Low-Dose Aronia melanocarpa Concentrate Attenuates Paraquat-Induced Neurotoxicity.

Herbicides containing paraquat may contribute to the pathogenesis of neurodegenerative disorders such as Parkinson's disease. Paraquat induces reactive oxygen species-mediated apoptosis in neurons, which is a primary mechanism behind its toxicity. We sought to test the effectiveness of a commercially available polyphenol-rich Aronia melanocarpa (aronia berry) concentrate in the amelioration of paraquat-induced neurotoxicity. Considering the abundance of antioxidants in aronia berries, we hypothesized that aronia berry concentrate attenuates the paraquat-induced increase in reactive oxygen species and protects against paraquat-mediated neuronal cell death. Using a neuronal cell culture model, we observed that low doses of aronia berry concentrate protected against paraquat-mediated neurotoxicity. Additionally, low doses of the concentrate attenuated the paraquat-induced increase in superoxide, hydrogen peroxide, and oxidized glutathione levels. Interestingly, high doses of aronia berry concentrate increased neuronal superoxide levels independent of paraquat, while at the same time decreasing hydrogen peroxide. Moreover, high-dose aronia berry concentrate potentiated paraquat-induced superoxide production and neuronal cell death. In summary, aronia berry concentrate at low doses restores the homeostatic redox environment of neurons treated with paraquat, while high doses exacerbate the imbalance leading to further cell death. Our findings support that moderate levels of aronia berry concentrate may prevent reactive oxygen species-mediated neurotoxicity.

Arginine deiminase in Staphylococcus epidermidis functions to augment biofilm maturation through pH homeostasis.

Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.

Three-dimensional printing and porous metallic surfaces: a new orthopedic application.

As-cast, porous surfaced CoCr implants were tested for bone interfacial shear strength in a canine transcortical model. Three-dimensional printing (3DP) was used to create complex molds with a dimensional resolution of 175 microm. 3DP is a solid freeform fabrication technique that can generate ceramic pieces by printing binder onto a bed of ceramic powder. A printhead is rastered across the powder, building a monolithic mold, layer by layer. Using these 3DP molds, surfaces can be textured "as-cast," eliminating the need for additional processing as with commercially available sintered beads or wire mesh surfaces. Three experimental textures were fabricated, each consisting of a surface layer and deep layer with distinct individual porosities. The surface layer ranged from a porosity of 38% (Surface Y) to 67% (Surface Z), whereas the deep layer ranged from 39% (Surface Z) to 63% (Surface Y). An intermediate texture was fabricated that consisted of 43% porosity in both surface and deep layers (Surface X). Control surfaces were commercial sintered beaded coatings with a nominal porosity of 37%. A well-documented canine transcortical implant model was utilized to evaluate these experimental surfaces. In this model, five cylindrical implants were placed in transverse bicortical defects in each femur of purpose bred coonhounds. A Latin Square technique was used to randomize the experimental implants left to right and proximal to distal within a given animal and among animals. Each experimental site was paired with a porous coated control site located at the same level in the contralateral limb. Thus, for each of the three time periods (6, 12, and 26 weeks) five dogs were utilized, yielding a total of 24 experimental sites and 24 matched pair control sites. At each time period, mechanical push-out tests were used to evaluate interfacial shear strength. Other specimens were subjected to histomorphometric analysis. Macrotexture Z, with the highest surface porosity, failed at a significantly higher shear stress (p = 0.05) than the porous coated controls at 26 weeks. It is postulated that an increased volume of ingrown bone, resulting from a combination of high surface porosity and a high percentage of ingrowth, was responsible for the observed improvement in strength. Macrotextures X and Y also had significantly greater bone ingrowth than the controls (p = 0.05 at 26 weeks), and displayed, on average, greater interfacial shear strengths than controls, although they were not statistically significant.

Small animal surgical and histological procedures for characterizing the performance of tissue-engineered bone grafts.

