John M. Clark

University of Washington Seattle, Seattle, Washington, United States

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Publications (15)42.84 Total impact

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    ABSTRACT: Regeneration of mammalian digit tips is well described; however, associated cellular or molecular events have not been studied in humans. We describe an in vitro human fetal model of response to digit tip amputation, and report expression of the transcription repressor Msx1 in the developing and regrowing human digit tip. Human fetal digits from specimens ranging from 53 to 117 days' estimated gestational age (EGA) were cultured in a defined serum-free medium with supplemented oxygen for time periods from 4 days to 4 weeks. Histology and immunohistochemistry were performed on paired control and tip-amputated digits. Regrowing tissue covered the cut end of the distal phalanx in digits up to 80 days' EGA. Msx1 expression was detected beneath the nail field in control digits to at least 70 days' EGA and at the regrowing tip of 57-day digits at 4 and 7 days post-amputation. Our results show that human fetal digits regrow tissue in vitro in response to tip amputation. This process appears spatially associated with Msx1 expression. Msx1 expression appears increased at the regrowing tip of 57-day digits by 4 days after amputation.
    Wound Repair and Regeneration 07/2006; 14(4):398-404. DOI:10.1111/j.1743-6109.2006.00139.x · 2.77 Impact Factor
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    ABSTRACT: Type I collagen fibrils in tendons and ligaments assume a sinusoidal wave shape, or crimp, which straightens only with tensile load. The load response of crimp has been studied primarily in isolated subunits and not in complex, intact structures. The purpose of our study was to determine if freeze substitution fixation of an entire ligament could preserve changes in crimp morphology induced by functionally relevant loading conditions. We hypothesized that, in ligaments prepared by freeze-substitution fixation under load, crimp would progressively extinguish with increasing loads, and nonuniform strain following partial section could be detected from crimp morphology. Tensile loads ranging from 0 to 220 N were applied to patellar ligaments of 16 fresh rabbit stifle joints using simulated isometric quadriceps pull through the patella. The loaded joints were flash frozen with isopentane cooled in liquid nitrogen, then fixed using freeze substitution. Another six ligaments were loaded to 150 N following incision of the anterior third and evaluated under polarized light microscopy for crimp distribution. Ligaments with no or low loads could be identified by the presence of crimp on mid-sagittal sections. Strain distribution was inhomogeneous, in that the ligament displayed a consistent pattern of collagen fiber recruitment among three morphologically distinct bands seen on coronal sections. At very low loads (about 18 N), the fibers in a central band were uncrimped; anterior and deep bands uncrimped at higher loads. The crimp in the entire specimen was extinguished at about 67 N, which correlates closely with the previously reported toe-region of the stress-strain curve of the rabbit patellar ligament. When the anterior third was transected, fibers within that segment retained a crimp in ligaments prepared under loads that ordinarily would ablate all crimp. These findings suggest that freeze fixation could be used to map the functional microstructure of ligaments or tendons.
    Journal of Orthopaedic Research 04/2006; 24(4):793-9. DOI:10.1002/jor.20081 · 2.97 Impact Factor
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    ABSTRACT: This study characterizes the healing response of the glenoid after spherical reaming and prosthetic humeral head replacement in a canine model of glenohumeral hemiarthroplasty. The right glenoid of twelve skeletally mature female dogs was reamed to a uniform radius of curvature, removing all cartilage down to bleeding subchondral bone. The glenoid was not resurfaced. The humeral head was replaced with a stemmed metal prosthesis. Post-surgery, the operated limbs were immobilized for seven days, with motion allowed ad libitum thereafter. Fluorescent bone labels were administered to identify bone formation. These procedures were not complicated by instability, infection or death. Six animals were euthanized at 10 week and six more at 24 week. The intact glenohumeral joints were evaluated by gross examination, assessment of glenoid concavity, and light microscopy of methylmethacrylate sections. At 10 week, vascular fibrous tissue partially covered the glenoid, maintaining a concave surface congruent with the prosthetic humeral head. New bone formed at the margin of the glenoid, and the density of the periarticular trabecular bone increased. At 24 week, the healing was more advanced; thick fibrocartilaginous tissue covered the entire glenoid surface. These results demonstrate that spherical glenoid reaming produced a consistent healing response characterized by remodelling of the reamed bony concavity to a congruent, living, smooth, securely attached interface articulating with the humeral prosthesis.
