Gerald A. M. Finerman

The Effect of Regional Gene Therapy with Bone Morphogenetic Protein-2-Producing Bone-Marrow Cells on the Repair of Segmental Femoral Defects in Rats*

BACKGROUND Recombinant human bone morphogenetic proteins (rhBMPs) can induce bone formation, but the inability to identify an ideal delivery system limits their clinical application. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone-marrow cells, which allow delivery of the BMP-2 to a specific anatomical site. The autologous BMP-2-producing bone-marrow cells then were used to heal a critical-sized femoral segmental defect in syngeneic rats. METHODS Femoral defects in five groups of rats were filled with 5 x 10(6) BMP-2-producing bone-marrow cells, created through adenoviral gene transfer (twenty-four femora, Group I); twenty micrograms of rhBMP-2 (sixteen femora, Group II); 5 x 10(6) beta-galactosidase-producing rat-bone-marrow cells, created through adenoviral gene transfer of the lacZ gene (twelve femora, Group III); 5 x 10(6) uninfected rat-bone-marrow cells (ten femora, Group IV); or guanidine hydrochloride-extracted demineralized bone matrix only (ten femora, Group V). Guanidine hydrochloride-extracted demineralized bone matrix served as a substrate in all experimental groups. Specimens that were removed two months postoperatively underwent histological and histomorphometric analysis as well as biomechanical testing. RESULTS Twenty-two of the twenty-four defects in Group I (BMP-2-producing bone-marrow cells) and all sixteen defects in Group II (rhBMP-2) had healed radiographically at two months postoperatively compared with only one of the thirty-two defects in the three control groups (beta-galactosidase-producing rat-bone-marrow cells, uninfected rat-bone-marrow cells, and guanidine hydrochloride-extracted demineralized bone matrix alone). Histological analysis of the specimens revealed that defects that had received BMP-2-producing bone-marrow cells (Group I) were filled with coarse trabecular bone at two months postoperatively, whereas in those that had received rhBMP-2 (Group II) the bone was thin and lace-like. Defects that had been treated with bone-marrow cells producing beta-galactosidase (Group III), uninfected bone-marrow cells (Group IV), or guanidine hydrochloride-extracted demineralized bone matrix only (Group V) demonstrated little or no bone formation. Histomorphometric analysis revealed a significantly greater total area of bone formation in the defects treated with the BMP-2-producing bone-marrow cells than in those treated with the rhBMP-2 (p = 0.036). Biomechanical testing demonstrated no significant differences, with the numbers available, between the healed femora that had received BMP-2-producing bone-marrow cells and the untreated (control) femora with respect to ultimate torque to failure or energy to failure. CONCLUSIONS This study demonstrated that BMP-2-producing bone-marrow cells created by means of adenoviral gene transfer produce sufficient protein to heal a segmental femoral defect. We also established the feasibility of ex vivo gene transfer with the use of biologically acute autologous short-term cultures of bone-marrow cells.

Estrogen Affects the Cellular Metabolism of the Anterior Cruciate Ligament A Potential Explanation for Female Athletic Injury

Investigations from this laboratory have established the presence of estrogen receptors in the human an terior cruciate ligament. This study further investigates the effects of 17β-estradiol on the cellular proliferation and collagen synthesis of fibroblasts derived from the rabbit anterior cruciate ligament. Fibroblast prolifera tion and collagen synthesis in response to near log concentrations of 17β-estradiol (at 0.0029, 0.025, 0.25, 2.5, and 25 ng/ml) were assessed by measuring [3H]thymidine and [14 C]hydroxyproline incorporation, respectively. Collagen synthesis was significantly re duced with increasing local estradiol concentration (P < 0.001). Declining collagen synthesis was first noted at a 17β-estradiol concentration of 0.025 ng/ml. Within normal physiologic levels of estrogen (0.025 to 0.25 ng/ml), collagen synthesis was reduced by more than 40% of control, and at pharmacologic levels of 2.5 and 25 ng/ml, by more than 50% of control. A signifi cant reduction of fibroblast proliferation was also ob served with increasing estradiol concentrations (P = 0.023). Clinically, alterations in anterior cruciate liga ment cellular metabolism caused by estrogen fluctua tions may change the composition of the ligament, rendering it more susceptible to injury.