Developing effective tissue-engineered constructs for bone regeneration requires careful assessment of the in vivo bone response to novel biomaterials, scaffold architectures, and biologically augmented, tissue-engineered constructs. Both the implant material and scaffold architecture are known to significantly effect the local tissue response (1-3). Consequently, in characterizing the performance of new bone implants, it is prudent to establish material-dependent and scaffold-architecture-dependent bone-growth phenomena, in addition to the effect of biological augmentation, e.g., preseeded cells, growth factors, and cell-attachment proteins. Here we describe rabbit transcortical pin and trephine defect models, which, in combination, yield a method to investigate such variables on bone regeneration. The necessary histological and histomorphometry procedures are also detailed.

The acoustic and structural properties of the human femur.

This study evaluates the variations in the acoustic properties of the human femur at ten evenly spaced locations along its length, as well as differences that exist within given transverse sections. Six pairs of human femora, three male and three female, were sectioned, ground, and polished, and scanned with a microprocessor-driven scanning acoustic microscope. Images with a resolution of approximately 140 microns were used to calculate the average acoustic impedances for each transverse cross section and each quadrant within a cross section. The mean acoustic impedance for all the cross sections was 7.69 +/- 0.18 Mrayls. Variations were observed among the cross sections, and the central sections (4-7) had values that were statistically greater than the other more distal and proximal sections. Within the cross sections, the posterior quadrant had a lower average acoustic impedance compared to the other quadrants and this was statistically significant (Tukey's multiple comparison test). The cross sections were further analyzed to determine several geometric parameters including the principal moments of inertia, polar moment of inertia, and the biomechanical shape index. The product of the acoustic impedance and the maximum moment of inertia provided a result that attempted to account for the acoustic property variation and the change in shape at the different section locations.

A methodology report of a randomized prospective clinical trial to assess velopharyngeal function for speech following palatal surgery.

Cleft lip and palate occurs in approximately 1 in every 750 live human births, making it one of the most common congenital malformations. Surgical closure of the palatal cleft does not always result in a velopharyngeal port capable of supporting normal speech. The University of Florida (UF), in collaboration with the University of São Paulo (USP), is engaging in a 5-year prospective, randomized controlled study to compare velopharyngeal function for speech outcomes between patients undergoing palatoplasty for complete unilateral cleft lip and palate performed using the von Langenbeck procedure with intravelar velarplasty and those receiving the Furlow double-reversing Z-plasty palatoplasty. The von Langenbeck procedure was selected as the time-tested standard against which the Furlow procedure could be judged. The Furlow procedure, a relatively new operation, has been reported to yield substantially higher rates of velopharyngeal competency for speech than have most other reported series and theoretically should result in less disturbance to midfacial growth. A total of 608 patients will be entered into one of two age categories. Inclusion of two age groups will allow a comparison of results between patients having surgery before 1 year of age (9-12 months) and patients undergoing surgery at approximately 1.5 years of age (15-18 months). Speech data will be collected and will be available for definitive analysis throughout the last 3 years of the study. Collection of preliminary growth data will require more than 5 years; growth analysis is anticipated to continue until all patients have reached maturity. The Hospital for Research and Rehabilitation of Patients with Cleft Lip and Palate at the University of São Paulo (USP-HPRLLP) in Bauru, Brazil, is uniquely situated for conducting this study. The well-equipped and modern facilities are staffed by well-trained specialists representing all disciplines in cleft-palate management. In addition, an already existing social services network throughout Brazil will ensure excellent follow-up of study cases. The clinical caseload at this institution currently exceeds 22,000, and more than 1200 new cases are added annually. This project represents a unique opportunity to obtain prospective data from a large number of subjects while controlling the variables that have traditionally plagued cleft-palate studies. This study is designed to determine which of the two proposed surgical procedures is superior in constructing a velum capable of affecting velopharyngeal competency for the development of normal speech.

Regional and temporal changes in the acoustic properties of fracture callus in secondary bone healing.

