The effects of radiofrequency energy probe speed and application force on chondrocyte viability.

OBJECTIVE: To determine the thermal effects of monopolar radiofrequency energy (mRFE) on bovine articular cartilage when it was moved at different speeds and using varying application forces. METHODS: Thirty-six fresh osteochondral sections divided into two groups (18 sections/group) were used in this study. The first group was tested at three speed rates of mRFE probe (1 mm/sec, 5 mm/sec and 10 mm/sec) at a constant force (50 g) applied to the probe tip. In the second group, three application forces of the probe tip were tested (25 g, 50 g and 75 g) at a constant speed (5 mm/sec) (n = 6/test). All tests were performed using a custom-built jig to control the mRFE (Vulcan EAS) probe during a 20-mm pass on each section. After treatment, viability of osteochondral sections was determined by confocal laser microscopy (CLM) combined with vital cell staining. RESULTS: There were not any significant differences in cartilage thickness of tested osteochondral sections among the different speeds or forces. During the mRFE probe treatments at different speeds, CLM demonstrated that probe application at the speed of 1 mm/sec caused significantly greater chondrocyte death than at the speeds of 5 and 10 mm/sec, whereas there were no significant differences in chondrocyte death among the variable application forces (p > 0.05). DISCUSSION: This in vitro study demonstrated that RFE thermal penetration correlated most closely with probe application speed than application force for this mRFE probe. CLINICAL RELEVANCE: Improper use of mRFE may cause thermal injury on articular cartilage.

Comparison of mechanical debridement and radiofrequency energy for chondroplasty in an in vivo equine model of partial thickness cartilage injury.

OBJECTIVE: The purpose of this study was to develop a long-term model of cartilage injury that could be used to compare the effects of radiofrequency energy (RFE) and mechanical debridement as a treatment. METHODS: Partial thickness fibrillation of patellar cartilage was created in 16 mature ponies. Three months after the initial surgery all injured patellae were randomly selected to receive one of the four treatments (n = 8/treatment): (1) control, (2) mechanical debridement with a motorized shaver, (3) TAC-CII RFE probe, and (4) CoVac 50 RFE probe. The ponies were euthanized 22 months after treatment. Macroscopic appearance of the cartilage surface was scored, vital cell staining was used to determine chondrocyte viability and light microscopy was used to grade the morphometric changes within the cartilage. Mechanical properties (aggregate modulus, Poisson's ratio and permeability) also were determined and compared to normal uninjured cartilage. RESULTS: There were no differences in the cartilage surface scores among the treatment groups and control samples (P > 0.05). The maximum depth of cell death and the percentage of dead area in control and mechanical debridement groups were significantly less than those in both RFE groups. There were no significant differences in maximum depth and the percentage of dead area between the two RFE treatment groups. Histologic scores demonstrated better cartilage morphology for the control and mechanical debridement groups than those of RFE groups. However, even with full thickness chondrocyte death, the matrix in the RFE treated sections was still retained and the mechanical properties of the treated cartilage did not differ from the mechanical debridement group. CONCLUSION: RFE caused greater chondrocyte death and more severe morphological changes compared to untreated degenerative cartilage and mechanical debridement in this model.

Comparison of three methods of gluteal muscle attachment to an allograft/endoprosthetic composite in a canine model.

This study used radiography, gait analysis, gluteal muscle mass, mechanical testing, and qualitative histology to compare three methods of gluteal muscle attachment to an allograft/endoprosthetic composite of the proximal 25% of the femur in an in vivo canine model. The three methods of gluteal muscle attachment were identical to those used clinically in human patients for hip revision and proximal femoral limb salvage: the host gluteal tendon sutured to the allograft tendon (tendon group), the host greater trochanter with intact gluteal tendons secured to the allograft with a cable-grip system (grip group), and periosteally vascularized proximal femoral bone onlay with intact tendons wrapped around the allograft (wrap group). On the basis of radiographs taken every 2 months, the tendon group had more graft fractures than did the grip or wrap group. Radiographic union of the graft-host bone junction occurred more rapidly and there was less graft resorption in the wrap group than in the other two groups. In all dogs, peak vertical ground-reaction forces in the treated limb decreased immediately after surgery and then slowly increased over the length of the study. The dogs in the wrap group regained normal weight-bearing on the treated limb more quickly than did those in the other groups. The constructs in the tendon group were weaker and less stiff immediately after surgery than were those in the other groups or in intact controls. Histologic analysis confirmed that the wrap technique resulted in complete union of the host bone-allograft junction more often than did the other techniques. The wrap method had the best functional outcome after 9 months when an allograft/endoprosthetic composite was used during total hip arthroplasty in this canine model.

