts of Type III and V. The hexuronate content of the tissue was found to be 2.62 +/- 0.40 micrograms hexuronate/mg dry weight of tissue. On incubation in vitro collateral ligament incorporated [35S]sulfate and [3H]acetate into proteoglycans and [3H]acetate into hyaluronate and glycoproteins. The rate of synthesis of proteoglycans by collateral ligament was shown on a weight basis to be greater than that of tendon but lower than that of articular cartilage. Analysis of the proteoglycans present in collateral ligament showed two populations of proteoglycans to be present. Approx. 20% of the total proteoglycans present were large chondroitin- and keratan sulfate-containing proteoglycans capable of forming aggregates with hyaluronate. The major species of proteoglycan present were small dermatan sulfate proteoglycans made up of a core protein with a molecular mass of 45,000 daltons with one dermatan/chondroitin sulfate glycosaminoglycan chain of 30,000 daltons attached. The N-terminal amino acid sequence of the core protein of this proteoglycan showed it to be analogous to the core protein of dermatan sulfate proteoglycan II.

Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment.

The protein composition in the extracellular matrix of cultured neonatal rat aortic smooth muscle cells has been monitored over time in culture. The influence of ascorbate on insoluble elastin and collagen has been described. In the absence of ascorbate, the cells accumulate an insoluble elastin component which can account for as much as 50% of the total protein in the extracellular matrix. In the presence of ascorbate, the amount of insoluble collagen increases, while the insoluble elastin content is significantly less. When ascorbate conditions are varied at different times during the culture, the extracellular matrices are altered with respect to collagen and elastin ratios. The decrease in elastin accumulation in the presence of ascorbate may be explained by an overhydroxylation of tropoelastin. Approximately 1/3 of the prolyl residues in the soluble elastin fractions isolated from cultures grown in the presence of ascorbate are hydroxylated. Since the insoluble elastin accumulated in these cultures contain the unique lysine-derived cross-links in amounts comparable to aortic tissue, this culture system proves ideal for studying the influence of extracellular matrix elastin on cell growth and metabolism.

The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study.

Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 X 10(6) cells/25 cm2 Falcon flask) with adequate nutrient media and pH control grow rapidly and form multilayers of cells with typical "hill and valley" organization. After 10 days growth insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become larger irregular refractile aggregates and later coalesced to form larger aggregates and small fibres. With light and electronmicroscopy, elastin was the predominant matrix protein formed, with the "hill regions" of cultures containing abundant elastin aggregates and some collagen. In 2-week-old cultures differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesis organelles and myofilaments and were in close association with large often coalescing elastin aggregates; compared to younger more superficial cells which contained more free polyribosomes less myofilaments, and were associated with fewer and small elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies, and lysosomes. Elastin aggregates and newly formed elastic fibres were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-week culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino-acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulphate (32%), chondroitin 4-sulphate (43%), keratan and heparan sulphate (30%), with only a trace of hyaluronic acid. This study indicates that primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthesize and deposit large amounts (mg) of insoluble elastin which aggregate and from elastic fibres in vitro.

Biomechanics of patellar tendon autograft for reconstruction of the anterior cruciate ligament in the goat: three-year study.

This study examined the biomechanics of anterior cruciate ligament-patellar tendon autografts for as long as 3 years after surgery. Twenty-seven adult female goats were tested; four served as controls and the others received an autograft to the right knee with each left knee serving as an additional control. The animals with grafts were tested at 0 week (n = 4), 6 weeks (n = 4), 12 weeks (n = 4), 24 weeks (n = 3), 1 year (n = 5), and 3 years (n = 3) after surgery. The anteroposterior laxity of the knee joint, load-relaxation, and structural and material properties of the graft were tested. The anteroposterior laxity was significantly greater than that of the controls for all groups except at 3 years. Load-relaxation was greater than that of the control anterior cruciate ligaments, but in the 1 and 3-year grafts load-relaxation was less than that of the patellar tendons with 5 minutes of sustained loading. Between 12 and 52 weeks, the stiffness and modulus of the grafts increased 3-fold, but the improvement was slow afterward. At 3 years, the strength and stiffness of the grafts were 44 and 49% those of the control ligaments, respectively; the modulus was 37 and 46% that of the control anterior cruciate ligaments and patellar tendons, respectively. The persistent inferior mechanical performance at 3 years suggests that anterior cruciate ligament grafts may never attain normal strength.

