Morphological and Clinical Studies ofMicrowounds in Ischemic Human Tissues

John E . Olerud, M .D.Veterans Administration Medical CenterSeattle, Washington 98195

Sponsor : Veterans Administration RehabilitationResearch and Development Service

Peripheral vascular disease (of morbidity and mortality in this country. Approxi-mately 30,000 lower extremity amputations are per-formed annually in the United States, primarily forPVD . Failure of wound healing is one of the principalproblems encountered in these patients, either non-healing of leg ulcers or nonhealing of attemptedsurgical intervention, such as grafting, debridement,or amputation . To date no systematic studies ofwound healing have been done in patients with PVDbecause of the invasive nature of the methods for thestudy of wound healing and the tenuous nature of theaffected extremities.

The method used in this study involves the creationof standardized microwounds on the extremities ofindividuals awaiting amputation necessitated by PVD.The wounds were made under sterile conditionsusing a Simlate-ll bleeding-time device at sites distalto the planned amputation at predetermined timesprior to amputation. The microvvounds were excisedfrom the amputation specimens immediately afteramputation, placed in Karnovsky's fixative, and em-bedded in Epon 812 for thin sectioning for light andelectron microscopy. Morphological events of dermaland epidermal wound healing were expressed as aratio of the morphologically apparent wound agedivided by the actual wound age and were related to

outcome, and clinical factors such as diabetic status.Microwounds have been studied in 15 subjects with

wounds created 7 days prior to amputation . In threeof those subjects additional time points were studiedat approximately 2, 12, 24, 48, and 72 hours as well as10, 14, 21, and 28 days prior to amputation . There wasno morbidity from the procedure . In general, theevents of healing were considerably retarded relative

Rehabilitation R&D Progress Reports 1e84

to normal . The dermal events of healing were signifi-cantly more retarded than the epidermal events.While the method appears to be a safe and effectivemeans of obtaining relatively standardized woundtissue for study of the healing process in ischemichuman tissue, it did not appear to be consistentlyuaeful as a predictor of amputation outcome at theselected level.

Future studies include expanding the number ofsubjects so that more meaningful analysis of clinicalfactors such as 7tpOo, diabetic status, and amputa-tion level may be made. Electron microscopy ofselected material will be done and immunohisto-chemical studies of human wounds are planned.

Effect of Stress and Motionon Repair of Hard and Soft Tissues

Sa"mL-xvVovPh .D . ; Richard D . Coutts, M .D .;Steven R . Garfin ; and Wayne uAkoson~D.Veterans Administration Medical CenterSan Diego, California 92161

Sponsor : Veterans Administration RehabilitationResearch and Development Service

This research program is designed to pursueWolff's Law on tissue healing and remodeling . Al-though initial immobilization after musculoskeletaltrauma is necessary for tissue healing to take place,the deleterious effects of prolonged and/or over-im-mobilization on the repair process and of the disuseatrophy of the surrounding tissues are also wellknown. We have chosen two projects as areas ofconcentration : (\)10 study the deleterious effects oflarge in1ornal fixation plate systems used to im-mobilize long bone fractures, and (ii) to evaluate theeffects of motion and stress on the repair of themedial-collateral ligaments.

In the internel fixation plate study it should benoted that by and large the currently used devices areeffective in treatment of long bone fractures . Howev-er, direct osteonal bone union healing under rigidfixation must be viewed, conceptually, as a slow andunnatural way to mend fractures . In addition, thelarge fixation systems shield the underlying healedbone from physiological stresses and can causeatrophy or osteoporosis . At plate removal, refracturesof bones are of significant clinical concern . In order togain a better understanding and to improve thepresent internal fixation plate systems, we havedeveloped new design criteria, i .e ., moderate bendingand torsional plate rigidities for adequate early im-mobilization in order to achieve callus fracture union,

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and a low plate axial rigidity to minimize the stress (orstrain) shielding of the underlying bone during thepost-union remodeling process. A tubular cross-sec-tional plate, i .e ., a flattened stainless steel tube filledwith polyethylene, has been made . This plate hasbeen applied to immobilized canine mid-shaft femo-ral osteotomy models and has been tested against thecnntnzl plate (rigid plate made out of stainless steel)iAt 6 and 9 months postosteotomy, there were signifi-cant advantages both in terms of mechanical andstructural properties of the healed bone underneaththe low uxial rigidity tubular plate . Morphometricevaluations of bones are in progress.