Effect of intramedullary polymethylmethacrylate and autogenous cancellous bone on healing of frozen segmental allografts.

This study was designed to compare bone mineral density, periosteal callus production, new bone formation, bone porosity, histologic appearance, and union of mid-diaphyseal segmental allografts of the femur stabilized with an interlocking nail technique in a canine model 6 months after the procedure. An in vivo study was performed to compare the effects of augmenting interlocking nail fixation with an interlocking nail alone, intramedullary polymethylmethacrylate, intramedullary polymethylmethacrylate and autogenous cancellous bone applied to the periosteal surface of the host-allograft junction, autogenous cancellous bone applied to the endosteal surface of the allograft, autogenous cancellous bone applied to the periosteal surface of the host-allograft junction, and autogenous cancellous bone applied to the periosteal surface of the host-allograft junction and to the endosteal surface of the allograft. There were no differences among treatments for bone mineral density at any time interval. Callus area 4 weeks after the procedure was greater along the lateral and cranial surfaces for treatments with periosteal cancellous bone (p < 0.05). New bone within the allograft segment did not differ among treatments and was reduced compared with the host-allograft junctions (p < 0.05). The amount and quality of bone tissue at the host-allograft junctions were greatest with treatments of intramedullary polymethylmethacrylate and autogenous cancellous bone applied to the periosteal surface of the host-allograft junction and of autogenous cancellous bone applied to the periosteal surface of the host-allograft junction and to the endosteal surface of the allograft (p < 0.05). The rate of bone union was lower, and there was a greater gap (non-bone tissue) remaining between host and allograft bone with treatment involving just intramedullary polymethylmethacrylate than with other treatments (p < 0.05). The results suggest that augmenting interlocking nail fixation with intramedullary polymethylmethacrylate by itself offers no advantage but that a combination of intramedullary polymethylmethacrylate and cancellous bone at the periosteal surface or of cancellous bone within the medullary canal and at the periosteal surface improves the quality of healing at 6 months.

Effect of nonablative laser energy on the joint capsule: an in vivo rabbit study using a holmium:YAG laser.

BACKGROUND AND OBJECTIVE: The nonablative application of holmium:yttrium-aluminum-garnet (Ho:YAG) laser energy to the joint capsule of patients with glenohumeral instability has been found to shrink capsular tissue and to help stabilize the joint. The purpose of this study was to evaluate the effect of nonablative laser energy on the short-term histological properties of joint capsular tissue in an in vivo rabbit model. STUDY DESIGN/MATERIALS AND METHODS: Eighteen mature New Zealand white rabbits were used in this study. One randomly selected stifle was treated with laser energy, and the contralateral stifle was sham-operated. Animals were euthanized immediately after surgery (day 0), at 7 days postsurgery and 30 days postsurgery. Specimens were processed for histology and transmission electron microscopy. RESULTS: Laser-treated samples at day 0 showed diffuse hyalinization of collagen with nuclear karyorrhexis of fibroblasts. Laser-treated tissue at 7 days postsurgery revealed fibroblast proliferation around and into acellular hyalinized regions of collagen. At 30 days postlaser treatment, areas of fused collagen were greatly reduced as large reactive fibroblasts migrated and secreted matrix. CONCLUSION: This study illustrates the short-term in vivo tissue response to nonablative laser treatment, where acellular hyalinized regions of collagen are infiltrated by fibroblasts that have used the treated collagen as the framework for migration and secretion of new collagen matrix in order for tissue repair to proceed.

The effect of thermal heating on the length and histologic properties of the glenohumeral joint capsule.