Long-term study of the biochemistry and biomechanics of anterior cruciate ligament-patellar tendon autografts in goats.

This study examined the change in type-III collagen concentration and hydroxypyridinium crosslink density of anterior cruciate ligament-patellar tendon autografts and their correlations with Young's modulus of the anterior cruciate autografts and anterior cruciate controls for as long as 3 years after surgery. Fifteen adult female goats (two control and 13 experimental) were tested. Each experimental animal received an anterior cruciate ligament-patellar tendon autograft to the right knee. These animals were tested at 0 (n = 2), 6 (n = 2), 12 (n = 2), and 24 (n = 1) weeks and 1 (n = 3) and 3 (n = 3) years after surgery. After mechanical testing, the anterior cruciate autograft and control tissues were analysed for type-III collagen concentration and hydroxypyridinium crosslink density. The results of sodium dodecyl sulfate gel electrophoresis showed a trend of initial increase in the percentage of type-III collagen in the anterior cruciate ligament autografts and a subsequent decrease after 24 weeks following surgery. There was a nonsignificant (p > 0.05) negative correlation between type-III collagen concentration and Young's modulus. The hydroxypyridinium crosslink density was highest at 1 year after surgery. There was a significant (p < 0.05) positive correlation between hydroxypyridinium crosslink density and Young's modulus in the anterior cruciate autografts and controls. This suggests that hydroxypyridinium crosslink density has a good linear relationship with the material strength of the anterior cruciate ligament autograft and hence could be used as an objective guide for rehabilitation with anterior cruciate autografts.

Ground reaction forces, bone characteristics, and tibial stress fracture in male runners.

PURPOSE: Tibial stress fracture is a common overuse running injury resulting from repetitive mechanical loading. This research project aimed to determine whether runners with a history of tibial stress fracture (TSF) differ in tibial bone geometry, tibial bone mass, and ground reaction force (GRF) parameters during running from those who have never sustained a stress fracture (NSF). METHODS: Forty-six male running athletes (23 TSF; 23 NSF) ranging in age from 18 to 42 yr were recruited for this cross-sectional study. A force platform was used to measure selected GRF parameters (peak and time to peak for vertical impact force, vertical active force, and horizontal braking force) during running at 4.0 m x s(-1). Tibial bone geometry (cross-sectional dimensions and area) was calculated from a computerized tomography (CT) scan at the junction of the middle and distal thirds. Dual energy x-ray absorptiometry (DXA) provided measurements of tibial bone area, bone mineral content (BMC), and bone mineral density (BMD). RESULTS: The TSF group had significantly smaller tibial cross-sectional area (P = 0.02) and DXA tibial bone area (P = 0.02), after adjusting for height and weight, than the NSF group. There were no significant differences between groups for GRF, tibial BMC, or tibial BMD. CONCLUSION: These findings support the contention that bone geometry plays a role in stress fracture development and that male athletes with smaller bones in relation to body size are at greater risk for this bony injury.

Collagen fibril populations in human anterior cruciate ligament allografts. Electron microscopic analysis.

We studied human anterior cruciate ligament allograft specimens by quantitative electron microscopy to analyze their collagen fibril populations. The specimens were procured at the time of second-look arthroscopy from the superficial region of the midzone of the anterior cruciate ligament grafts after synovial clearage. The grafts used for the anterior cruciate ligament reconstructions were from fresh-frozen allogenic Achilles, tibialis anterior or posterior, or peroneus longus or brevis tendons and had been implanted 3 to 96 months previously. By 12 months after surgery, the anterior cruciate ligament allografts consisted predominantly of small-diameter collagen fibrils (30 to 80 nm), which resulted in a unimodal pattern in the collagen fibril profile. The number of large-diameter fibrils (90 to 140 nm) within the allogenic tendon grafts had decreased. This predominance of small-diameter collagen fibrils persisted in almost all specimens older than 12 months. The anterior cruciate ligament allografts had collagen fibril profiles that did not resemble normal tendon grafts or normal anterior cruciate ligaments, even several years after surgery.