    Journal of Orthopaedic Research 02/2005; 23(1):18-26. DOI:10.1016/j.orthres.2004.06.019 · 2.97 Impact Factor
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    ABSTRACT: We tested the hypothesis that articular cartilage adjacent to experimental osteochondral defects is not subject to unusual strains under load. A 2.5-mm drill hole was made in the medial femoral condyle of 15 knees from 10 adult rabbits. Experimental joints were loaded with simulated quadriceps force, then frozen under load and preserved by freeze-substitution fixation. Deformation in the region of the defect was evaluated by scanning electron and light microscopy and compared with nondrilled and nonloaded control knees. To simulate blood clot, alginate was placed into some defects before loading. In loaded knees, articular cartilage at the edge of the drill hole was abnormally flattened and folded into the defect. Opposing tibial cartilage or meniscus intruded into the femoral defect beyond the cement line. Alginate did not prevent incursion of opposing cartilage. In this standard drill-hole model, the articular cartilage defect is occupied by the opposing surface when a joint is loaded. Any tissue growing or surgically implanted in the defect is subject to loading and displacement, therefore complicating attempts to characterize the healing or regenerative potential in similar drill-hole models. Deformation of cartilage at the defect edge suggests load concentration or increased compliance. Either phenomenon would contribute to subsequent degeneration of the cartilage adjacent to defects.
    Clinical Orthopaedics and Related Research 02/2005; DOI:10.1097/01.blo.0000145990.58146.3d · 2.88 Impact Factor
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    ABSTRACT: Exact reconstruction of an osteochondral defect by autogenous transplantation (mosaicplasty) is difficult given the variation in joint surface contour. Clinical and experimental studies do not show the extent to which incongruity can be tolerated in autografting. Grafted articular cartilage will hypertrophy to correct the incongruity created by recession of the transplanted surface. Controlled laboratory study. To test the response of grafts to incongruities, osteochondral autografts were transplanted from the trochlea to the femoral condyle in adult male sheep stifle joints. In groups of 6 animals, graft surfaces were placed flush, countersunk 1 mm or countersunk 2 mm, then histologically analyzed 6 weeks after surgery. Cartilage thickness, condition of the articular surfaces, and preservation of hyaline characteristics were the primary features compared. Bony union, vascularization, and new bone formation were present in all grafts. Cartilage-to-cartilage healing did not occur. In flush specimens, cartilage changed minimally in thickness and histologic architecture. The specimens countersunk 1 mm demonstrated significant cartilage thickening (54.7% increase, P <.05). Chondrocyte hyperplasia, tidemark advancement, and vascular invasion occurred at the chondroosseous junction, and the surface remained smooth. Cartilage necrosis and fibrous overgrowth were observed in all grafts countersunk 2 mm. Minimally countersunk autografts possess a capacity for remodeling that can correct initial incongruities while preserving hyaline characteristics. Grafts placed deeper do not restore the contour or composition of the original articular surface. If preservation of normal hyaline cartilage is the objective, thin grafted articular cartilage can remodel, but the tolerance for incongruity is limited and probably less than that reported for an intra-articular fracture.
    The American Journal of Sports Medicine 01/2005; 32(8):1842-8. · 4.70 Impact Factor
  • The Journal of Bone and Joint Surgery 12/2003; 85-A(11):2249-51; author reply 2251. · 4.31 Impact Factor
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    John M. Clark, Anthony Norman, Hubert Nötzli
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    ABSTRACT: Changes in the 3-dimensional arrangement of the articular cartilage matrix during growth of the rabbit tibial plateau were studied. Knees from newborn, and 1, 2 and 6 wk-old rabbits were compared with those of adults by light and electron microscopy. The specimens were fixed, embedded en bloc in epoxy resin and sectioned vertically/coronally through the point where the articular cartilage was thickest in the adult medial tibial plateau. At birth, the proximal tibial epiphysis was cartilaginous, but nascent articular cartilage was recognisable as a densely cellular layer covering the tibial condyle. Within 30 microns of the articular surface, the chondrocytes were flattened and collagen fibres ran among these cells in a direction parallel to the surface. Deeper in the articular cartilage, rounded cells were evenly distributed within a random collagen fibril network. At the centre of the plateau, the tangential layer changed little during growth, whereas the subjacent cellular layer grew in thickness and steadily achieved a more vertical character in the organisation of its constituent collagen and cellular elements. At 1 wk, cells were separated into clusters by acellular regions filled with collagen fibrils. At 2 wk, cells within the forming radial zone were aligned in columns bracketed by vertical collagen fibres. Continuity of these vertical fibres with those in the tangential surface layer was evident at this age. The chondrocytes were surrounded by fibrous capsules typical of chondrons. By 6 wk, the bases of the radial collagen fibres in the very centre of the condyle had calcified, as had the adjacent hypertrophic hyaline cartilage. A solid subchondral plate and tidemark did not appear until skeletal maturity. From birth to age 6 wk, maximum thickness of the layer identified as primordial articular cartilage increased from 0.13 mm to 0.70 mm, and was 1.5 mm in the adult. Throughout growth, however, the thickness of the tangential layer in the centre of the plateau never exceeded 0.05 micron. In the patella, femoral head and peripheral tibial plateau, cartilage development followed the same general sequence. In contrast to the central tibial plateau, the tangential layer also grew in thickness, but at a slower rate than that of the radial zone. At all ages, the developing articular cartilage was structurally distinct from the deeper hyaline cartilage which contributed to growth of the ossification centre through enchondral ossification. The collagen matrix of articular cartilage acquires a characteristic, orderly 3-dimensional structure soon after birth. Growth in cartilage thickness occurs primarily through enlargement of the radial zone.