The next phase of this study will include the longerterm (12 and 15 months) experimental animals aswell as additional animal groups to study the effectsof plate remmove! (recovery of bone) . It is hoped ourdata will enable the clinician to choose a less rigidplate, e.g ., tubular plate, and to select appropriatetiming of plate removal on a rational basis.

In the ligament repair study, dogs and rabbits havebeen used as experimental animals, and a variety oftreatment conditions, ranging from rigid immobiliza-tion and cage activity to immobilization plus cageactivity, have been imposed to determine which set ofconditions will enhance the speed and strength ofligament ropa!: In addition, ligament healing versussurgical repair studies have also been conducted.

The quality of the healing and repaired ligamentsare being evaluated by morphological, bionnechuni-ca!,and biochemical techniques . The advanced bio-mechanical testing procedures developed in our labo-ratory permit the studies of the properties of therepair line separately from, and in addition to, thebone-ligament composites . This is of great impor-tance because the responses to the treatment regi-mens from various elements of the ligament nonopoo-ite,viz., area of repair, area of ligament proximal anddistal to repair, and ligament insertion sites aredifferent . In addition, we are measuring the joint laxityquantitatively using our newly developed apparatus.Cyclic and viscoelastic characteristics of ligamentsare also being evaluated.

Currently, the healing MCLs of rabbits up to 40weeks have been studied, but those for the dogs arelimited to 6 weeks. Preliminary data indicate that earlybut limited motion is advantageous, and that thehealed ligament is not comparable as the normal(even up to 40 weeks postoperative) in terms ofstrength, stiffness, and joint laxity. Additional sacrificeperiods for dog MCLs up to 26 weeks are in progress.Bioohennioal measurements of water content, GAG,cell cellularity and collagen typing, and collagen

cross-links of repairs and normal sites of the MCLsfrom sacrificed animals are also in progress . It ishoped that as a result of this data, additional treat-ment programs including exercise training can beincorporated so that determination of treatment pro-grams on the healing ligaments will be possiblem

Transcutaneous Oxygen Tensionas Predictor of Wound Healing

Frederick A . Matsen UI, M .D. ; Ernest M. Burgess, M .D .;and Craig R . Wyss, Ph .D.Limb Viability LaboratoryUniversity of WashingtonDepartment of OrthopaedicsSeattle, Washington 98195

Sponsor : Veterans Administration RehabilitationResearch and Development Service

Transcutaneous oxygen tension has been studiedfor its usefulness in predicting wound healing poten-tial in disvascular limbs. This noninvasive measure oflocal cutaneous perfusion has been correlated withthe severity of peripheral vascular disease as deter-mined by clinical symptoms . The relationship be-tween local cutaneous perfusion, clinical symptoms,and sensory nerve function is currently under investi-gation.

Segmental transcutaneous P0 2 measurementswere made on the limbs of diabetic and nondiabeticpatients admitted to the Amputation and VascularServices at the Seattle Veterans Administration Medi-calperipheral vascular disease . Nondiabetic limbs withtranscutaneous P0 2 values on the foot or below-the-knee of less than 20 mm Hg were significantly morelikely to have rest pain or ulcers, to need an amputa-tion, and to have failure of amputation healing thanwere those limbs with transcutaneous P0 2 valuesabove 40 mm Hg . Diabetic patients show similarresults, although many had ulcers coincident withtranscutaneous P0 2 values greater than 40 mm Hg,suggesting factors other than inadequate cutaneousoxygen delivery may result in ulceration of diabeticlimbs.

