Solid Digestion of Demineralized Bone as a Method To Access Potentially Insoluble Proteins and Post-Translational Modifications.

Bone proteomics is an expanding field for understanding protein changes associated with disease as well as characterizing and detecting proteins preserved in fossil bone. Most previous studies have utilized a protocol with demineralization and extraction approach to isolate and characterize proteins from bone. Through near-complete EDTA demineralization, followed by solid digestion of the remaining bone pseudomorph, a total of 92 protein accessions were detected from dog bone. In the EDTA, 14 unique proteins were found, including osteocalcin, an important bone protein. Osteocalcin was not found in the solid digestion samples, demonstrating the importance of examining the demineralization supernatant. The solid-digestion samples were analyzed both with (11 unique accessions) and without (16 unique accessions) alkylation, resulting in a total of 78 protein accessions. In addition to the diversity of proteins detected, various post-translational modifications were observed, including phosphorylation and glycosylation. The solid-digestion approach will allow for characterization of proteins that are insoluble and would otherwise be missed by traditional bone protein extraction alone. All data are available at ftp://massive.ucsd.edu/MSV000081399 .

The fusion rate of demineralized bone matrix compared with autogenous iliac bone graft for long multi-segment posterolateral spinal fusion.

BACKGROUND: Although autogenous iliac bone graft (AIBG) remains the gold standard for spine fusion, harvesting morbidity has prompted the search for alternatives especially for multi-segment fusion. This study aimed to evaluate the efficacy of using demineralized bone matrix (DBM) as a substitute of AIBG for long instrumented posterolateral fusion (≧ three-level fusion). METHODS: A total of 47 consecutive patients underwent laminectomy decompression, and multi-level instrumented posterolateral fusions were reviewed. Group 1 comprised 26 patients having DBM with autologous laminectomy bone (ALB). Group 2 consisted of 21 patients having AIBG with ALB. The fusion success evaluation was based on findings using the 12-month anteroposterior and dynamic plain radiographs. RESULTS: Gender, age, and the number of fusion levels were similar for both groups. 21 of 26 (80.8%) patients in group 1 and 18 of 21 (85.7%) patients in group 2 were observed to achieve solid bony fusion. There was no statistical difference in the fusion success (p = 0.72). Blood loss was significantly more in group 2 (p = 0.02). The duration of the hospital stays and operative times being longer for group 2, but the difference was not significant. CONCLUSIONS: DBM combined with ALB and osteoconductive materials is as effective as an autologous iliac bone graft with respect to long multi-segment posterolateral fusion success. DBM can be used as an effective bone graft substitute and may decrease morbidities associated with iliac bone graft harvest.

In vitro effect of mineralized and demineralized bone allografts on proliferation and differentiation of MG-63 osteoblast-like cells.

Due to the extensive use of bone allografts in bone reconstruction and periodontal therapy as suitable alternatives to autografts, they are now marketed under different commercial brands. Considering the controversial reports regarding the osteoinductive properties of bone allografts, this study sought to assess the effect of type (mineralized/demineralized), amount and particle size of several allografts on the proliferation and differentiation of MG-63 osteoblast-like cells. MG-63 cells (24-h culture) were exposed to 20 and 40 mg amounts of nine different commercially available freeze-dried bone allografts. After 24 and 72 h of incubation, the effect of water-soluble allograft released materials on cell viability and proliferation was assessed using methyl thiazol tetrazolium (MTT) assay after 24 and 72 h of exposure. Cell differentiation and mineralization was assessed by real-time quantitative reverse transcription PCR and alizarin red staining after 72 h of exposure. The amount and particle size of understudy allografts had significant effects on cell viability after 24 h of exposure (in contrast to 72 h). Higher rate of proliferation was seen in non-differentiated or slow-differentiated groups. The amount and particle size factors had no significant effect on the amount of calcified nodules or the expression of osteogenic marker genes in most groups. Faster and more distinct differentiation and mineralization was noted in mineralized compared to demineralized groups during the 3-day study period. Based on the results, the understudy mineralized (non-demineralized) bone allografts had greater effect on osteogenic differentiation of the MG-63 cells and showed more in vitro osteoinductive activity compared to partially demineralized and fully demineralized types.

