Fresh-frozen Bone Research Articles

Fresh-frozen human bone graft for repair of defect after adenomatoid odontogenic tumour removal

The aim of this paper was report the clinical, radiographic, and histological case of adenomatoid odontogenic tumour (AOT) in adolescent woman as well as present the reconstructive treatment of AOT using fresh-frozen human bone graft with guided bone regeneration. AOT is a benign, noninvasive lesion with slow but progressive growth. Biopsy and microscopic examination confirmed the presence of an AOT. Treatment was conservative and the prognosis was excellent. The patient has been followed-up for without recurrence. The use of fresh-frozen human bone graft can be a safe choice for reconstruction of the bone defects to treat AOT.

Overdentures on implants placed in bone augmented with fresh frozen bone

Introduction In the last decade several studies have been performed to evaluate the clinical outcome of one or two stage loaded implants supporting overdentures. Aim Since fresh frozen bone (FFB) has an ever-increasing number of clinical applications and few reports are available on implants inserted into FFB, we performed a retrospective study on fixtures inserted in FFB and bearing overdentures. Methods In the period between December 2003 and December 2006, 17 patients (14 females and 3 males with a median age of about 56 years) were grafted and 60 implants inserted thereafter. A total of 17 overdentures were delivered: 8 in the mandible and 9 in the maxilla. Multiple implant systems were used: 22 Double etched, 7 SLA, 9 Anodic oxidized, and 22 CaPo4 ceramic-blasted. Implant diameter ranged from 3.25 to 4.3 mm and length from 11.5 to 16.0 mm. Implants were inserted to replace 23 incisors, 9 cuspids, 20 premolars and 8 molars. Results No implants were lost (i.e. survival rate = 100%) and no differences were detected among the studied variables. Kaplan Meier algorithm and Cox regression did not reveal any statistical differences among the studied variables also as regards the success rate. Conclusion Implants inserted FFB and bearing overdentures have a high survival rate and success rates, which are comparable to those of implants inserted in non-grafted bone. FFB bone is a reliable material for alveolar ridge augmentation. No difference was detected among removable prostheses supported by 2 or more implants.

The effects of embalming using a 4% formalin solution on the compressive mechanical properties of human cortical bone

Background The use of formalin fixed bone tissue is often avoided because of its assumed influence on the mechanical properties of bone. Fixed bone tissue would minimise biological risks and eliminate preservation issues for long duration experimental tests. This study aimed to determine the short- and long-term effects of embalming, using a solution with 4% formalin concentration, on the mechanical properties of human cortical bone. Methods Three-millimetre cylindrical specimens of human cortical bone were extracted from two femoral diaphyses and divided in four groups. The first group was used as control, the remaining three groups were left in the embalming solution for 48 h, 4 week, and 8 week, respectively. Compressive mechanical properties, hardness and ash density were assessed. The last was used to check the homogeneity among the four groups. Findings No significant differences were found among the four groups in yield stress, ultimate stress and hardness. The specimens stored for 8 week in the embalming solution had significant lower Young’s modulus (−24%), higher yield strain (+20%) and ultimate strain (+53%) compared to the other groups. Interpretation On a short-term perspective, embalming did not affect the compressive mechanical properties, nor hardness of human cortical bone, whereas a long-term preservation (8 week) did significantly affect Young’s modulus, yield strain and ultimate strain in compression. Preserving bone segments for up to 4 week in an embalming solution with low formalin concentration seems to be an interesting alternative when collecting and/or managing fresh or fresh-frozen bone segments for biomechanical experiments is not possible.

DECREASING STRAINS AT THE IMPLANT-BONE INTERFACE: A NEW DESIGN TO REDUCE THE RISK OF PIN LOOSENING

Two autopsy retrievals of acetabular reconstruction with different bone graft substitutes were analyzed. One patient underwent surgery with hydroxyapatite (HA) granules, and the other with demineralized bone chips. Incorporation occurred with the biomaterial bone-graft substitute HA primarily in the superior part of the acetabulum, where the HA was in contact with host cortical bone. No bony union occurred with the demineralized bone chips because of motion and migration of the acetabular cup. No bone incorporation of either bone-graft substitute was observed in the medial wall defects in both patients. There were two important findings: (1) the 3- to 8-mm depth and 70% to 75% surface of union of the HA bone graft substitute to host cortical bone was the same as seen with morselized, fresh-frozen bone graft; and (2) union of graft material to host bone was dependent on secure fixation of the acetabular construct.