Healing of below-the-knee amputations was cor-related with transcutaneous P0 2 values at that level,and amputation healing showed a strong correlationwith below-the-knee transcutaneous P0 2 . All patientswith below-the-knee transcutaneous P0 2 valuesabove 40 mm Hg healed, 94 percent of those withvalues between 20 and 40 mm Hg healed, and thehealing rate where thetranmcutaneous P0 2 value was

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less than 20 mm Hg

47 percent.Transcutaneous oxygen tension and laser-doppler

velocimetry were compared over heated and un-heated skin in which perfusion pressure was reducedby limb elevation . Results indicate that laser-dopplerand transcutaneous P0 2 measurements do reflectchanges in local perfusion pressure when made overwarmed skin, but not when made over unwarmedskin.

The multiprobe transcutaneous oxygen tensionmonitor has allowed simultaneous measurements ateight sites and has enabled spatial mapping of cuta-neous perfusion in disvascular limbs before and afteramputation . These studies continue to elucidate thevalue of transcutaneous P0 2 measurement in theclinical assessment of the local circulatory status ofskina

Studies of Factors

Affecting Orthopaedic Infections

Katharine Merritt, PhD.University of CaliforniaOrthopaedic Research LaboratoriesDavis, California 95616

Sponsor : National Institutes of Health(National Institute of Arthritis, Diabetes, and Digestiveand Kidney Diseases)

The research being undertaken is designed toinvestigate problems of infections in orthopaedicpatients. One aspect of the study is to better under-stand factors leading to wound contamination in

open fractures and any infections that might followsuch contamination . Various factors, including theuse of internal fixation devices, methods of irrigation,and time between injury and treatment, as well asunderlying diseases, are being compared with infec-tion rates and with level of tissue contamination . Ananimal model involving open fractures of the hamsterfemur using an osteotonny saw has been developedto better understand the role of internal fixation ininfection . Studies have been done with Staphylococ-cus aureus and will be done with Proteus and anAnaerobic Streptococcus.

Studies also are being undertaken in the hamster toinvestigate host reponses, such as sensitivity reac-tions, to the implant on infection rates . Finally, rapiddiagnostic techniques are being investigated for diag-nosis of osteomyelitis and septic arthritis usingblood, joint fluid, and aspirates . These studies shouldprovide information on the management of patientslikely to develop infectionsm

Stimulation of Repair of Cortical BoneTransplants by Implantation ofPiezoelectric Materials

George Van B . Cochran, M .D ., M .Sc .D . ; Marvin W. Johnson, Ph .D .;Wendell Williams, PhD . ; avdBpk Lee, M .DVeterans Administration Medical CenterCastle Point, New York 12511

Sponsor : Veterans Administration RehabilitationResearch and Development Service

Introduction—Microampere DC currents have beenused extensively to stimulate bone formation . Tradi-tionally, these currents are delivered to implantedelectrodes by external power sources or implantedbatteries . The purpose of this project is to develop anovel approach to m!ectrical stimulation of bonehealing and regeneration (including incorporation ofbone transplants) by employing direct delivery offaradic, microampere currents generated during phy-siologic loading by a piezoelectric rna1erial incor-porated in a less rigid internal fixation device . From atheoretical standpoint, assuming simple tension orcompression on a piezoelectric material, the chargegenerated imO=/\du I SI , where A is the material area,

dn i is the piezoelectric constant, and S I is the stress.The current produced by application and release ofload is bipolar, and the magnitude varies with fre-quency of loading . For 1 Hz loading, with the currentoutput rectified by a full-wave bridge, the averagecurrent would be 2Ad 31 S 1 . For PZT-5 (d 31 = 270 p[/N),

if A=1 cm 2 and S 1 =6x10 x 6 N/nn 2 (id= 100 microstrain),the average current generated would be 0 .33 [LA.