Controlled fractures were created in the right femora of 17 male Sprague-Dawley rats. The fractured limbs were harvested at 2, 4, 6, and 8 weeks after fracture, fixed, and embedded in polymethylmethacrylate. Midsagittal sections from each animal were evaluated with a scanning acoustic microscope, a device that generates an acoustic impedance map of the scanned material. The impedance of the fracture callus was measured in six regions on each specimen. These regions were chosen in an effort to distinguish between the impedance of the callus formed through intramembranous or endochondral ossification, and we found that the time course of increasing impedance differed for the fracture callus formed through the two pathways. Additionally, we found a significant difference in the mean impedance of the callus at each time period (p < or = 0.0013 for all comparisons), which resulted in an extremely linear relationship (r2 = 0.999) between mean callus impedance and healing time. This experimental model has become a popular choice for the investigation of fracture healing. As such, an accurate determination of the mechanical properties of the fracture callus is often sought. We propose that the implementation of scanning acoustic microscopy in the study of fracture healing may determine the changes in the material properties more accurately than conventional testing methods.

A prospective, clinical study evaluating arthroscopic ACL reconstruction using the semitendinosus and iliotibial band: 2- to 5-year follow up.

Seventy-three ACL-deficient knees were reconstructed using a modified Zarins and Rowe arthroscopic procedure utilizing the semitendinosus tendon and iliotibial band (ITB). Fifty of 73 patients returned for 2- to 5-year follow up. On follow-up visit each patient completed a questionnaire pertaining to subjective results, including level of pain and return to, activities. Physical examination included the Lachman test, anterior drawer, pivot shift, and anterior subluxation tests (using the Stryker KT 1000), each graded 0 to 3. All patients have shown a statistically significant improvement between preoperative and postoperative knee scores (P < .0001). According to the knee scoring scale, 34 patients (68%) were clinically rated as excellent, 12 (24%) as good, 4 (8%) as fair, with no poor results. All patients have returned to pre-injury jobs (or school), while most resumed some level of sporting activities. No patients complained of patella femoral symptoms postoperatively. Complications included pain over the staple which was used for fixing the ITB to the tibia, requiring removal in 5 patients. These preliminary results are encouraging, with 92% of the patients rating good to excellent. This modified procedure offers advantages over other procedures, as it is entirely arthroscopic with minimal incisions, does not damage the extensor mechanism, and permits an enhanced rehabilitation program.

Biomechanical and histological analysis of an HA coated, arc deposited CPTi canine hip prosthesis.

The interfacial shear strength and bone tissue response was investigated for an arc deposited (AD) commercially pure titanium implant surface, with (AD/HA) and without (AD) plasma-sprayed hydroxyapatite (HA) coating. Ten purpose bred coonhounds received bilateral femoral stem implantation (AD and AD/HA) in the proximal femurs (hemiarthroplasty). The femoral prosthesis consisted of a modular CoCr alloy head, modular Ti-6Al-4V neck, and a 10-mm diameter cylindrical Ti-6Al-4V femoral stem. The AD surface had 30-35% greater surface roughness than the AD/HA surface. The HA coating had a purity greater than 90% and a crystallinity greater than 65%. After 6, 12, and 24 weeks, the implants were retrieved and analyzed with mechanical testing, qualitative and quantitative histology, and electron microscopy. The AD/HA implants had equivalent interfacial shear strengths to the AD implants at all time periods. The AD/HA implants had significantly greater linear bone contact than the AD implants. The 6-week implants had significantly thicker cortical bone than the 12- and 24-week implants. The HA coating was very stable in vivo, evidenced by no thickness reduction at any time period. Qualitatively, the AD/HA implants primarily had bone contacting the implant surface with little fibrous tissue present, and the AD implants had bone and fibrous tissue contacting the implant surface. The electron microscopy analysis showed that the mechanically tested implants exhibited a mixed failure mode at the bone, HA coating, and titanium interfaces.

Evaluation of poly(DTH carbonate), a tyrosine-derived degradable polymer, for orthopedic applications.