Pulse-lavage washing is an effective method for defatting of morselized allograft bone in the operating theater

Background and purpose Incorporation of fresh-frozen allograft bone and safety aspects associated with this procedure can be improved by removing blood and lipids from the bone. We investigated in a quantitative manner how efficient pulse lavage might be for removal of adipose tissue from morselized allograft bone.Methods Depending on the study, the washing was performed with an average of 0.8 L or 1.6 L of sterile saline at room temperature. Fat content of the morselized bone samples was determined using hexane elution. The efficiency of pulse lavage alone was compared with that after an additional wash in 12 L of warm water (55°C). Unprocessed controls were also included for comparison.Results Pulse lavage with 0.8 L saline alone removed 80% of the fat from the bone, whereas 95% of the fat was removed when washing was performed with 1.6 L of saline. The cleansing efficacy was improved further when an additional wash with warm water was used.Interpretation Our results indicate that pulse-lavage washing alone at room temperature is an effective method for defatting of morselized allograft bone, but an additional wash with warm water improves the cleansing efficiency. Pulse lavage is easily available and simple to use in the operating theater.

Arthroscopic Anterior Cruciate Ligament Reconstruction Using Fresh-Frozen Bone Plug–Free Allogeneic Tendons: 10-Year Follow-up

To evaluate the long-term outcomes following arthroscopic-assisted anterior cruciate ligament (ACL) reconstruction using fresh-frozen allogeneic tendon. Sixty-one athletically active patients (mean age at surgery, 20.9 years) who had arthroscopic-assisted ACL reconstruction using fresh-frozen free tendon allograft underwent physical examination, instrumented laxity measurement, radiographs, and thigh muscle strength at 10 to 14 years (mean, 11.5) postoperatively, and these data were compared with results of similar measurements obtained at 2 years postoperatively. Lachman test and pivot shift test were maintained as negative in 53 (87%) and 52 (85%) patients at follow-up, respectively. Quantitative measurements using the KT-2000 knee arthrometer showed 1.6 +/- 1.3 (mean +/- SD) mm in the side-to-side difference, and no more than 3 mm in 56 patients (92%) at final follow-up. All but one was assessed as normal or nearly normal by International Knee Documentation Committee score. Patient activity level was decreased at the long-term follow-up, but this change was mainly associated with changes in social context (e.g., graduation from school) rather than with knee limitations. None of the patients experienced deep infection or graft rejection. Degenerative joint disease on radiographs was seen in 13 out of 15 patients (87%) whose menisci had been excised, whereas it was observed in 12 out of 46 (26%) whose menisci had been preserved. A series of 61 patients with fresh-frozen bone plug-free tendon allografts using a 2-incision technique for arthroscopic ACL reconstruction results in long-term knee stabilization and functioning among young active individuals while simultaneously avoiding graft harvest site morbidity. Level IV, therapeutic case series.

Lipid oxidation may reduce the quality of a fresh-frozen bone allograft. Is the approved storage temperature too high?

Background International standards indicate that bone allografts for human use should be stored in a –40°C or colder environment and the storage time should be up to 5 years. Bone allografts contain lipids which oxidate and become toxic with time.Material and methods We determined lipid oxidation in femoral head bone allografts by headspace gas chromatography at different storage temperatures and storage times.Results We found that lipid oxidation in fresh-frozen bone allograft was influenced by storage temperature and storage time. Lipid oxidation was significantly more rapid at –30°C than at –70°C. Even at –70°C, however, some oxidation occurred but it was quite minimal after 3 years.Interpretation Because of the negative effects of lipid oxidation, we recommend a storage temperature of –70°C or lower for bone allografts.

Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections

To examine the stability of bone matrix proteins for crystal dislocation, the immunolocalization of type I collagen, bone sialoprotein, and osteopontin was investigated during different stages of fixation and decalcification. Four-week-old rat femurs were rapidly frozen, and were sectioned without fixation or decalcification. Thereafter, following or bypassing fixation in 4% paraformaldehyde, these sections were decalcified in 5% EDTA for 0-5 min. Before decalcification, marked radiopacity of bone matrix was observed in contact microradiography (CMR) images, and electron probe microanalysis (EPMA) demonstrated intense localization for phosphorus and calcium. In fixed and unfixed sections without decalcification, immunolocalization of bone matrix proteins were almost restricted to osteoid. After 1 min of decalcification, reduced radiopacity was apparent in the CMR images, and less phosphorus and calcium was observed by EPMA, which completely disappeared by 5 min decalcification. After 3-5 min of decalcification, unfixed sections showed that these proteins were immunolocalized in bone matrix, but were not detectable in osteoid. However, fixed sections demonstrated that these were found in both bone matrix and osteoid. The present findings suggest that bone matrix proteins are embedded in calcified matrix which is separated from the aqueous environment and that they hardly move, probably due to firm bonding with each other. In contrast, matrix proteins in osteoid are subject to loss after decalcification because they may be bound to scattered apatite crystals, not to each other.