    Journal of Anatomy 09/1997; 191 ( Pt 2)(2):215-21. DOI:10.1046/j.1469-7580.1997.19120215.x · 2.23 Impact Factor
  • John M. Clark, Peter T. Simonian
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    ABSTRACT: Specimens of articular cartilage from human knees with gross evidence of malacia (dull appearance and/or softness) or fibrillation (exposed fibrous strands and/or staining with India ink) were prepared for scanning electron microscopy (SEM) and compared to cartilage from apparently intact regions. Vertical cryofractures were made through the center of each specimen, so the matrix collagen structure and its relationship to surface features could be examined. Soft, dull, malacic cartilage was characterized by the presence of numerous clefts among the collagen fibers within the most superficial region of the cartilage. In one form of this condition, these clefts did not extend through the articular surface. In a second form, usually observed where the tangential zone was normally thin or absent, the free ends of radial collagen fibers were exposed, but the deeper layers were intact. Two forms of fibrillation were also identified. The first is created by separation of the superficial lamellae which curl up from the tangential layer and form frondlike projections above the normal plane of the joint surface. In the second, deep radial fibers are exposed by vertical fissures. This second form is associated with advanced damage to the joint. The early stages of cartilage failure are characterized by debonding among the major collagen fiber tracts. This process may initiate in the deep tangential zone where the radial fibers cross into the surface. The patterns of the degenerative changes are dictated by the original architecture of the collagen matrix. The microscopic findings do not correlate adequately with conventional gross grading. SEM provides useful information about injured articular cartilage.
    Microscopy Research and Technique 05/1997; 37(4):299-313. DOI:10.1002/(SICI)1097-0029(19970515)37:4<299::AID-JEMT5>3.0.CO;2-G · 1.17 Impact Factor
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    ABSTRACT: Exercise-associated amenorrhea is the cessation of menses in a woman following onset of training or an increase in training intensity. Its physiologic basis is characterized by consistently low levels of gonadotropin and ovarian hormones, but the underlying cause of this phenomenon is unknown. Although osteopenia has been described in amenorrheic women athletes, it has been primarily a laboratory diagnosis. Several recent studies have described a significantly lower bone mineral density (BMD) in the lumbar spine of amenorrheic athletes. Marcus et al. also reported an increased number of metatarsal and tibial stress fractures in a group of amenorrheic women. We report here the first case of a nontraumatic femur fracture in an amenorrheic athlete. A 32-yr-old white female, with four prior fibular stress fractures, suffered a left femoral shaft fracture during the 13th mile of a half-marathon. The fracture was successfully internally fixed. Biochemical studies showed no metabolic abnormality. Bone mineral density of the lumbar spine, femoral neck, tibia, and fibula were below the mean for both eumenorrheic and amenorrheic female athletes. Exercise-associated amenorrhea is a medical problem that may have serious implications for both competitive and high-intensity recreational female athletes.