The polymerization of desaminotyrosinetyrosylhexyl ester (DTH) with phosgene gives rise to poly(DTH carbonate), a new pseudopoly(amino acid). To evaluate the performance of this bioabsorbable material in orthopedic applications, the tissue responses elicited by compression-molded pins of poly(DTH carbonate) and clinically used polydioxanone pins (PDS; Orthosorb) were compared. The two types of pins were implanted in the paravertebral muscle and in the metaphyseal proximal tibia and distal femur of 10 White New Zealand Rabbits for 1, 2, 4, and 26 weeks. The tissue response was evaluated using histologic staining of soft- and hard-tissue sections, fluorescent bone marker of incorporation, and backscattered electron imaging. In soft tissue, both poly(DTH carbonate) and PDS elicited a mild inflammatory response resulting in encapsulation. During the disintegration phase, the PDS implants triggered a foreign body response involving the phagocytosis of polymeric debris by histiocytes and giant cells. No such response was observed for poly(DTH carbonate). In hard tissue, close bone apposition was observed throughout the 26-week test period for poly(DTH carbonate) implants. At the 26-week time point, the poly(DTH carbonate) implants exhibited surface erosion and were penetrated by new bone. In contrast, an intervening fibrous tissue layer was always present between the PDS pins and the bone. At 26 weeks, the PDS implants had partially resorbed and a foreign body response characterized by infiltration in several of the implantation sites. This study indicates that poly(DTH carbonate) and PDS exhibit fundamentally different interactions with hard tissue, and that poly(DTH carbonate) is a promising orthopedic implant material.

The application of scanning acoustic microscopy in a bone remodeling study.

Scanning acoustic microscopy (SAM) was used in the evaluation of bone remodeling around a cylindrical unicortical defect. SAM is a technique for the nondestructive evaluation of materials, and has only recently been employed as an orthopaedic research tool. The utility of SAM was demonstrated by using it to measure an elastic property known as acoustic impedance. Specifically, the acoustic impedance of bone formed by remodeling around a cylindrical defect was measured. The defects were filled with either a low modulus "void" or rigid inclusion to create various states of stress in the bone in the vicinity of the defect. After six months of implantation of the inclusions in the sheep metatarsal, new bone formation on periosteal and endosteal surfaces about the defect region was observed. These regions of new bone were less stiff and had 18.0 +/- 6.5% lower acoustic impedance than the pre-existing bone in the intracortical region of the metatarsal. There was no difference in the degree of new bone formation about void and rigid inclusions. Both underwent significant adaptational changes in response to the elevated stress about the defect. These changes affected the basic structure of the bone cross-section at the level of the defect and effectively reduced the stress levels about the defect. By using SAM to measure acoustic impedance, it was seen that little internal remodeling occurred in the intracortical region. Hence, the primary mechanism of strain-induced bone remodeling observed in this experimental model was surface remodeling.

The porous-coated anatomic (PCA) total hip arthroplasty: a review of 73 uncemented cases with 2-year follow up.

A total of 73 hips in 62 patients who underwent non-cemented total hip arthroplasties (THA) with porous-coated anatomic (PCA, Howmedica) prostheses have been reviewed. A complete evaluation including preoperative and postoperative Harris hip scores (HHSs), a complete radiographic study, and complication determination has been completed with greater than 2-year follow up. Diagnoses included osteoarthritis (74.0%), rheumatoid arthritis (4.1%), avascular necrosis (15.1%), and posttraumatic arthritis following fracture (6.8%). The complication rate was 10.9% (8 complications/73 arthroplasties); 20 patients (35.6%) received bilateral implants. The mean preoperative HHS for the entire group was 38.7, and the mean postoperative score, irrespective of postoperative time, was 93.6. Key findings regarding the consecutive radiographic review of each arthroplasty included: 9.6% lucencies, 43.8% stress shielding, stable fibrous ingrowth in 74.0%, tip sclerosis in 19.2%, and cortical widening in 4.1%. The finding of loose beads from the pore ingrowth area of the prosthesis was minimal (7 beads in 6 patients). These results demonstrate the efficacy of the PCA prosthesis.

Materials and design concepts for an intervertebral disc spacer. II. Multidurometer composite design.