No effect of platelet-rich plasma with frozen or processed bone allograft around noncemented implants

We compared processed morselized bone allograft with fresh-frozen bone graft around noncemented titanium implants. Also, the influence of platelet-rich plasma (PRP) in combination with bone allograft was evaluated. Analysis was based on implant fixation and histomorphometry. PRP was prepared by isolating the buffy coat from autologous blood samples. Bone allograft was used fresh-frozen or processed by defatting, freeze drying, and irradiation. Cylindrical hydroxyapatite-coated titanium implants were inserted bilaterally in the femoral condyles of eight dogs. Each implant was surrounded by a 2.5-mm concentric gap, which was filled randomly according to the four treatment groups--group 1: fresh-frozen bone allograft; group 2: processed bone allograft; group 3: fresh-frozen bone allograft + PRP; group 4: processed bone allograft + PRP. Histological and mechanical evaluation demonstrated no influence of bone allograft processing. Even though the level of platelet in PRP was 7.7 times that found in whole blood, we found no improvement of bone formation or implant fixation by adding PRP.

The influence of bone allograft processing on osteoblast attachment and function

In order to assess the influence of eight different sterilisation and disinfection methods for bone allografts on adhesion, proliferation, and differentiation of human bone marrow stromal cells (BMSC), cells were grown in culture and then plated onto pieces of human bone allografts. Following processing methods were tested: autoclavation (AUT), low-temperature-plasma sterilisation of demineralised allografts (D-LTP), ethylene oxide sterilisation (EtO), fresh frozen bone (FFB), 80 °C-thermodisinfection (80°C), γ-irradiation (Gamma), chemical solvent disinfection (CSD), and Barrycidal ®-disinfection (BAR). The seeding efficiency was determined after one hour to detect the number of attached cells before mitosis started. The cell viability was determined after 3, 7, and 21 days. Tests to confirm the osteoblastic differentiation included histochemical alkaline phosphatase staining and RT-PCR for osteocalcin. Human BMSC showed greatest attachment affinities for D-LTP-, 80°C-, and CSD-allografts, whereas less cells were found attached to AUT-, EtO-, FFB-, Gamma-, and BAR-probes. Cell viability assays at day 3 revealed highest proliferation rates within the FFB- and 80°C-groups, whereas after 21 days most viable cells were found in D-LTP-, 80°C-, CSD-, and Gamma-groups. BAR-treatment showed a considerably toxic effect and therefore was excluded from all further experiments. Highest AP-activity and gene expression of osteocalcin were detected in the D-LTP-group in comparison with all other groups. In summary, our results demonstrate that cell adhesion, final population, and function of BMSC are influenced by different disinfection and sterilisation methods. Therefore, processing-related alterations of BMSC-function may be important for the success of bone grafting. The experimental setup used in the present work may be useful for further optimisation of bone allograft processing.

Assessment of bone graft substitutes in autopsy implant retrievals of the acetabulum in total hip arthroplasty

Two autopsy retrievals of acetabular reconstruction with different bone graft substitutes were analyzed. One patient underwent surgery with hydroxyapatite (HA) granules, and the other with demineralized bone chips. Incorporation occurred with the biomaterial bone-graft substitute HA primarily in the superior part of the acetabulum, where the HA was in contact with host cortical bone. No bony union occurred with the demineralized bone chips because of motion and migration of the acetabular cup. No bone incorporation of either bone-graft substitute was observed in the medial wall defects in both patients. There were two important findings: (1) the 3- to 8-mm depth and 70% to 75% surface of union of the HA bone graft substitute to host cortical bone was the same as seen with morselized, fresh-frozen bone graft; and (2) union of graft material to host bone was dependent on secure fixation of the acetabular construct.

Impaction bone grafting with freeze-dried irradiated bone. Part I. Femoral implant stability: cadaver experiments in a hip simulator.