Methods—As a pilot study in vivo, miniature (36x 6mm) replicas of three-hole internal fixation plateswere constructed ofde!rin bonded to a non-electrod-ed PZT-5 ceramic bimorph oro strip of polyvinylidenefluoride (PVDF) . Four plates were implanted in each ofeight dogs, one active plate fixing a 1 .5-cm bone graftin the ulna, another overlaying two 4-mm drill holesin the radius, and two depoled (electrically inactive)plates on the contralateral bones as controls . Theentire free surface of the piezoelectric material was indirect contact with bone ; plastic screws were used forfixation . Results were assessed histologically after 8weeks . In a later in vitro experiment, four-hole Keviar/epoxy composite plates were constructed with astrain-gauged ceramic bimorph incorporated on top.For testing, the plate was attached to a canine femurwith four insulated steel screws and the bone loadedin cantilever bending . Similar experiments were con-ducted vvith a bimorph bonded to a titanium plate . Togenerate current, repetitive loads producing 100 mi-

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Wound and Fracture Healing

crostrain in the bimorph were applied at 1 and 2 Hz(load duration =0.5 s; rise time, 0 .2 s; plateau, 0.1 s;decay, 0 .2 s) . Bimorph output (surfaces versus vane)was connected to a full-wave diode rectifier. Averagecurrent was determined from voltage drop across a1 M ci resistor and a 0 .47 !IF capacitor shunting therectifier output.

Results —The in vitro experiments showed that withrectification, continuous DC currents of 0 .25 to 0 .4 RAcould be generated during a series of deformations.Without rectification, polarity varied with sign of thestrain and bipolar currents were generated . The ear-lier in vivo study failed to show significant tissuereactions or differences on bone healing/remodelingbeneath the free surface of active versus depoledbinoorphaor PVOF

Discussion and Future Plans—Our limited in vivostudy suggested that the charged surface ofo piezo-electric material, in itself, has no dramatic effect onosteogenesis, at least in a configuration in whichcharge density is low and fluctuations with stressare nearly symmetrical . Further work of this typeis planned to confirm these negative results. Also,the effect of contact with piezoelectric materialson streaming potentials in underlying bone will bestudied.

In the alternate mode, however, the in vitro testsdemonstrated the feasibility of designing piezoelec-tric internal fixation plates that, during normal phyui-oal activity, could generate DC currents exceeding the0.075 Ila !evel identified recently as the minimum forfaradic stimulation of osteogenesis . These experi-ments generated approximately 0 .3 p,u and an in-crease by a factor of 10 to 20 could be expected inclinical applications utilizing a proportionally largerarea of piezoelectric material subjected to largerstrains . This design concept offers considerable po-tendal for research and development with respect tocomponents, configurations, and applications for in-tornal fixation . Materials ranging from titanium tofiber/plastic composites could be selected to enhancemechanical and stress coupling properties, whilepiezoelectric materials ranging from tissue compat-ible ceramics to polymers (PVDF) could be fabricatedand poled to attain optimal current output and polari-ty in specific anatonnioa! locations . Conceivably, o!eo-trioal charge also could be generated by externalultrasonic energy during non-active periods.

In the next stage of this study, instrumented proto-type piezoelectric plates will be implanted in dogs andcurrent measurements made during walking . In thefinal stage, using rabbit and canine models, studies of

effect of these piezoelectrically generated currents onbone formation will made using implanted a!en-trodeanonnooted directly to the rectified plate outputs

Effects of

on and Motionin the Injured Tendon

Albert J . BanesUniversity of North CarolinaChapel Hill, North Carolina 27514

Sponsor : National Institutes of Health(National Institute of Arthritis, Diabetes, and Digestiveand Kidney Diseases>

For the coming support year, the objectives are:(i) to establish the pattern of tryptic peptides ofcollagen from tendons of exercised chickens andcompare these to the established patterns for im-mobilized and control birds ; (ii) to continue isolationof three major cross-link peptides that elute at thesame position for collagens from normal, immobi-lized, and exercised birds, and to perform amino acidand sequence analysis to assign a location for thesepeptides within the collagen molecule ; (iii) to con-tinue isolation of tendon cells from sheath, synovium,and tendon fibroblasts.