Resistant nonunions and partial or complete segmental defects of long bones. Treatment with implants of a composite of human bone morphogenetic protein (BMP) and autolyzed, antigen-extracted, allogeneic (AAA) bone.

Twenty-five patients with resistant nonunions including partial or complete segmental defects were treated with a composite alloimplant of human bone morphogenetic protein (h-BMP) and autolyzed, antigen-free, allogeneic bone (AAA). The series consisted of 16 females and nine males; average age was 45 years. Preoperative symptoms averaged 30 months (range, five to 83 months); 22 of 25 patients had failed multiple attempts at electrical stimulation. Twenty-three of 25 patients had an average of three prior failed surgical attempts at union (range, one to ten). There were ten segmental defects with an average length of 4 cm (range, 2-9 cm). The composite implant was incorporated as an onlay in 15 extremities and as an inlay graft supported by internal fixation in ten extremities. Seven patients received supplementary autogeneic cancellous bone grafting. Average healing time was six months (range, three to 14 months). Average follow-up time was 21 months (range, five to 82 months). Functional results were rated as excellent, 14; good, five; and fair, five. One failed to unite because of a recurrent infection. Union was obtained in 24 of 25 patients. There were five failures of the original operation that required reoperations; union eventually occurred in four of five extremities by repeat composite grafting and replacement of the failed internal fixation. Bony union between host bone and the composite implant began at an average of eight weeks postoperatively. Present results indicate that h-BMP/AAA composite implants represent adjunctive treatment of difficult nonunions. The h-BMP/AAA composite implants may be implanted in either partial or complete segmental defects of long bones.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology and matrix composition during early tendon to bone healing.

This study outlines the early morphologic phenomenon of tendon to bone healing in the rabbit model. Twelve skeletally mature, male New Zealand White rabbits received transplantation of the hallucis longus tendon into a 2-mm calcaneal bone tunnel. The morphologic characteristics of the healing tendon to bone interface were evaluated at 1, 2, 4, and 6 weeks after surgery by the use of conventional histology and immunohistochemical localization of collagen Types I, II, and III. Histologic analysis illustrated progressive maturation and reorganization of the tendon to bone interface with subsequent development of tissue collagen fiber continuity between the tendon and bone. Initially, diffuse immunolocalization of all three collagen types was observed within the scar tissue filling the space between the tendon and bone. During a 6-week period, reorganization of the scar tissue into an interface occurred, similar to an indirect insertion. Although a definitive fibrocartilage region did not form, Type II collagen was localized at the remodeling insertion site throughout the first 6 weeks of repair. In addition, Type III collagen fibers, resembling Sharpeys fibers, were noted to span this interface. The characterization of the insertion between tendon and bone is important to the understanding of healing in commonly used orthopaedic grafting procedures, such as anterior cruciate ligament reconstructions.

Direct in vitro measurement of forces in the cruciate ligaments. Part I: The effect of multiplane loading in the intact knee.

Specially designed load-transducers that measured the resultant forces exerted by the posterior and anterior cruciate ligaments on their respective femoral and tibial insertions were applied to eighteen fresh-frozen cadaveric knees for a series of controlled loading experiments. The mean force in the posterior cruciate ligament at 5 degrees of forced hyperextension of the knee was 23 per cent of the mean force in the anterior cruciate ligament. When the knee was hyperflexed by application of 10.0 newton-meters of bending moment to the tibia, the mean force in the posterior cruciate ligament was 55 per cent of that in the anterior cruciate ligament. Quadriceps tendon pull increased the force in the posterior cruciate ligament in twelve of the fourteen specimens to which it had been applied, at 80 and 90 degrees of flexion only. The force generated in the posterior cruciate ligament by applied internal tibial torque was greatest when the knee was in 90 degrees of flexion; the force in the anterior cruciate ligament was greatest when the knee was fully extended. External tibial torque generated force in the posterior cruciate ligament in only eight specimens, and only at 80 and 90 degrees of flexion. The levels of force that were generated in the posterior cruciate ligament by applied varus and valgus bending moment were greatest at 90 degrees of flexion of the knee; the levels of force in the anterior cruciate ligament were greatest with the knee in full extension. With the knee flexed 90 degrees and the tibia in neutral rotation, fifty newtons of applied posterior tibial force increased the mean force in the posterior cruciate ligament by 58.4 newtons; at full extension, no increase in the force in the ligament was recorded, indicating that tensed capsular structures were absorbing the applied load. When the tibia was internally or externally rotated by applied tibial torque, the increases in the force in the ligament from applied posterior tibial force were sharply diminished.