Processed freeze-dried irradiated allografts seem to be used less than instead of fresh-frozen allografts for impaction bone grafting in revision hip arthroplasties. Although biologically acceptable, their use is discouraged because of their questionable mechanical properties following freeze-drying and irradiation procedures. To address this question, we impacted freeze-dried grafts in 6 cadaveric femurs and loaded with a cemented Charnley prosthesis. The routinely used fresh-frozen allografts were used as controls in the contralateral side. These constructs were compared simultaneously in a walking hip simulator for their stability during 900,000 loading cycles. The mechanical parameters were axial inducible displacement and subsidence of the implant. The former parameter was lower in the implant mounted on freeze-dried impacted grafts than that mounted on the fresh-frozen bone. The latter parameter was also lower in the freeze-dried group. At the end of the test, we found no implant loosening in either group and their 'pull out' resulted in cement-prosthesis debonding, which showed the mechanical integrity of the impacted grafts. Freeze-dried grafts provide more stable fixation of the stem than fresh-frozen morselized grafts, when tested in a hip simulator.

Experimental and Clinical Motion Analysis of the Bipolar Hip Prosthesis

To determine the motion distribution of bipolar hip prosthesis, we performed experimental and clinical analyses. Materials and Methods: In the experimental studies, component motion was examined according to surface lubrication, cup angle, and acetabular reaming in fresh-frozen cadaveric bone. In the clinical examination, thirty patients with a bipolar hip prosthesis were examined by fluoroscopy. Results: In the experimental condition when both joints were well lubricated, motion occurred mainly at the inner joint. However, slight motion of the outer joint appeared in terminal abduction if the acetabular cup angle was low and joint pressure was applied. When the only the outer joint was lubricated, motion occurred mostly at the outer joint. When both joints were not lubricated, motion occurred at outer joint unless the acetabular reaming was performed. In every situation having acetabular reaming, motion occurred only at the inner joint. Clinically, motion distribution varied between patients. However, we found a predominant contribution was made by the outer joint, in contrast to theoretical prediction, and the persistence of the combined motion of two joints in the majority of the patients studied (67-70 percent). Conclusion: We found that the motion was combined in both outer and inner joint in two-thirds of patients, and that outer motion was more predominated.

Separation of the Raman Spectral Signatures of Bioapatite and Collagen in Compact Mouse Bone Bleached with Hydrogen Peroxide

We studied the effectiveness of hydrogen peroxide in removing visible laser-induced photoluminescence from the Raman spectra of compact bone of mice. In testing various bone sample preparations, we found that hydrogen peroxide bleaching was most effective when applied directly to fresh or fresh-frozen bone samples. The extent of the reduction in photoluminescence in the peroxide-bleached bone was such that the Raman spectrum could be readily recorded with 532-nm laser excitation. A comparison of bone samples before and after hydrogen peroxide bleaching shows that the Raman shifts of all the collagen and bioapatite bands are unaffected by the peroxide bleach. Moreover, the low spectral backgrounds of the peroxide-treated bone samples permit the Raman spectra of these two major components of bone to be fully separated. The Raman spectrum of collagen-rich periosteum was subtracted from the Raman spectrum of compact bone, which isolated the Raman spectral signature of the bioapatite fraction of the bone. This derived spectrum of bioapatite was then used, through spectral subtraction, to generate the spectral signature of the collagen component of the bone. All the major and minor Raman bands of collagen and bioapatite can be identified in these separate spectra.

The Nucleus Contour electrode array: a radiological and histological study.

To evaluate the handling and insertion trauma of the recently developed Nucleus perimodiolar Contour electrode array (Cochlear Ltd., Pty, Lane Cove, New South Wales, Australia) in human temporal bones compared with the Nucleus standard straight electrode array. E-perimental control group. Twenty-nine fresh-frozen bones were implanted with different electrode arrays by an experienced cochlear implant surgeon, and evaluated both radiologically and histologically. Intracochlear insertion of the standard Nucleus straight electrode array was found to be atraumatic, confirming previous findings in the literature. Insertion of the Nucleus Contour electrode array resulted in instances of localized basilar membrane penetration causing the electrode array to move from the scala tympani into the scala vestibuli. However, this trauma did not result in any observable damage to the osseous spiral lamina or the modiolus. Basilar membrane penetration was observed in six of eight cochlear bones when a standard cochleostomy size (approximately 0.8 mm) and site (anterior and superior to the round window) were used. However, when the surgical technique was modified to use a slightly larger cochleostomy ( approximately 1.8 mm) situated closer to the round window and employ a partial stylet withdrawal technique during electrode insertion, the frequency of penetrations was restricted to two of seven bones. This trauma rate is comparable to that observed with other cochlear implants designs. Following our results, the design of the Nucleus Contour electrode appears to fulfill the safety requirements for an intracochlear electrode array, provided that the surgical insertion technique is modified in the manner outlined.