    Medicine &amp Science in Sports &amp Exercise 01/1992; 23(12):1323-5. DOI:10.1249/00005768-199112000-00001 · 4.46 Impact Factor
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    ABSTRACT: In a previous experiment, sodium fluoride in a biodegradable polymer matrix was introduced into the femoral canal of the rabbit and bone formation was compared with contralateral controls. We noted significant bone formation, but only in the distal third of the periosteal surface of the femur. This experiment was performed to distinguish fluoride-induced periosteal bone formation from that due to the reactive osteogenic changes associated with local injury caused by the process of implantation. A proximal approach on the right leg and a distal approach on the left were used for the insertion of the implants in rabbits. Femurs were removed after 30 days and tested for stiffness and load to failure. The cross-sectional area of mineralized bone was determined at proximal, midshaft, and distal locations. Fluorescent bone tissue growth labels were injected at weekly intervals to measure the rate of new periosteal bone formation. The results were compared with a control group that received sham implants. Results showed no difference between measured properties in right and left femurs in the control group or in those exposed to fluoride. A significant increase was found in the fluoride group in load to failure, along with cross-sectional area of mineralized bone, and periosteal growth rates compared with the control group, but no difference was seen in stiffness. No difference was detected between the response proximally and distally in the fluoride group regardless of the location of insertion. There were no detectable changes in serum fluoride level after implantation of the poly L-lactic acid/sodium fluoride matrix. These results show that fluoride exerts its osteogenic effects equally at proximal, midshaft, and distal regions of diaphyseal bone and is uninfluenced by the site of local injury due to insertion of the implant.
    Journal of Orthopaedic Research 11/1991; 9(6):890-901. DOI:10.1002/jor.1100090616 · 2.97 Impact Factor
  • John M. Clark
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    ABSTRACT: To determine if articular cartilage collagen fiber organization differs with location on the tibial plateau, specimens from dogs, humans, and rabbits were studied by scanning electron microscopy. Joint surfaces were fixed, dehydrated, and fractured radially so that the periphery could be compared with the center on single specimens. Generally, fibers were more tightly packed in the lateral side than in the medial and the periphery as compared with the center, where the cartilage was consistently thicker and the radial zone was dominant and composed of straight vertical fibers. In the periphery, the tangential and transitional zones were better developed and contributed up to 50% of the cartilage depth in comparison to only 5% centrally. The soft, dull, malacic appearance of the center results from lack of a true surface layer of tangential collagen fibers.
    Journal of Orthopaedic Research 03/1991; 9(2):246-57. DOI:10.1002/jor.1100090213 · 2.97 Impact Factor
  • John M. Clark, Eric Rudd
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    ABSTRACT: To study the distribution of cells in the surface layer of articular cartilage, rabbit hip and knee specimens were stained with silver and studied by scanning electron microscopy (SEM). The cartilage was treated en bloc using the Gomori methenamine silver technique, which stains the nuclei of exposed cells with reduced silver. The intact surface was then studied with a binocular microscope and SEM in the backscatter mode Only those cells within 30 microns of the surface stained, permitting that population to be imaged selectively. Depressions in the surface were related to groups of cells in clusters or rows bounded by collagen fibers. This study demonstrates the effectiveness of backscatter imaging in the study of chondrocytes. The relationship between surface contours and underlying cells is more complex than previously described.
    Journal of Orthopaedic Research 03/1991; 9(2):275-83. DOI:10.1002/jor.1100090216 · 2.97 Impact Factor
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    ABSTRACT: This paper presents a theoretical analysis of the equilibrium mechanics of bending and twisting fiber geometries in ligaments and tendons. The theory predicts that the bending of loaded fibers is necessarily accompanied by large transverse pressures and pressure gradients. The predicted pressures are especially large at the bone tunnel entries of ligament grafts, where they can equal or exceed the applied tensile loads. Experimental measurements of internal pressures confirm these predictions.
    Journal of Biomechanics 02/1991; 24(10):943-9. DOI:10.1016/0021-9290(91)90172-J · 2.50 Impact Factor
  • John M. Clark, John A. Sidles
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    ABSTRACT: The anterior cruciate ligaments (ACL) of dogs, humans, and rabbits were studied by light and scanning electron microscopy after fixation in situ. In all species, the ACL was composed of multiple 20 microns-wide collagen fiber bundles separated by columns of cells in fibrous capsules. These bundles were in turn grouped into fascicles of varied size. The fascicles were surrounded by thin membranous sheets that ran through the ligament forming single or multiple layers between fascicles. Splaying of the ACL at insertion was created by increased volume in the cellular intervals. Bending of the fiber bundles occurred in this region--which corresponds to the fibrocartilaginous zone. We propose that the cell layers accommodate compressive forces and the membranes allow slipping among fascicles without compromising blood supply.
    Journal of Orthopaedic Research 03/1990; 8(2):180-8. DOI:10.1002/jor.1100080205 · 2.97 Impact Factor