Potential sexual transmission of Trypanosoma cruzi in mice.

Infection with the protozoan parasite Trypanosoma cruzi, the etiologic agent of human Chagas disease, results in life-long infection. Infective trypomastigotes circulate in the bloodstream and have the capacity to infect any cell type, including reproductive tissue. This study sought to assess the potential for sexual transmission of T. cruzi in an experimental mouse model. The conditions used in this study, in which acutely infected males and immunosuppressing the females, created a worst-case scenario allowing for the greatest chance of measuring transmission through intercourse. Male BALB/c mice were infected and mated with uninfected females, and the females were subsequently examined for T. cruzi tissue parasitism. A single transmission event of 61 total matings was observed, indicating a low but non-zero risk potential for male-to-female sexual transmission of T. cruzi.

Biomechanical properties and histology of db/db diabetic mouse Achilles tendon.

Foot ulcers are a severe complication of diabetic patients resulting from nerve and tendon pathologic alterations. In diabetic patients the tendons are thicker, shorter and have increased stiffness. We examined C57BL/KsJ (BKS.Cg-Dock7(m) +/+ Lepr (db) /J) (db/db) mice tendons to determine whether they are an animal model for human diabetic tendon changes. We hypothesized that the Achilles tendons of db/db diabetic mice would be thicker, stiffer, fail at lower loads and stresses, and have degenerative changes compared to control mice. Biomechanical and histologic analyses of the Achilles tendons of 16 week old db/db and control male mice were performed. There was a significant increase in tendon diameter and significant decreases in maximum load, tensile stress, stiffness and elastic modulus in tendons from diabetic mice compared to controls. Mild degenerative and neutrophil infiltration was observed near the tendon insertions on the calcaneous in 25% of db/db mice. In summary, hyper-glycemia and obesity lead to severe changes in db/db mice will be a useful model to examine mechanisms for tendon alterations.

Soft-tissue allografts terminally sterilized with an electron beam are biomechanically equivalent to aseptic, nonsterilized tendons.

BACKGROUND: Allograft safety is contingent on effective sterilization. However, current sterilization methods have been associated with decreased biomechanical strength and higher failure rates of soft-tissue allografts. In this study, electron beam (e-beam) sterilization was explored as an alternative sterilization method to preserve biomechanical integrity. We hypothesized that e-beam sterilization would not significantly alter the biomechanical properties of tendon allograft compared with aseptic, nonsterilized controls and gamma-irradiated grafts. METHODS: Separate sets of forty fresh-frozen tibialis tendon allografts (four from each of ten donors) and forty bisected bone-patellar tendon-bone (BTB) allografts (four from each of ten donors) were randomly assigned to four study groups. One group received a 17.1 to 21.0-kGy gamma radiation dose; two other groups were sterilized with an e-beam at either a high (17.1 to 21.0-kGy) or low (9.2 to 12.2-kGy) dose. A fourth group served as nonsterilized controls. Each graft was cyclically loaded to 200 N of tension for 2000 cycles at a frequency of 2 Hz, allowed to relax for five minutes, and then tested in tension until failure at a 100%/sec strain rate. One-way analysis of variance testing was used to identify significant differences. RESULTS: Tibialis tendons sterilized with both e-beam treatments and with gamma irradiation exhibited values for cyclic tendon elongation, maximum load, maximum displacement, stiffness, maximum stress, maximum strain, and elastic modulus that were not significantly different from those of nonsterilized controls. BTB allografts sterilized with the high e-beam dose and with gamma irradiation were not significantly different in cyclic tendon elongation, maximum load, maximum displacement, stiffness, maximum stress, maximum strain, and elastic modulus from nonsterilized controls. BTB allografts sterilized with the e-beam at the lower dose were significantly less stiff than nonsterilized controls (p = 0.014) but did not differ from controls in any other properties. The difference in stiffness likely resulted from variations in tendon size rather than the treatments, as the elastic moduli of the groups were similar. CONCLUSIONS: The biomechanical properties of tibialis and BTB allografts sterilized with use of an e-beam at a dose range of 17.1 to 21.0 kGy were not different from those of aseptic, nonsterilized controls or gamma-irradiated allografts. CLINICAL RELEVANCE: E-beam sterilization can be a viable method to produce safe and biomechanically uncompromised soft-tissue allografts.