The purpose of this study was to evaluate the effect of temperature on shrinkage and the histologic properties of glenohumeral joint capsular tissue. Six fresh-frozen cadaveric shoulders were used for this study. Seven joint capsule specimens were taken from different regions from each glenohumeral joint and assigned to one of seven treatment groups (37 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees, 80 degrees C) using a randomized block design. Specimens were placed in a tissue bath heated to one of the designated temperatures for 10 minutes. Specimens treated with temperatures at or above 65 degrees C experienced significant shrinkage compared with those treated with a 37 degrees C bath. The posttreatment lengths in the 70 degrees, 75 degrees, and 80 degrees C groups were significantly less than the pretreatment lengths. Histologic analysis revealed significant thermal alteration characterized by hyalinization of collagen in the 65 degrees, 70 degrees, 75 degrees, and 80 degrees C groups. This study demonstrated that temperatures at or above 65 degrees C caused significant shrinkage of glenohumeral joint capsular tissue. These results are consistent with histologic findings, which revealed significant thermal changes of collagen in the 65 degrees, 70 degrees, 75 degrees, and 80 degrees C groups. To verify the validity of laser application for shrinkage of joint capsule, studies designed to compare these findings with the effects of laser energy must be performed.

The effect of nonablative laser energy on joint capsular properties. An in vitro histologic and biochemical study using a rabbit model.

The purpose of this study was to evaluate the effect of laser energy at nonablative levels on joint capsular histologic and biochemical properties in an in vitro rabbit model. The medial and lateral portions of the femoropatellar joint capsule from both stifles of 12 mature New Zealand White rabbits were used. Specimens were divided into three treatment groups (5 watts, 10 watts, and 15 watt) and one control group using a randomized block design. Specimens were placed in a 37 degrees bath of lactated Ringer's solution and laser energy was applied using a holmium:yttrium-aluminum-garnet laser in four transverse passes across the tissue at a velocity of 2 mm/sec with the handpiece set 1.5 mm from the synovial surface. Histologic analysis revealed thermal alteration of collagen (fusion) and fibroblasts (pyknosis) at all energy densities, with higher laser energy causing significantly greater morphologic changes over a larger area (P < 0.05). Application of laser energy did not significantly alter the biochemical parameters evaluated, including type I collagen content and nonreducible crosslinks (P > 0.05). This study demonstrated that nonablative laser energy caused significant thermal damage to the joint capsular tissue in an energy-dependent fashion, but type I collagen content and nonreducible crosslinks (P > 0.05). This study demonstrated that nonablative laser energy caused significant thermal damage to the joint capsular tissue in an energy-dependent fashion, but type I Collagen content and nonreducible corsslinks were not significantly altered.

The effect of nonablative laser energy on the ultrastructure of joint capsular collagen.

This study was designed to evaluate the effect of laser energy at nonablative levels on the ultrastructure of joint capsular collagen. The femoropatellar joint capsules of six mature New Zealand white rabbits were harvested immediately after death. Specimens were divided into three treatment groups (5, 10, and 15 watts) and one control group. Laser energy was applied using a holmium: YAG laser. Transmission electron microscopy showed significant ultrastructural alterations in collagenous architecture for all laser treatment groups, with increased fibril cross-sectional diameter for each of the treated groups. The fibrils began to lose their distinct edges and their periodical cross-striations at subsequently higher energy densities. A morphometric analysis showed that each subsequently higher laser energy caused a significant increase in collagen fibril diameter. Ultrastructural alteration of collagen fibril architecture caused by the thermal effect of laser energy is probably the dominant mechanism of laser-induced tissue shrinkage.

Treatment of inflammatory arthritis by synovial ablation: a comparison of the holmium: YAG laser, electrocautery, and mechanical ablation in a rabbit model.

BACKGROUND AND OBJECTIVE: Although the majority of patients with immune-mediated inflammatory arthritis are managed conservatively, some patients may require synovectomy if they have persistent pain secondary to chronic unresponsive swelling of a joint. In this study, three methods of synovial ablation using laser energy, electrocautery, and mechanical debridement were compared in an in vivo chronic synovitis rabbit model. STUDY DESIGN/MATERIALS, AND METHODS: In the first phase of this study, the optimal laser energy/pulse frequency combination for synovial ablation in this model was determined. In the study's second phase, 48 mature rabbits were then divided into four equal groups: laser synovectomy, electrocautery synovectomy, mechanical synovectomy, and control. Chronic synovitis was induced in both stifles of all treatment groups and in the right stifle of the control rabbits. Synovectomy was performed on one stifle of each rabbit; the contralateral stifle served as a sham-operated control. Six rabbits per group were euthanized 2 weeks and 3 months after surgery, respectively. RESULTS: There were no differences among groups in synovial fluid parameters, except at 2 weeks, when the electrocautery group had significantly more white blood cells than the laser and mechanical debridement groups. Histologic examination revealed that mechanical debridement resulted in significantly more synovial hemorrhage, capillary dilatation, plasma cell infiltration, lymphocyte infiltration, joint capsular defects, and poorer synovial ablation than ablation achieved with either laser energy or electrocautery. CONCLUSION: Laser energy and electrocautery achieved similar results when used for ablative purposes, although electrocautery did not achieve the same degree of synovial ablation as laser energy.