Novel intracochlear electrode positioner: effects on electrode position.

Spiral ganglion cells, located in the cochlear modiolus, are widely thought to be the neural elements stimulated by cochlear implants (CIs). Positioning CI electrodes adjacent to the modiolus may allow delivery of more focused, discrete fields of electrical stimulation, reducing both current requirements and the channel interaction associated with simultaneous stimulation. In order to provide greater consistency in positioning CI electrodes against the modiolus, Advanced Bionics Corporation has designed a silicone intracochlear positioner that is inserted alongside the electrode. Placed against the outer wall of the scala tympani, it directs the electrode array toward the modiolus. Five fresh-frozen temporal bones were studied to validate enhanced electrode placement. Electrode position was documented with image-enhanced digital fluoroscopy, and the distance to the modiolus was measured with computer-assisted morphometrics. The electrode positioner was highly effective in positioning the electrodes closer to the modiolus (p < .001).

Investigating severe neutropenia with a simple bone marrow immunofluorescence test.

Severely neutropenic patients who are suspected of having circulating autoantibodies to neutrophils and/or myeloid precursors present a diagnostic problem. This report describes the use of a simple bone marrow immunofluorescence test (BMIFT) to demonstrate autoantibodies reactive with the patient's own bands, neutrophils and myeloid precursors represented on fresh-frozen bone marrow aspirate smears. Positive results were seen in two of 13 evaluable marrows from children with primary neutropenia and in eight of 44 evaluable marrows from adults with primary or secondary neutropenia. The BMIFT is simple enough to perform in most laboratory environments and provides serological information to support a diagnosis of immune neutropenia when other means of investigation are precluded.

Comparative Biomechanical Stability of Titanium Bone Fixation Systems in Metacarpal Fractures

The biomechanical properties of apex bending and torsional rigidity of 11 different titanium mini- and microplates (Leibinger and Synthes) were examined to evaluate the effects of plate design and thickness and screw size and design. Two hundred sixteen fresh-frozen human cadaveric metacarpal bones underwent a midshaft transverse osteotomy followed by application of one of the following plates: Synthes 1.5-mm and 2.0-mm five-hole linear plates; Leibinger linear 1.2-mm 5-hole, 1.7-mm 4-hole, 2.3-mm 4-hole plates; or Leibinger three-dimensional 1.2-mm 4-hole, 1.2-mm 8-hole, 1.7-mm 4-hole, 1.7-mm 8-hole, 2.3-mm 4-hole, and 2.3-mm 8-hole plates. The specimens were subjected to a three-point bending test with apex dorsal or apex volar loading or torsional loading. Analysis of variance statistical analysis revealed that increasing plate thickness and, more significantly, three-dimensional design were associated with increased rigidity.

The use of fresh frozen allogeneic bone for maxillary and mandibular reconstruction

The use of fresh frozen bone (FFB) alone, or in combination with autogenous bone (AB), for bony augmentation of the maxilla and mandible in preparation for dental reconstruction with endosseous implants has been studied. Ten patients received FFB±AB for augmentation of a severely atrophic mandible ( n=6) or for reconstruction of a jaw defect secondary to trauma or tumor resection ( n = 4). Average follow-up was 26.3 ± 5.4 months. At the time of implant placement, the bone grafts were found to be firm in consistency, well incorporated, and well vascularized in all 10 patients. Twenty-nine endosseous implants were placed an average of 8.3±3.1 months following bone grafting. One implant failed and was replaced, and one implant remains buried as a nonfunctional unit. All patients have been restored prosthetically by means of 28 of the 29 implants. This preliminary study indicates that FFB may be used alone or in combination with autogenous bone for augmentation or reconstruction of the atrophic maxilla and mandible. The resultant ridge is adequate to support loaded endosseous implants. A potential disadvantage is the minimal risk of disease transmission.

Ossification of decalcified and nondecalcified homologous bone grafts.

This two-year study examined the process of reossification and survival of decalcified and nondecalcified bone grafts implanted under the scalp tissue of 15 rabbits. Both radiologic and histologic examination showed that the nondecalcified fresh-frozen bone specimens became totally viable and recalcified while largely retaining their initial size. The decalcified bones, however, were totally absorbed within a period of four months. Possible reasons for failure of the decalcified bone to reossify are discussed.