Repeated freeze-thaw cycles do not alter the biomechanical properties of fibular allograft bone.

BACKGROUND: Allograft tissues can undergo several freeze-thaw cycles between donor tissue recovery and final use by surgeons. However, there are currently no standards indicating the number of reasonable freeze-thaw cycles for allograft bone and it is unclear how much a graft may be degraded with multiple cycles. QUESTIONS/PURPOSES: We therefore asked whether (1) the mechanical properties of fibular allograft bone would remain unchanged with increasing numbers of freeze-thaw cycles and (2) histologic alterations from increased numbers of freeze-thaw cycles would correspond to any mechanical changes. METHODS: Fibular allograft segments were subjected to two, four, and eight freeze-thaw cycles and compared biomechanically and histologically with a control group (one freeze-thaw cycle). Two freeze-dried treatments, one after being subjected to one freeze-thaw cycle and the other after being subjected to three freeze-thaw cycles, also were compared with the control group. RESULTS: For all segments, the average ultimate stress was 174 MPa, average modulus was 289 MPa, average energy was 2.00 J, and the average stiffness was 1320 N/mm. The material properties of the freeze-thaw treatment groups were similar to those of the control group: ultimate stress and modulus were a maximum of 16% and 70% different, respectively. Both freeze-dried treatments showed increased stiffness (maximum 53% ± 71%) and energy to failure (maximum 117% ± 137%) but did not exhibit morphologic differences. There were no alterations in the histologic appearance of the bone sections in any group. CONCLUSIONS: Fibular allograft segments can be refrozen safely up to eight times without affecting the biomechanical or morphologic properties. Freeze-dried treatments require further study to determine whether the detected differences are caused by the processing. CLINICAL RELEVANCE: Cryopreserved cortical allografts are thawed by surgeons in preparation for procedures and then occasionally discarded when not used. Refreezing allograft tissues can result in a cost savings because of a reduction in wasted graft material.

Demineralization removes residual alendronate in allograft bone procured from donors with a history of bisphosphonate use.

BACKGROUND: Bisphosphonate-associated osteonecrosis (BON) of the jaw is a growing concern in the dental community, but the possible presence of residual bisphosphonates in demineralized allograft bone from bisphosphonate-using tissue donors and the clinical implications of using such bone are unclear. The objectives of this study are to determine whether alendronate remained in demineralized bone matrix (DBM) procured from donors with a documented history of oral bisphosphonate use and to examine whether the demineralization process removes alendronate from allograft bone. METHODS: A gas chromatography?mass spectrometry method was developed and validated to quantify residual alendronate in allograft bone. Alendronate levels in DBM procured from tissue donors with a history of oral bisphosphonate use were compared to alendronate levels in DBM procured from donors without a history of bisphosphonate use. In addition, mineralized and demineralized bone was soaked in alendronate at concentrations of 0.002, 2.0, and 2,000 ng/mg bone and analyzed to examine the effect of the demineralization process. RESULTS: Residual alendronate was not detected in the DBM from either group, nor was it detected in any of the DBM samples soaked in alendronate solutions. Soaked mineralized bone contained measureable alendronate, but the substance was removed by demineralization. CONCLUSIONS: The demineralization process effectively removed residual alendronate from allograft bone. These results may relieve anxieties regarding the use of DBM in dental patients because it is unlikely to trigger BON of the jaw.

Osteoinductivity of demineralized bone matrix is independent of donor bisphosphonate use.