The latter cells from immobilized and exercisedbirds will be subjected to tension and compression invitro. The 35S-methionine labeling patterns will becompared to that of the normal birds . Particularattention will be given to the amounts of collagen,actin, and tubulin produced by the cells . The cellculture studies have taken an unusual turn . Popula-tions of cells have been isolated : cells from sheath,synovium, and tendon proper are different in mor-phology, protein profile, rate of division, and adher-ence to substrate . On the other hand, tendon fibro-blasts from a physically small tendon (flexor profun-dus) versus a large tendon (flexor to the gastro-cnemius) appear to have minor differences in proteinsynthesis patterns . Consideration of physical stresson tendons led to the creation of a new culture platethat can accommodate up to 200 percent stretch . Thelatter has been in testing for 10 weeks as of thiswriting . Tendon cells in culture seem to respond to0.1 percent compression in vitro by decreasing tubu-lin and increasing actin concentrations . Experimentswith tension are in progress.

With respect to peptide sequencing, it is anticipatedhaving at least three major peptides that containeither reducible or 3-hydroxypyridinium (HP) cross-links quantitated and compared among the groups inthe next 6 months . The absorbence and HP patternshave been completed . Currently being used is a

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170-minute, 0 .1M PO4 2.85 pH, acetonitrole gradientto elute collagen tryptie peptides . As little as 10 ug ofcollagen need to be injected to establish absorbenceand fluorescence patterns (HP), but more material isrequired for radioactivity detection . It is anticipatedthat the amounts and location of HP cross-link sites intendon collagen from exercised birds will be differentfrom those of the immobilized and control counter-parts. The latter patterns are different ; peptide purifi-cation and analysis are underwaym

Flexor Tendon Healing : Restorationof the Gliding Surface

Richard GelbermanUniversity of CaliforniaSan Diego, California 92103

Sponsor : National Institutes of Health(National Institute of Arthritis, Diabetes, and Digestiveand Kidney Diseases)

Preliminary results from our laboratory suggestedthat primary tendon healing could be stimulated byprotective passive motion which prevented tendonsheath adhesion ingrowth . This effect resulted from acellular proliferation of the epitenon layer. An im-proved mechanism of repair was anticipated as aresult of carefully applied and protected early motiontechniques. The objectives of the first year of thisgrant period were to establish a model and obtaindata on the intrinsic capacity for flexor tendons toheal in the absence of tendon sheath adhesion in-growth.

An initial study was carried out in conjunction withDr. Paul Manske at Washington University in St . Louisin which flexor-tendon segments from New Zealandwhite rabbits were lacerated, immobilized, and placedin tissue culture media . The tendon segments werestudied by light and electron microscopy at 3-, 6-, 9-,and 12-week intervals . A characteristic sequence ofrepair was noted, including epitenon thickening, cel-lular differentiation, cell migration, and phagocytosisin each of the lacerated tendons. This response wassimilar to previously observed in vivo tendon healingand indicated that tendons have the inherent capacityto heal in the absence of adhesion growth . Anadditional study of the healing characteristics of fourdifferent species was explored in a tissue culturesystem . Tendon segments were studied by light andelectron microscopy at 3, 6, 9, and 12 weeks . A similarpattern of repair was seen in each model, but the rateof healing differed significantly.

Other research efforts in this period have included

the construction of a continuous passive motiondevice so that the effects of continuous motion couldbe compared with previous results using intermittentpassive motion and immobilization techniques . Thedevice has been constructed, and anirnal experi-mentation is ready to begin . Finally, we have estab-lished an in vivo tendon healing model in whichtritiated thymidine is used to tag cells participating inthe tendon healing processm

A Study of Untertrochanteric FractureFixation Methods

A. Ogden, F .R .C.S . ; A.C . Nicol' Ph]D . ; K. Garufis ' NIa Ch.B . ; and

I .G . Kelly, F .R.C .S.Bioengineering UnitWolfson CentreGlasgow G4 ONVK Scotland

Sponsor : University of Strathclyde

This summary reports the findings of a three-partpilot project to determine the optimum technique forthe fixation of unstable intertrochanteric fractures ofthe femur in elderly patients with osteoporosis.

Atota\ of 121 patients with unstable \ntertronhun-teriofraotunem were studied. Eighty-two patients weretreated with Kuntscher-Y nails (KY) and 39 weretreated with a Honey Capener (HC) fixed-angle nailplate. Most complications were seen with the use ofthe HC neil plate . These were nail breakage (five),bending with loss of reduction (ten), and detachmentof the plate (four) . Most cases of failure could berelated to a significant degree of osteoporosis whichwas estimated using Singh's classification.