Atrophic nonunion can be predicted with dual energy x-ray absorptiometry in a canine ostectomy model.

This study demonstrated that dual energy x-ray absorptiometry can be used to distinguish between normal union and atrophic nonunion, with high sensitivity and high negative predictive value, by 8 weeks after surgery in a canine model. Eighteen adult mixed-breed dogs were divided into two equal groups: normal union and atrophic nonunion. In the normal union group, a 5 mm mid-diaphyseal transverse ostectomy was performed in the right tibia, and the bone was stabilized with a unilateral external fixator. In the atrophic nonunion group, a 5 mm mid-diaphyseal ostectomy was performed; the distal 1.5 cm of the bone ends, including the periosteum, were frozen twice to -20 degrees C using liquid nitrogen and thawed slowly twice; and the bone was stabilized with a unilateral external fixator. The members of the research team were blinded to the group assignments until after all dogs were killed and all data were acquired. Radiography and dual energy x-ray absorptiometry of both tibiae were performed at weeks 1, 2, 3, 4, 6, 8, 10, 12, 14, and 16 after surgery. All dogs were killed at 16 weeks, and the torsional stiffness and maximum torque of both tibiae of five dogs in each group were determined. Mechanically, the tibiae in the normal union group had significantly higher maximum torque (43-fold higher) and torsional stiffness (86-fold higher) than the tibiae in the atrophic nonunion group. The sensitivity and negative predictive value of dual energy x-ray absorptiometry for predicting nonunion were 100% by 8 weeks after surgery. The specificity and positive predictive value reached 78 and 82%, respectively, by 16 weeks. Radiographic scores were significantly higher for the union group than for the nonunion group beginning at 2 weeks after surgery. The earliest time after surgery that radiography classified an ostectomy as a nonunion was significantly later (4.6 +/- 1.2 weeks) than for dual energy x-ray absorptiometry (2.6 +/- 1.4 weeks). Before these results can be extrapolated to human applications, further animal studies must be performed to evaluate clinically or experimentally induced fractures, or both, rather than the well defined ostectomies that were performed in this study.

The effect of nonablative laser energy on joint capsular properties. An in vitro mechanical study using a rabbit model.

To evaluate the effect of laser energy at nonablative levels on the mechanical properties of joint capsular tissues, we tested the femoropatellar joint capsules of 12 mature New Zealand White rabbits. Specimens were divided into three treatment groups (5, 10, and 15 watts) and one control group. All specimens were first nondestructively mechanically tested to determine stiffness and viscoelastic properties and then treated with laser energy or served as a control. Shrinkage was recorded and mechanical testing was repeated. The application of laser energy resulted in 9%, 26%, and 38% reduction in capsular tissue length for the 5, 10, and 15 watt groups, respectively. Tissue shrinkage was significantly and strongly correlated with energy density. Laser energy caused a significant decrease in tensile stiffness only in the 10 and 15 watt groups. Laser energy did not change the relaxation properties at any energy density. This study demonstrates that significant capsular shrinkage can be achieved with the application of nonablative laser energy without detrimental effects to the viscoelastic properties of the tissue; although at higher energy densities, laser energy did lessen capsular stiffness properties. The results of this study should be interpreted with caution until in vivo studies are performed.

Comparison of allograft/endoprosthetic composites with a step-cut or transverse osteotomy configuration.