Modern Approaches to Studies of New Osteogenic Biomaterials on the Model of Regeneration of Critical-Size Cranial Defects in Rats.

Osteoinductive characteristics of new osteoplastic materials based on demineralized bone matrix of xenogenic origin with high and controlled degree of purification were studied on the model of regeneration of critical-size cranial defects in rats using modern approaches, including histological analysis, evaluation of morphological parameters of the bone tissue obtained by micro-computed tomography, and estimation of bone tissue growth rate using in vivo fluorochrome label. Demineralized bone matrix and, to a much greater extent, its activated form containing modified recombinant growth factor rhBMP-2 with high content of the dimeric form exhibited osteoinductive activity.

Development of an improved bone washing and demineralisation process to produce large demineralised human cancellous bone sponges.

Shaped demineralised bone matrices (DBM) made from cancellous bone have important uses in orthopaedic and dental procedures, where the properties of the material allow its insertion into confined defects, therefore acting as a void filler and scaffold onto which new bone can form. The sponges are often small in size, <1.0 cm(3). In this study, we report on an improved bone washing and demineralisation process that allows production of larger DBM sponges (3.375 or 8.0 cm(3)) from deceased donor bone. These sponges were taken through a series of warm water washes, some with sonication, centrifugation, 100 % ethanol and two decontamination chemical washes and optimally demineralised using 0.5 N hydrochloric acid under vacuum. Demineralisation was confirmed by quantitative measurement of calcium and qualitatively by compression. Protein and DNA removal was also determined. The DBM sponges were freeze dried before terminal sterilisation with a target dose of 25 kGy gamma irradiation whilst frozen. Samples of the sponges were examined histologically for calcium, collagen and the presence of cells. The data indicated lack of cells, absence of bone marrow and a maximum of 1.5 % residual calcium.

Comparative study of indigenously prepared and imported, demineralized, freeze-dried, irradiated bone allograft in the treatment of periodontal infrabony defects.

Demineralized freeze-dried bone allograft (DFDBA) has been used extensively in periodontal therapy. Questions have been raised however, about the osteogenic potential of the variety of grafts available. In India the cost factor is another important consideration. The aim of this study therefore was to evaluate the clinical efficiency of the low priced, indigenously prepared DFDBA obtained from the Tata Memorial Hospital (TMH) Tissue Bank, in periodontal regeneration in infrabony periodontal defects, as compared to DFDBA obtained from the Pacific Coast Tissue Bank (DEMBONE). The latter was used as the control. 16 patients with bilaterally similar periodontal infrabony defects were selected, and randomly allotted to the test and control groups. At baseline, using standardized protocol, recession, probing depths (PD), and clinical attachment levels (CAL) were measured, following which periodontal surgery was carried out, with placement of the respective graft materials. Patients were recalled after 6 months for re-assessment. Statistically significant improvement was obtained for PD reduction and CAL gain for both groups (p < 0.05). However, no significant difference was observed between the test and control groups. It was therefore concluded that both the materials from different tissue banks are equally effective clinically, with the test material being additionally cost effective.

Production of an osteoinductive demineralised bone matrix powder without the use of organic solvents.