BACKGROUND: Demineralized bone matrix is commonly used as a bone graft substitute, either alone or to supplement an osteoconductive material, because of its osteoinductive properties. The aging of the population has led to an increase in the number of prospective donors of demineralized bone matrix who have taken bisphosphonates to prevent osteoclast-mediated bone resorption. The aim of this study was to determine whether oral bisphosphonate usage affects the osteoinductivity of demineralized bone matrix from donors. METHODS: Sex-matched and age-matched pairs of samples were provided by four tissue banks (three or four pairs per bank). Demineralized bone matrix donors without bisphosphonate treatment had a mean age (and standard deviation) of 69.1 ± 2.5 years, and donors with bisphosphonate treatment had a mean age of 68.9 ± 2.0 years. Each pair included one donor known to have taken bisphosphonates and one who had not taken bisphosphonates. Demineralized bone matrix previously confirmed as osteoinductive was the positive control, and heat-inactivated demineralized bone matrix was the negative control. Demineralized bone matrix incubated with 1 mL of phosphate-buffered saline solution containing 0, 0.002, 2.0, or 2000 ng/mL of alendronate was also tested. Gelatin capsules containing 15 mg of demineralized bone matrix were implanted bilaterally in the gastrocnemius muscle of male nude mice (eight implants per group). The mice were killed thirty-five days after implantation, and hind limbs were recovered and processed for histological analysis. Osteoinductivity was measured with use of a qualitative score and by histomorphometry. RESULTS: Nine of fifteen samples from donors who had had bisphosphonate treatment and ten of fifteen samples from patients who had not had bisphosphonate treatment were osteoinductive. Qualitative mean scores were comparable (1.7 ± 0.4 for those without bisphosphonates and 1.9 ± 0.7 for those with bisphosphonates). Osteoinductive demineralized bone matrix samples produced ossicles of comparable size, regardless of bisphosphonate usage. Histomorphometric measurements of the area of new bone formation and residual demineralized bone matrix were also comparable. The addition of alendronate to control demineralized bone matrix did not affect its osteoinductivity. CONCLUSIONS: Demineralized bone matrix samples from donors treated with bisphosphonates and donors not treated with bisphosphonates have the same ability to induce bone formation. However, it is not known if the quality of the new bone is affected, with subsequent consequences affecting bone remodeling.

Biomechanical and biologic effects of meniscus stabilization using triglycidyl amine.

The susceptibility of meniscus allografts to enzymatic degradation may be reduced through tissue stabilization. We have previously reported on an epoxide-based crosslinker, triglycidyl amine (TGA), which can be used alone or with a bisphosphonate (MABP) to stabilize heterograft heart valves and reduce their pathologic calcification. Our objective was to evaluate the effects of TGA and TGA-MABP pretreatment on an orthopedic allograft involving meniscus crosslinking, degradation, calcification, and compressive properties. Ovine menisci treated with TGA or TGA-MABP for up to seven days and glutaraldehyde crosslinked controls were examined in vitro for degree of crosslinking, resistance to degradation by collagenase, and material property changes. Likewise treated menisci were implanted in rats for eight weeks and examined for calcium content and biomechanical changes. TGA treatment for three days significantly reduced collagen loss by 88% and increased thermal denaturation temperatures (Ts) above 80 degrees C versus Ts of 70 degrees C or less for non-crosslinked meniscus. In vitro, TGA and TGA-MABP significantly increased aggregate modulus by 19% and 32% compared to native controls, respectively. TGA decreased permeability by 53% while TGA-MABP increased it by 303%. In vivo, TGA significantly reduced explant calcification by 42% compared to glutaraldehyde, and including MABP reduced it by 90%. Analyses revealed that TGA and TGA-MABP stabilized menisci had significantly lower modulus and permeability values than glutaraldehyde controls by at least 28% and 86%, respectively. It is concluded that TGA crosslinking of meniscus increases resistance to both collagenase degradation and pathologic calcification, while demonstrating comparable or improved biomechanical properties versus glutaraldehyde controls.