The main function of the intervertebral disc is to transmit and attenuate compressive and torsional forces, and stabilize the intervertebral joint. Unfortunately, the disc may be displaced or damaged due to trauma or disease causing the nucleus to herniate and protrude into the vertebral canal or intervertebral foramen. Pressure on the spinal nerve may cause pain or paralysis in the area of its distribution. At present, the surgical procedures used to alleviate this condition include disc excision, and/or spinal fusion. A more desirable situation would involve removing the nucleus pulposus and part or all of the annulus fibrosis and implanting a suitable biofunctional equivalent. Such a prosthesis should attenuate stresses and prevent abnormal stress at adjacent intervertebral joints. Maintenance of normal disc height would prevent impingement of the posterior facet joints and facet joint syndrome. In a previous companion paper (J. Applied Biomat. 5:125-132; 1994), the mechanical behavior of disc prostheses manufactured from fiber reinforced, elastomeric thermoset resins were examined. In order to develop devices which were more practical from a manufacturing standpoint and extremely reproducible, the fiber reinforced thermoset resins were replaced by multi-durometer thermoplastic elastomeric materials. In the present paper, the mechanical properties of thermoplastic multicomponent designs have been investigated.

The acoustic properties of normal and imbedded bovine bone as measured by acoustic microscopy.

The acoustic impedance of bovine femoral cortical bone was measured with a scanning acoustic microscope used in the reflection mode. The bone was measured in the unimbedded state and after plastic imbedding. The acoustic impedance of the unimbedded specimens was also measured with a standard transmission ultrasonic technique. For the unimbedded bovine specimens there was a good correlation between the bulk transmission impedance and the reflection surface impedance (r2 = 0.976) and the values were 9.32 and 9.29 MRayls, respectively. Plastic imbedding produced a consistent and statistically significant increase in the acoustic impedance of the bone (9.71 MRayls). This experiment verified the use of acoustic microscopy as a quantitative materials analysis technique and it demonstrated the potential for material property analysis of imbedded bone.

Investigation of an organic delivery system for demineralized bone matrix in a delayed-healing cranial defect model.

The osteogenic potential of demineralized bone matrix (DBM) has been demonstrated in multiple animal models and clinical applications. A particulate form of DBM is generally used to fill defects because it is easily packed into a defect site without operative planning or shaping. One potential disadvantage in the use of a particulate is the migration of particles from the defect site. To stem this, glycerol was added to DBM to improve handling properties. A study was undertaken to compare two forms of DBM with glycerol, with DBM particulate and autograft in a bony defect site. The model chosen consisted of bilateral 8-mm trephine defects created in the parietal skull of 40 rabbits. Animals were sacrificed at 2 and 8 weeks. Results demonstrated both cartilage and bone induction with the three forms of DBM, with 75-90% of the linear width of the defect filled with new bone by 2 weeks. This was similar to the autograft (86%) and significantly greater than the unfilled defects (26%) at 2 weeks. The addition of glycerol to the DBM did not affect the inductive capacity, and produced a response similar to that of DBM particulate alone or autogenous bone.

The biomechanics and histopathology of chemically processed patellar tendon allografts for anterior cruciate ligament replacement.

A study was initiated to examine chemically processed patellar tendon allografts in sheep anterior cruciate ligament repairs, both mechanically and histologically. One group of animals received frozen, untreated allografts, one group received frozen grafts that were processed with a chloroform-methanol solvent extraction technique, and one group received frozen tendons treated with a permeation-enhanced extraction technique. All animals were operated on unilaterally, with the contralateral knee acting as a normal, intact control. Histologic analysis after 2 months of implantation revealed similar enhanced cellular repopulation in both chemically treated ligament allografts compared with the more hypocellular, untreated grafts. At 6 months the chloroform-methanol group demonstrated a more aggressive chronic cellular response with numerous thick-walled vessels relative to the untreated and permeation-enhanced grafts. Mechanical testing after 6 months of implantation showed statistically similar anterior drawer resistance in all grafted knees, yet the two chemically extracted grafts had significantly less stiffness than untreated anterior cruciate ligament grafts. Both treatment groups also tended to be weaker than the untreated allografts. All anterior cruciate ligament reconstructions showed excessive anterior drawer laxity and, regardless of treatment, had lower strength and less stiffness than normal anterior cruciate ligament tissue at the 6-month period.