Human bone morphogenetic protein (hBMP)

Abstract Human bone morphogenetic protein (hBMP) was chemically extracted from demineralized gelatinized cortical bone matrix by means of a CaCl2·urea inorganic-organic solvent mixture, differential precipitation in guanidine hydrochloride, and preparative gel electrophoresis. hBMP is isolated in quantities of 1 mg/kg of wet weight of fresh bone, and has the amino-acid composition of an acidic polypeptide. The mol wt is 17 to 18 k-Da (kilodaltons). Implants of the isolated 17-kDa protein are very rapidly adsorbed and produce a smaller volume of bone than protein fractions consisting of 24-, 17-, and 14-kDa proteins. Since the isolated 24- and 14-kDA components lack hBMP activity, the kinetics of the bone morphogenetic processes including the function of other proteins as carrier molecules, await investigation.

Direct in vitro measurement of forces in the cruciate ligaments. Part II: The effect of section of the posterolateral structures.

Specially designed load-transducers were applied to eight fresh-frozen cadaveric knee specimens in order to measure resultant forces in both cruciate ligaments as the knees were subjected to straight varus-valgus bending moment and to tibial torque (with and without a superimposed posterior tibial force). The forces in the ligaments and tibial rotation were recorded at seven angles of flexion of the knee, between 0 and 90 degrees, before and after section of the posterolateral structures. Ligamentous section increased angulation of the tibia when varus moment was applied to the knee; the large increases in lateral opening of the knee joint were accompanied by increases in the force in the anterior cruciate ligament at all angles of flexion and increases in the force in the posterior cruciate ligament between 45 and 90 degrees of flexion. When valgus moment was applied, there were no significant changes in valgus angulation or the resultant force in either cruciate ligament after ligamentous section. Ligamentous section increased rotation of the tibia when external torque was applied to the knee. The increased external rotation was accompanied by decreases in the force in the anterior cruciate ligament between 0 and 20 degrees of flexion of the knee and increases in the force in the posterior cruciate ligament between 45 and 90 degrees of flexion. In the studies involving applied internal tibial torque, after ligamentous section, rotation of the tibia increased slightly between 60 and 90 degrees of flexion. The force in the anterior cruciate ligament increased between 0 and 20 degrees of flexion, while the force in the posterior cruciate ligament was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomechanical Consequences of Replacement of the Anterior Cruciate Ligament with a Patellar Ligament Allograft. Part II: Forces in the Graft Compared with Forces in the Intact Ligament*