Human peritoneal membrane reduces the formation of intra-abdominal adhesions in ventral hernia repair: experimental study in a chronic hernia rat model.

BACKGROUND: Adhesions leading to intestinal obstructions and fistulae are severe complications related to the intraperitoneal placement of synthetic meshes. This study evaluated the efficacy of human peritoneal membrane (HPM) in a chronic hernia repair rat model as an anti-adhesive solution for preventing the development of intra-abdominal adhesions. MATERIALS AND METHODS: The mechanical properties of HPM and human fascia lata (HFL) were evaluated prior to in vivo implantation. Twenty rats underwent midline laparotomy, which led to the development of chronic hernias 28 d later. Then, animals underwent incisional hernia repair in an underlay fashion (n=5/mesh group) with compressed poly(tetra-fluoro-ethylene) (cPTFE), onto which HPM or HFL were affixed pre-repair, along with two additional controls. The extent and tenacity of intra-abdominal adhesions were determined through qualitative gross evaluations and quantitative tensiometry at 30 d post-repair. The host tissue response was evaluated histologically. RESULTS: In hydrated state, the elastic properties of HPM were superior to HFL. Repairs with HPM had significantly less surface area covered by adhesions, with significantly lower tenacity compared with all other groups. Furthermore, intra-abdominal adhesions developed in the presence of HPM were associated with omentum only, and were distributed around the perimeter of the exposed cPTFE. HPM served as an active tissue remodeling template, replacing the traditional foreign body encapsulation with an anatomically and physiologically superior outcome. CONCLUSIONS: HPM significantly reduces the extent and tenacity of intra-abdominal adhesion formation, and represents a bioprosthetic template that encourages structural and functional neo peritonealization.

Human peritoneal membrane controls adhesion formation and host tissue response following intra-abdominal placement in a porcine model.

BACKGROUND: Even with the advent of bioresorbable barriers, complications due to visceral adhesions following surgery continue to occur. The use of a homologous adhesive barrier such as human peritoneal membrane (HPM) could prevent adhesions formation and enhance wound healing. This study evaluates HPM as an effective adhesive barrier in a porcine model simulating a ventral hernia procedure. MATERIALS AND METHODS: Through a midline laparotomy, meshes (10 cmx10 cm) were sewn onto the intact peritoneum of a pig, on each side of a midline incision in superior and inferior positions (4 randomized meshes/pig, n=9 pigs). The pigs were survived for 90 d. The meshes used were: HPM, compressed polytetrafluoro-ethylene (cPTFE), cPTFE+HPM, and polyester-collagen composite (PX). Exploratory laparoscopy was performed at 30 and 90 d to evaluate the extent of visceral adhesions. At necropsy, the extent and tenacity of visceral adhesions as well as material-abdominal wall integration were evaluated. Finally, host tissue response was assessed through scoring of inflammation, foreign body reaction, and mesothelialization. RESULTS: HPM and PX led to the least extent and tenacity of visceral adhesions compared to cPTFE and cPTFE+HPM, but integrated less strongly within the adjacent abdominal wall. PX displayed the most robust foreign body reaction among all prosthetic materials, while HPM scored similarly to the native peritoneum. The extent of mesothelialization was similar throughout the materials tested. CONCLUSIONS: The HPM barrier which promotes long-term peritoneal remodeling could diminish postsurgical intraperitoneal adhesions following hernia repair.

Tensile stimulation of murine stem cell-collagen sponge constructs increases collagen type I gene expression and linear stiffness.