This study was designed to compare the biomechanical and functional characteristics of allograft/endoprosthetic composites of the proximal 25% of the femur repaired with either a transverse or a step-cut osteotomy, using a canine model (10 dogs, five with each type of osteotomy). Serial radiography and weight-bearing studies were performed monthly, and mechanical testing was done 6 months after surgery. The femora were tested in torsion and compared with the contralateral control (insertion of a femoral component but no osteotomy). At 6 months, the composites with a step-cut osteotomy had 36% greater structural stiffness than the composites with a transverse osteotomy (p < 0.005) and 121% greater maximum torque at failure than the controls (p < 0.005), without greater structural stiffness. Evaluation of peak vertical ground reaction forces revealed significantly greater weight-bearing on the experimental limb in dogs with a transverse osteotomy. The results of this relatively short-term study were mixed. Despite the increased structural stiffness of the allograft/endoprosthetic composite with a step-cut osteotomy, the dogs with this type of reconstruction had decreased weight-bearing throughout the course of the study. The step-cut osteotomy may augment the stability of the allograft/endoprosthetic composite, allowing faster healing (as demonstrated by the results of mechanical testing), but in some way, not understood, may cause pain in the reconstructed limb. Longer term studies are needed to answer these questions and to determine whether alteration of the traditional transverse osteotomy has any advantage.

Comparison of healing of allograft/endoprosthetic composites with three types of gluteus medius attachment.

This study compared three methods of gluteus medius tendon attachment to an allograft/endoprosthetic composite of the proximal 25% of the femur in a canine model. The three methods were bone to bone, tendon to bone, and tendon to tendon attachment. In an in vivo study, 24 dogs were assigned to three groups of eight dogs each, and serial radiography and weight-bearing analyses were performed throughout the study. The dogs were killed at 6 months, and the specimens were tested in tension to failure and were analyzed histologically. In an in vitro study, each repair was done on six limbs, with a contralateral limb serving as a control for each. In these specimens, the bone to bone attachments were significantly stronger (99.1% of the controls) than the tendon to bone attachments (71.8% of the controls) and the tendon to tendon attachments (40.0% of the controls); there were no differences in tensile stiffness among the three types of attachment. By 6 months, the tensile strength of the tendon to tendon attachments increased significantly and that of the tendon to bone attachments decreased significantly. There were no significant differences in tensile strength among the three types of attachment. The tensile stiffness of the bone to bone attachments (91.0% of the controls) was significantly greater than that of the tendon to bone attachments (40.8% of the controls) but not significantly different from that of the tendon to tendon attachments (63.2% of the controls). The bone to bone attachment was associated with increased bone resorption, bone remodeling, and bone porosity, accompanied by thinner allograft cortices, when compared with the other types of attachment. In dogs with a bone to bone attachment, weight-bearing increased more slowly than in dogs with either of the other two attachments. These changes associated with the bone to bone attachment may merely be secondary to healing of the bone to bone attachment to the greater trochanter; therefore, they may only be temporary phenomena or they may be the portents for long-term complications. Longer term studies of at least 1-2 years must be performed before these questions can be answered.

Dual-energy x-ray absorptiometry and force-plate analysis of gait in dogs with healed femora after leg-lengthening plate fixation.

Dual-energy x-ray absorptiometry was used to measure bone mineral density of four regions in healed femora of nine dogs after fracture fixation with a leg-lengthening plate. Six to 85 months (mean, 46 months) after surgery, the bone mineral density of healed femora was not significantly different from the contralateral uninjured femora (P > .05; power = 0.8 at delta = 15%). Radiolucencies around the proximal screws, apparently associated with screw loosening, were seen on radiographic views of the healed femora of three dogs. In one of these dogs, one screw in the proximal metaphysis had broken. Force-plate analysis of gait was also performed on dogs at the time of bone mineral density measurement. Peak vertical force was decreased in the pelvic limb with the healed fracture compared with the contralateral unoperated limb (P < 0.05). Clinically apparent lameness in three dogs did not appear to be associated with altered bone mineral density and may have been caused by hip osteoarthritis, a nondisplaced hairline diaphyseal fracture, and screw loosening in conjunction with extensive post-traumatic soft tissue injury.

Dual-energy X-ray absorptiometry of canine femurs with and without fracture fixation devices.