Seventeen fresh-frozen knee specimens from cadavera were instrumented with a load-cell attached to a mechanically isolated cylinder of subchondral bone containing the tibial insertion of the anterior cruciate ligament. The forces in the intact anterior cruciate ligament were recorded as the knee was passively extended from 90 degrees of flexion to 5 degrees of hyperextension without and with several constant tibial loads: 100 newtons of anterior tibial force, ten newton-meters of internal and external tibial torque, and ten newton-meters of varus and valgus moment. The anterior cruciate ligament was resected, and a bone-patellar ligament-bone graft was inserted. The knee was flexed to 30 degrees, and the graft was pre-tensioned to restore normal anterior-posterior laxity. The knee-loading experiments were repeated at this level of pre-tension (laxity-matched pre-tension) and at a level that was forty-five newtons greater than the laxity-matched pre-tension (over-tension). During passive extension of the knee, the forces in the graft were always greater than the corresponding forces in the intact anterior cruciate ligament. Over-tensioning of the graft increased the forces in the graft at all angles of flexion. At full extension, the mean force in the anterior cruciate ligament was fifty-six newtons; the mean force in the graft at laxity-matched pre-tension was 168 newtons, and it was 286 newtons in the over-tensioned graft. Greater pre-tensioning may be required when the knee demonstrates apparent tightening of the graft in flexion. The mean forces in the graft generated during all constant loading tests were greater than those for the intact anterior cruciate ligament over the range of flexion. When the graft was over-tensioned, the forces generated by the anterior tibial force and by varus and valgus moment increased but those generated by internal and external tibial torque did not. There was no significant change in the mean tibial rotation as a function of the angle of flexion of the knee after insertion of the graft; normal tibial rotation of the knee during passive extension (the so-called screw home mechanism) was eliminated. CLINICAL RELEVANCE: When a patellar ligament allograft was pre-tensioned to restore normal anterior-posterior laxity, the forces in the graft were markedly greater than those in the intact anterior cruciate ligament. Thus, the penalty of increased forces in the graft must be accepted if anterior-posterior laxity is to be restored. Of particular concern are the large forces in the graft generated by loading states, such as external tibial torque and varus moment, which normally generate minimum force in the intact anterior cruciate ligament. In terms of force magnitude, internal torque applied to an extended knee is likely to be the most dangerous loading state for a patient who has a patellar ligament graft. There is a current trend toward early postoperative mobilization and intensive rehabilitation after substitution of the anterior cruciate ligament with a graft. Although this approach results in an excellent range of motion, the surgeon should be aware that a return to full activity could produce forces in the graft that are many times greater than those in the intact anterior cruciate ligament. For this reason, early return to full activity may not be indicated until full biological maturation of the graft.

Repair of segmental defects of the tibia with cancellous bone grafts augmented with human bone morphogenetic protein: a preliminary report

Human bone morphogenetic protein (hBMP) is a bone cell differentiation-inducing factor. Six patients with traumatic segmental three- to 17-cm tibial defects developed solid union by implantation of hBMP and autogeneic cancellous grafts and stabilization. There were no allergic, infectious, or surgical complications. If hBMP augmentation in biodegradable delivery systems can be established by a prospective, randomized, double-blind investigation, the incidence of successful bone graft operations for treatment of large segmental defects would be measurably improved.

Outcome after total hip arthroplasty: Comparison of a traditional disease-specific and a quality-of-life measurement of outcome

The purpose of this study was to examine the relationship between the Harris Hip Score (HHS), a traditional method of patient assessment of a total hip arthroplasty (THA), and the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36), a commonly used health-related quality-of-life survey. One hundred forty patients returning for routine clinical follow-up evaluation of a primary THA were asked to fill out the SF-36 quality-of-life survey, as well as questions concerning their perceptions of their THA. The patients surgeon assessed the THA with the traditional HHS. The correlations between the HHS and the SF-36 domains were highest in the physical component summary scores for male patients of all ages and female patients 65 years of age or older. The correlations were lower for the mental component summary scores of all patients, but particularly in female patients younger than 65. When the SF-36 scores were compared with age and sex-matched population norms, both age and sex were found to be important. Men younger than 65 had scores lower than norms in the physical function domains, but were comparable in the mental health domains. The older men had scores comparable to the norms in all domains. Female patients of all ages, however, had lower scores in the physical function domains. The greatest differences were noted in the female patients younger than 65. The HHS is commonly used to assess disease-specific pain and function in THA patients; however, the results of this study suggest that the SF-36 health survey can capture additional important quality-of-life domains that are influenced by a THA and that these domains are influenced by the age and sex of the patient. The combination of a disease-specific scoring system and a quality-of-life survey would allow a more global assessment of a THA in all patients. Studies evaluating the results of THAs should either assess the results of male and female patients separately when sample size is sufficiently large or use sex as a possible covariate in a multivariate analysis.