The long-term biomechanical and viscoelastic performance of repairing anterior cruciate ligament after hemitransection injury in a goat model.

To test the healing of the partially torn anterior cruciate ligament, we transected the posterolateral bundle in 11 adult female goats and tested the ligaments at 12, 24, and 52 weeks and 3 years after surgery. As early as 12 weeks after surgery translucent fibrous tissue covered the wound. The differences in anteroposterior laxity between right and left knees measured at 45 degrees and 90 degrees of flexion were not significantly different at each period. Results of Instron testing of the posterolateral bundle revealed the normalized changes in load-relaxation and Young's modulus were not significantly different at each period, but the ultimate tensile strength and stiffness at 3 years were significantly higher than at 12 weeks (P < 0.05) Failure started at the repair site for the 12-week group, but at 24 and 52 weeks the failure occurred throughout the ligament. At 3 years, the specimens failed with bony avulsion, indicating the repaired tissue was not the weakest link of the bone-ligament-bone complex. This study shows that under favorable conditions, partial anterior cruciate ligament injuries are capable of repair. What is more important, the high ultimate tensile strength and stiffness of the 3-year repaired tissue indicate full structural repair of such an artificial transection injury may be possible.

A prospective study of injuries in basketball: a total profile and comparison by gender and standard of competition.

The study aimed to determine prospectively a basketball injury profile, including severity of injury, and to compare the injury profile by gender and standard of competition. Trained observers viewed basketball games, noting the occurrence of injuries, and confirmed injuries by questioning all players on site after the game. Injured players completed a questionnaire and the progress of their injury was monitored by telephone interview. A total of 10,393 basketball participations were observed. An overall injury rate was documented of 18.3 per 1,000 participations (24.7 per 1,000 playing hours), and was comparable by gender and standard of competition. Serious injuries (missing one or more weeks of play) occurred at a rate of 2.89/1,000 participations; with the ankle joint the most common serious injury (1.25/1,000 participations), followed by the calf/anterior leg (0.48/1,000 participations) and knee joint (0.29/1,000 participations). The severity of the injury was significantly associated with the body region injured, with more serious injuries incurred to the lower limb than other body regions (p <.05). The severity of the injury incurred was not related to the standard of competition, gender, age, height, number of games played per week, amount of training undertaken, type of injury, or the mechanism of injury (p> .05).

Case Study: Rehabilitation of a Surgically Repaired Medial Collateral Knee Ligament Using a Limited Motion Cast and lsokinetic Exercise*.

Recent studies indicate that early mobilization of surgically repaired collateral ligaments leads to a more rapid gain in tensile strength compared to immobilized ligaments. Other advantages of early mobilization include prevention of muscle atrophy, joint stiffness, and articular cartilage fibrillation. Because of these observations, a professional Australian Rules footballer with a complete rupture of the right medial collateral ligament of his knee joint, was commenced, after surgical repair, on an early controlled mobilization program using a limited motion cast (LMC) and isokinetic exercise. Rehabilitation was commenced after 2 weeks of immobilization in a LMC, and progressed through four stages, all coupled with isokinetic exercise. Weeks 2-6, exercise in LMC through 20-60 degrees ; weeks 7-8, exercise out of LMC through 15- 1 OOO; weeks 8-9, exercise through a full range of movement, 0-130'; weeks 10- 12, exercise through a full range plus inner range 0-30' repetitions. The footballer returned to full competition approximately 12 weeks postsurgery with no medial collateral laxity and almost normal Cybex IP parameters. J Orthop Sports Phys Ther 1986;7(4):154-158.

Hamstring muscle injuries.

Probably the most common lower limb injury in amateur and professional athletics is strain of the hamstring muscles. Its frequent mismanagement stems from the inability of the athlete and his medical advisors to assess accurately the severity of the damage, coupled with the often premature return to competition which can result in a demoralising repeat muscle tear.

In vitro studies of elastin metabolism.