The effect of 5 fracture fixation methods on bone mineral density (BMD) measurement of femurs, using dual-energy X-ray absorptiometry (DXA) was determined in a canine model. Six regions of interest were measured, including the entire femur, the diaphysis of the femur, and small regions centered over the middiaphysis of the bone (lateral middiaphyseal cortex, medial middiaphyseal cortex, middiaphyseal medullary canal, and total middiaphysis). Eight unpaired femurs were collected and scanned by use of DXA before (5 separate scans/femur) and after (5 separate scans/femur) fixation by use of 1 of 5 fixation methods. These fixation methods included: intramedullary (IM) nail; IM nail and cerclage wires; IM nail and external skeletal fixation; locked IM nail; and a dynamic compression plate (DCP). All implants were made of stainless steel. The IM nail fixation devices caused significant decreases in the DXA measurement of BMD in the small regions of interest, compared with femurs without fixation devices (mean decrease, 37.3%; P < 0.05). The locked nail caused similar, but larger, decreases in the DXA measurement of BMD, compared with the IM nail fixation methods (P < 0.05). Plate fixation caused a small, but significant (P < 0.05), decrease (2.8%) in the DXA measurement of BMD in the large regions of interest, but when all regions were averaged, it did not cause significant change in this measurement, compared with femurs without fixation devices.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of increasing gap width on localized densitometric changes within tibial ostectomies in a canine model.

Dual-energy X-ray absorptiometry (DXA) was used to quantitate the localized densitometric changes that occur early (0-16 weeks) in a tibial ostectomy model of three different gap widths in 15 dogs. Dogs were divided into three equal groups. A 5-mm (group 1), 15-mm (group 2), or 25-mm (group 3) unilateral tibial ostectomy was performed and stabilized with a unilateral external skeletal fixator in each dog. DXA of the gap tissue was performed at 0, 14, 30, 60, 90, and 120 days after surgery. Regions of interest (ROIs) included the entire gap (groups 1, 2, 3) and ROIs within the gap a defined distance from the proximal or distal cortical bone ends: 0-2.5 mm (groups 1, 2, 3); 2.5-5.0 mm (groups 2, 3), 5.0-7.5 mm (groups 2, 3), 7.5-10.0 mm (group 3), and 10.0-12.5 mm (group 3). Bone mineral density (BMD) significantly changed over time in all three groups (P < 0.0001). The BMD of the 5-mm gap increased over the 4-month study period and reached normal middiaphysial tibial BMD by 90 days after surgery. The BMD of the 15-mm gap also increased after surgery but reached a plateau at a BMD of approximately 0.45 g/cm2 (48% of middiaphyseal BMD) at 60 days after surgery. The BMD of the 25-mm gap increased to a small extent during the first 30 days after surgery and then gradually decreased during the study period. Overall, the 5-mm gap had the highest BMD, followed by the 15-mm gap and the 25-mm gap (P < 0.0001).

Dual energy x-ray absorptiometry of implanted femora after cemented and press-fit total hip arthroplasty in a canine model.

The effect of a press-fit and cemented titanium alloy endoprosthesis on the measurement of bone mineral density (BMD) of the proximal femur in a canine model with dual energy x-ray absorptiometry (DXA) was determined. Seven regions of interest, corresponding to zones 1-7 according to Gruen et al., were measured. Eight unpaired femora were scanned with DXA before implantation (five separate scans per femur), after press-fitting with a titanium alloy femoral component (five separate scans per femur); and after cementing with the same component (five separate scans per femur). When the titanium alloy endoprosthesis was press-fit, the BMD of five of seven regions of the proximal femur increased significantly (range, 2.7-23.1%; mean, 11.2%), although the mean precision error of this measurement was not altered (before implantation, 1.1%; after press-fit 1.8%). Cementing of the implant caused a variable effect on BMD, resulting in a decrease in distal regions where cortical bone was relatively thin compared with the cement mantle (regions 3, 4, and 5) and in an increase in the other regions. Cementing of the implant significantly increased the mean precision error of measurement to 7.2% (range, 1.1-12.6%).

Recovery of soluble dietary fiber is dependent on the method of analysis.

The effects of different methods on the distribution of total neutral sugars (TNS), uronic acids (UA), and beta-glucans (beta G) between the soluble (S) and insoluble (I) fractions of dietary fiber (DF) were determined for peas, kidney beans, oat bran, rice, and macaroni. Incorporation of a protease step into the Theander method "A" modestly increased, and addition of a pepsin digestion further increased the proportion of total fiber recovered in the S fraction. The effect of extraction method on the distribution of TNS, UA, and beta G between the S and I fractions varied with the food. The three methods measured the same total DF in a food and 1-3% starch in the I fraction of peas and kidney beans. Use of dimethyl sulfoxide to solubilize starch, or elevated temperature to extract S components, had no effect on the distribution of DF between S and I fractions of peas and macaroni. Incomplete protein hydrolysis did not always lower Klason lignin and excluding lignin from the fiber complex did not always substantially increase the S fraction.