A similar study using Richards sliding screws wasconducted on 86 patients with unstable intertrochan-teric fractures . There was a 4 .7 percent incidence ofcomplications which was associated with a moderatedegree of osteoporosis.

Clinical experience suggests the mechanical superi-ority of the Richards screws and KY nails but alsohighlights osteoporosis as a limiting factor in the useof these internal fixation devices.

Mechanical tests were therefore performed onthese two implants using an Instron testing machine.In order to investigate the effect of osteoporosis,parallel tests were conducted with the use of adjuvantbone cement.

Paired femoral specimens with a standardized frac-ture were tested with and without the addition ofcement . The degrees of osteoporosis were assessedfor the specimens using Singh's classification . Thetotal strength of the Richards screw fixation ranged

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Wound and Fracture Healing/Properties of Muscle

from 280 N to 1670 N for uncemented specimens and7GONto275ONforcement addition . A similar twofoldincrease was also found for the KY nail.

These results would appear to validate the use ofadjuvant bone cement in the repair of fractures inoateoporoticbone~

The Effects of Pulsed Galvanic Currentson the Healing of Soft Tissue Injuries

mei!Spk4hnkz, Ph .D.

New York University Medical CenterDepartment of Rehabilitation MedicineNew York, New York 10016

Sponsor : National Institute of Handicapped Research

About 2 years ago, a Ph .D. candidate working in thislab reported that pulsed galvanic stimulation seemedto speed the healing of surgically divided Achillestendons in rats . This past year, using tensile strengthas the measure of healing, we confirmed thoseobservations . Equipment limitations, however, pre-cluded our ability to measure tensile strength above1500 gms. Since this value is well below the loadneeded to break a normal or fully-healed tendon, wemust currently limit our comments about healing toonly the early stages . We hope ultimately to be able toappraise the long-term effects as well.

In addition to the tensile strength studies, weaccumulated considerable electron microscopic dataconcerning the ultrastructure of normal tendon andtendon subjected to immobilization by casting ordenervation . The observations, which suggest strong-ly that tendon is far from being an inert structure,hopefully will be reported at an upcoming conferencein Boston. Basically, the findings are that immobiliza-tion leads to atrophy of collagen fibers . This suggestsnot only a turnover of collagen but the ability oftendon to meet the demands placed on it in much thesame way that bone and muscle do.

Plans for the coming year include attempts toupgrade this project's materials-testing abilities(which will permit the monitoring of healing in moredetail) and to continue with the electron microscopestudioam

[See also VI. Biomechanics, A. Joint Studies,2 . Lower Limb, Biomechanics of Anterior CruciateRepairs]

~~0~~

Properties oJ~ Muscle

Control of Muscle Pro ein MetabolismDuring Exercise

East Carolina UniversityGreenville, South Carolina 27834

Sponsor : National Institutes of Health(National Institute of Arthritis, Diabetes, and Digestiveand Kidney Diseases)

The broadchanges that occur in protein metabolism as a resultof exercise and to establish the underlying biochemi-cal mechanisms that bring about these changes . Inresearch already completed, we have clearly estab-lished that an acute bout of exercise (either running orswimming) causes a decrease in the rate of muscleprotein synthesis and increased rates of protein de-gradation in muscle and liver . Preliminary evidencefrom our lab and reports from other investigatorssuggest that an acute exercise bout also increases therate of amino acid oxidation.

During the next granting period, we propose tofurther study the effect of an acute exercise bout onprotein synthesis, protein degradation, and aminoacid oxidation . To determine mechanisms involved inthe decrease in protein synthesis in muscle, we planto determine the effect of exercise on the variouscomponents of protein synthesis : charging of t-RNA,initiation of peptide synthesis, and peptide elonga-tion. An experiment will be conducted to investigatewhether an acute bout of exercise increases alanineproduction by the isolated epitrochlearis muscle . Inaddition, we plan to continue investigating the bio-chemical regulation of leucine oxidation in muscleduring exercisem

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