The objectives of this study were to determine how tensile stimulation delivered up to 14 days in culture influenced type I collagen gene expression in stem cells cultured in collagen sponges, and to establish if gene expression, measured using a fluorescence method, correlates with an established method, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Using a novel model system, mesenchymal stem cells were harvested from six double transgenic mice in which the type I and type II collagen promoters were linked to green fluorescent protein-topaz and enhanced cyan fluorescent protein, respectively. Tissue-engineered constructs were created by seeding 0.5 x 10(6) mesenchymal stem cells onto type I collagen sponge scaffolds in a silicone dish. Constructs were then transferred to a custom pneumatic mechanical stimulation system housed in a standard incubator and stimulated for 5 h=day in tension for either 7 or 14 days using a repeated profile (2.4% peak strain for 20 s at 1 Hz followed by a rest period at 0% strain for 100 s). Control specimens were exposed to identical culture conditions but without mechanical stimulation. At three time points (0, 7, and 14 days), constructs were then prepared for evaluation of gene expression using fluorescence analysis and qRT-PCR, and the remaining constructs were failed in tension. Both analytical methods showed that constructs stimulated for 7 and 14 days showed significantly higher collagen type I gene expression than nonstimulated controls at the same time interval. Gene expression measured using qRT-PCR and fluorescence analysis was positively correlated (r = 0.9). Linear stiffness of stimulated constructs was significantly higher at both 7 and 14 days than that of nonstimulated controls at the same time intervals. Linear stiffness of the stimulated constructs at day 14 was significantly different from that of day 7. Future studies will vary the mechanical signal to optimize type I collagen gene expression to improve construct biomechanics and in vivo tendon repair.

Using functional tissue engineering and bioreactors to mechanically stimulate tissue-engineered constructs.

Bioreactors precondition tissue-engineered constructs (TECs) to improve integrity and hopefully repair. In this paper, we use functional tissue engineering to suggest criteria for preconditioning TECs. Bioreactors should (1) control environment during mechanical stimulation; (2) stimulate multiple constructs with identical or individual waveforms; (3) deliver precise displacements, including those that mimic in vivo activities of daily living (ADLs); and (4) adjust displacement patterns based on reaction loads and biological activity. We apply these criteria to three bioreactors. We have placed a pneumatic stimulator in a conventional incubator and stretched four constructs in each of five silicone dishes. We have also programmed displacement-limited stimuli that replicate frequencies and peak in vivo patellar tendon (PT) strains. Cellular activity can be monitored from spent media. However, our design prevents direct TEC force measurement. We have improved TEC stiffness as well as PT repair stiffness and shown correlations between the two. We have also designed an incubator to fit within each of two electromagnetic stimulators. Each incubator provides cell viability like a commercial incubator. Multiple constructs are stimulated with precise displacements that can mimic ADL strain patterns and record individual forces. Future bioreactors could be further improved by controlling and measuring TEC displacements and forces to create more functional tissues for surgeons and their patients.

Meniscal material properties are minimally affected by matrix stabilization using glutaraldehyde and glycation with ribose.

Knee meniscus replacement holds promise, but current allografts are susceptible to biodegradation. Matrix stabilization with glutaraldehyde, a crosslinking agent used clinically to fabricate cardiovascular bioprostheses, or with glycation, a process of crosslinking collagen with sugars such as ribose, is a potential means of rendering tissue resistant to such degradation. However, stabilization should not significantly alter meniscal material properties, which could disturb normal function in the knee. Our objective was to evaluate the effects of glutaraldehyde- and glycation-induced matrix stabilization on the material properties of porcine meniscus. Normal untreated meniscus specimens were tested in confined compression at one of three applied stresses (0.069, 0.208, 0.347 MPa), subjected to either a glutaraldehyde or glycation stabilization treatment, and then re-tested to measure changes in tissue aggregate modulus, permeability, and compressive strain at equilibrium. Changes in these properties significantly increased with glutaraldehyde concentration and exposure time to ribose. One glutaraldehyde and three glycation treatments did not alter aggregate modulus or compressive strain at equilibrium compared to controls (p > 0.10). However, all treatments increased permeability by at least 108% compared to controls (p < 0.001). This study reveals a dose-dependent relationship between meniscal material properties and certain stabilization conditions and identifies treatments that minimally affect these properties. Further research is necessary to determine whether these treatments prevent enzymatic degradation before and after surgical implantation in the knee.