A biomechanical analysis of four humeral fracture fixation systems.

A biomechanical study was initiated to compare four fracture fixation devices: the AO dynamic compression plate, a distal fin locking nail, a solid locked intramedullary nail, and paired flexible nails for humeral fracture fixation. Eighteen pairs of fresh-frozen, intact humeri were harvested, standardized midshaft transverse osteotomies were created in each specimen, and left and right specimens were fixed with plates and nails, respectively. The bending properties of the plate-fixed humeri were significantly greater than the nailed humeri in both the anteroposterior and mediolateral planes on a paired basis. The distal fin nail and solid locked nail had comparable bending properties, and both had bending properties superior to those measured for the paired flexible nails. The torsional properties of humeri fixed with plates and solid locked nails were equivalent, except for rigidity and stiffness, which were superior for the nail. Both fixation methods resulted in torsional properties significantly greater to those measured for humeri fixed with paired flexible nails or a distal fin nail.

Mechanical evaluation of a canine intervertebral disc spacer: in situ and in vivo studies.

An elastomeric intervertebral disc spacer with hydroxyapatite ingrowth surfaces was implanted in a canine model. We studied (a) the mechanical behavior of motion segments at time 0 and at 3, 6, and 12 months and (b) the effect of the interface between the spacer and vertebral bone on implant stability and bone ingrowth. A polymeric spacer was designed with compressive and torsional properties similar to those of the isolated canine lumbar disc. Implantation of the spacer in canine cadaver motion segments permitted in situ biomechanical evaluation at time 0. An in vivo study permitted continuous neurological monitoring of animals, with evaluation of mechanical behavior, stability, and ingrowth at 3, 6, and 12 months. Mechanical testing of cadaver motion segments with the spacer in situ resulted in decreased compressive and torsional stiffnesses, averaging 25 and 42%, respectively. This decrease was due to a combination of the surgical insult to the annulus and decortication of adjacent vertebral endplates. In the in vivo study, all 12 animals tolerated the surgery well and none had permanent neurological impairment. The measured parameters indicated that behavior of the spacer-motion segment composite appeared to return to normal within 3-6 months. However, despite use of a porous hydroxyapatite on the implant surface, there was no significant bone ingrowth. Instead, a layer of dense fibrous connective tissue was formed at the spacer-vertebral bone interface. Early migration of five of the 12 spacers resulted in eccentric loading patterns with consistent reactive osteophyte formation.

Mechanics of interbody spinal fusion. Analysis of critical bone graft area.

Bone graft subsidence is a serious complication of interbody spinal fusion. In this study, 66 mechanical tests were performed on 35 thoracic vertebral bodies to investigate the in situ mechanics of interbody spinal fusion. The relationships among trabecular bone density, bone strength, and size of bone graft area were analyzed. All vertebral bodies were scanned by quantitative computer tomography (QCT) to determine their bone density before mechanical testing. The decorticated trabecular beds of the vertebral bodies, void of all posterior elements, were loaded in a manner similar to that which occurs after surgical interbody fusion. That is, rectangular blocks of polymethylmethacrylate, representing bone grafts, were used to transfer controlled compressive loads to the decorticated vertebral trabecular surface. Both destructive and nondestructive tests were performed. The relationship between QCT bone density and trabecular bone strength was related by a power function, and, on average, the bone density and trabecular bone strength were 0.137 g/cm3 and 3.97 MPa, respectively. Eighty percent of the vertebral bodies with graft covering 25% of the total end plate area or less failed at loads less than 600 N, while 88% of the vertebral bodies with 30% or greater covered were able to carry a load greater than 600 N. The results suggest that the intrinsic behavior of trabecular bone loaded within the vertebral body is little different from the behavior of the whole body, that QCT bone density is indicative of bone strength, and that interbody graft area should be significantly greater than 30% of the total end plate area to provide a margin of safety.(ABSTRACT TRUNCATED AT 250 WORDS)