Neonatal pig and rat aortas were studied for their ability to synthesize elastin in an in vitro situation. Smooth muscle cells from the rat aorta produced excellent multilayered cultures and produced soluble elastin (tropoelastin), insoluble elastin, and small amounts of collagen. BAPN proved to be toxic to these cells, adversely affecting the level of extracellular protein production. Tissue minces from pig aorta continued to synthesize elastin for two hours after removal. However, a 24 hour study indicated that elastin synthesis had almost completely shut down and that collagen synthesis continued in an apparently normal fashion. It is concluded that in vitro elastin synthesis is an extremely sensitive process easily altered by culture conditions and the addition of extraneous substances such as BAPN, and also highly influenced by the past history of the smooth muscle cells involved.

Ankle injuries in basketball: injury rate and risk factors.

OBJECTIVES: To determine the rate of ankle injury and examine risk factors of ankle injuries in mainly recreational basketball players. METHODS: Injury observers sat courtside to determine the occurrence of ankle injuries in basketball. Ankle injured players and a group of non-injured basketball players completed a questionnaire. RESULTS: A total of 10 393 basketball participations were observed and 40 ankle injuries documented. A group of non-injured players formed the control group (n = 360). The rate of ankle injury was 3.85 per 1000 participations, with almost half (45.9%) missing one week or more of competition and the most common mechanism being landing (45%). Over half (56.8%) of the ankle injured basketball players did not seek professional treatment. Three risk factors for ankle injury were identified: (1) players with a history of ankle injury were almost five times more likely to sustain an ankle injury (odds ratio (OR) 4.94, 95% confidence interval (CI) 1.95 to 12.48); (2) players wearing shoes with air cells in the heel were 4.3 times more likely to injure an ankle than those wearing shoes without air cells (OR 4.34, 95% CI 1.51 to 12.40); (3) players who did not stretch before the game were 2.6 times more likely to injure an ankle than players who did (OR 2.62, 95% CI 1.01 to 6.34). There was also a trend toward ankle tape decreasing the risk of ankle injury in players with a history of ankle injury (p = 0.06). CONCLUSIONS: Ankle injuries occurred at a rate of 3.85 per 1000 participations. The three identified risk factors, and landing, should all be considered when preventive strategies for ankle injuries in basketball are being formulated.

In vitro response of chondrocytes to mechanical loading. The effect of short term mechanical tension.

Chick epiphyseal chondrocytes were grown in high density cultures for 14 days, after which the cell layers were placed in a cyclical stretching apparatus and subjected to a strain of 5.5% at a frequency of 0.2 Hz. There was a 1.4- and 1.7-fold increase in the incorporation of 35SO4 and 14C-glucosamine, respectively, into glycosaminoglycans in cultures subjected to mechanical loading for 24 h. No significant change was observed in the hydrodynamic size of the proteoglycans synthesized by chondrocytes subjected to mechanical loading. In this time period there was no increase in 3H-glycine incorporation into acid-insoluble protein, but there was a 2.4-fold increase of 3H-thymidine into DNA in cultures subjected to tensional strain. Concomitant with these changes, the cellular levels of cyclic AMP increased 2.2 times in the mechanically loaded cultures. This is discussed as a possible mechanism whereby chondrocytes respond to mechanical stimuli.

A comparison of the injuries sustained by female basketball and netball players.

A prospective and reliable method of injury surveillance was implemented to document a comprehensive injury profile in female basketball and netball. The study further aimed to compare the injury profiles of the two sports. Trained observers viewed basketball and netball games, noting the occurrence of injuries. Injuries were confirmed by questioning all players on site after the game. Injured players completed a questionnaire and the progress of their injury was monitored by telephone interview. A total of 16,162 player participations were observed; 6,972 for basketball and 9,190 for netball. Comparable injury rates were observed for female basketball and netball players; 18.22 and 17.30 injuries per 1,000 participations, respectively. The ankle, hand and knee were the body parts injured most frequently in both sports, whilst head and neck injuries were prevalent in basketball only. Netball players sustained severe injuries at a rate 3.3 times that of female basketball players. The major and severe injuries occurred at an average of one injury in 625 games in female basketball and one in 250 games in netball. The ankle, knee and calf/shin were the body parts most frequently involved in the more serious injuries.