Structural and mechanical properties of the glenohumeral joint posterior capsule.

The purpose of this study was to quantify regional variations in material properties of the glenohumeral joint posterior capsule and to compare these data with the anterior band of the inferior glenohumeral ligament (AB-IGHL). Mechanical properties were determined for individual bands of the AB-IGHL, superior posterior capsule (SUP-PC), middle posterior capsule (MID-PC), and inferior posterior capsule (INF-PC). Significant differences in tissue thickness were found among the 3 posterior capsular regions and the AB-IGHL. The AB-IGHL was thicker than the MID-PC (P=.03) and INF-PC (P=.01), and the SUP-PC was thicker than the INF-PC (P=.02). Except for significant differences in failure strains, material properties were not significantly different among the 4 tissue regions. There were no significant differences between tissue bands in modulus (P=.2), maximum stress (P=.46), or strain energy density (P=.62). Specimens failed primarily near the glenoid insertion (75%), with 4 specimens failing at the humeral insertion and 2 others failing in the tissue's mid substance.

Functional tissue engineering parameters toward designing repair and replacement strategies.

Abnormal joint kinematics and loads induced after soft tissue injuries are assumed to contribute to long-term degenerative joint disease and osteoarthritis. Controlling abnormal kinematics after repair and reconstruction of these injured structures would seem to be important for limiting wear of the articular cartilage surfaces. In this paper, we propose to expand the paradigm of functional tissue engineering to more fully characterize normal joint function and to establish design parameters for soft tissue repair and reconstruction to ultimately protect joint surfaces after surgery. Structure-function relationships are examined for tissues of increasing complexity, from tendons to menisci. Emphasis is placed on understanding normal in vivo function of tissues by conducting biomechanical experiments in vitro that better mimic in vivo conditions. This process yields nine classes of functional tissue engineering parameters: differential fiber length, in vivo force and displacement, variations in relative attachment site locations, loading from adjacent structures, fiber interactions, types of insertion, regional variations in material properties, nonparallel fiber orientations, and complex loading within the structure. These functional tissue engineering parameters are useful not only for understanding the function of normal tissues but for more effectively designing their repair and replacement. This paper concludes with a discussion of research directions that investigators might take to establish tissue-specific functional tissue engineering parameters for improving joint function and reducing articular surface degradation and osteoarthritis.

Effects of matrix stabilization when using glutaraldehyde on the material properties of porcine meniscus.

Meniscus transplantation frequently is one of the only options available for treating symptomatic younger patients with tibiofemoral pain and early arthrosis after a prior meniscectomy. However, clinical results indicate that current meniscal allografts may undergo degenerative changes due to enzymatic degradation during the remodeling phase. The objective of this study was to evaluate the effects of glutaraldehyde-induced matrix stabilization on the material properties of porcine meniscus prior to surgical implantation. Protocols for fabricating heart-valve replacements were examined, followed by an exploration of the effects of reducing glutaraldehyde concentration and exposure time. Cylindrical meniscus specimens were tested in uniaxial confined compression under a 0.196 MPa compressive stress, and aggregate modulus (H(A)), permeability (k), and compressive strains at equilibrium (epsilon(eq)) were calculated from the creep response. Compared to controls, the mean values for H(A) and k increased, on average, by 213 and 709%, respectively, and epsilon(eq) decreased by 57% for all "heart-valve" treatments. Reducing tissue exposure time to glutaraldehyde had little effect, but decreasing glutaraldehyde concentration to 0.02% resulted in tissues with material properties no different from the untreated controls. We conclude that minimal concentrations of glutaraldehyde (less than 0.2%) should be used in future studies to preserve normal meniscus properties.