Demineralised bone matrix (DBM) is produced by grinding cortical bone into a powder, sieving the powder to obtain a desired size range and then demineralising the powder using acid. Protocols for the production of DBM powder have been published since 1965 and the powder can be used in lyophilised form or it can be mixed with a carrier to produce a paste or putty. The powder is generally produced from cortical bone which has been processed to remove blood, bone marrow and bone marrow components, including fat. Removal of fat is accomplished by incorporating incubation in an organic solvent, often chloroform, chloroform/methanol or acetone. The use of organic solvents in a clean room environment in a human tissue bank is problematic and involves operator exposure and the potential for the solvent to be trapped in air filters or recirculated throughout the clean room suite. Consequently, in this study, we have developed a cortical bone washing step which removes fat/lipid without the use of an organic solvent. Bone was prepared from six femoral shafts from three donors by dissecting soft tissue and bisecting the shaft, the shafts were then cut into ~9-10 cm lengths. These struts were then taken through a series of hot water washes at 56-59 °C, centrifugation and decontamination steps. Washed cortical struts were then lyophilised before being ground with a compressed air milling machine. The ground bone was sieved, demineralised, freeze-dried and terminally sterilised with a target dose of 25 kGy gamma irradiation. The DBM powder was evaluated for residual calcium content, in vitro cytotoxicity and osteoinductivity by implantation into the muscle of an athymic mouse. Data indicated that in addition to removing in excess of 97% DNA and extractable soluble protein, the washing protocol reduced lipid 10,000-fold. The processed bone was easily ground without clogging the grinder; the sterilised DBM powder was not cytotoxic but was osteoinductive in the animal model. Therefore, we have developed a method of producing osteoinductive DBM without the need to use organic solvents.

Anisotropy in bone demineralization revealed by polarized far-IR spectroscopy.

Bone material is composed of an organic matrix of collagen fibers and apatite nanoparticles. Previously, vibrational spectroscopy techniques such as infrared (IR) and Raman spectroscopy have proved to be particularly useful for characterizing the two constituent organic and inorganic phases of bone. In this work, we tested the potential use of high intensity synchrotron-based far-IR radiation (50-500 cm(-1)) to gain new insights into structure and chemical composition of bovine fibrolamellar bone. The results from our study can be summarized in the following four points: (I) compared to far-IR spectra obtained from synthetic hydroxyapatite powder, those from fibrolamellar bone showed similar peak positions, but very different peak widths; (II) during stepwise demineralization of the bone samples, there was no significant change neither to far-IR peak width nor position, demonstrating that mineral dissolution occurred in a uniform manner; (III) application of external loading on fully demineralized bone had no significant effect on the obtained spectra, while dehydration of samples resulted in clear differences. (IV) using linear dichroism, we showed that the anisotropic structure of fibrolamellar bone is also reflected in anisotropic far-IR absorbance properties of both the organic and inorganic phases. Far-IR spectroscopy thus provides a novel way to functionally characterize bone structure and chemistry, and with further technological improvements, has the potential to become a useful clinical diagnostic tool to better assess quality of collagen-based tissues.

Efficacy of two different demineralised bone matrix grafts to promote bone healing in a critical-size-defect: a radiological, histological and histomorphometric study in rat femurs.

PURPOSE: The aim of the study was to compare two different demineralised bone matrices used clinically regarding their ability to induce bone healing in a critical-size-defect rat model. METHODS: We stabilised 4 mm femur defects with a custom-made plate and filled them either with demineralised bone matrix (DBM) or DBX (DBX Putty®). Bone morphogenetic protein 2 (BMP-2)-loaded collagen and an empty defect served as controls. The outcome was followed after 21 and 42 days by radiology (Faxitron; microCT) and histology. RESULTS: Defect healing did not occur in any animal from the empty control, DBM or DBX group. Residuals of the implanted material were still found after six weeks, but only limited callus formation was visible. In contrast, the BMP-2 control demonstrated enhanced formation of callus tissue and undisturbed healing. After 21 days, 11 out of 16 and after 42 days, 7 out of 8 BMP-2-treated animals showed complete defect bridging by cancellous bone tissue. CONCLUSIONS: Demineralised bone grafts were not capable of defect reconstruction; only BMP-2 was able to provide sufficient stimulus to induce uneventful bridging under the specific experimental conditions.

Demineralized bone matrix and platelet-rich plasma do not improve healing of osteochondral defects of the talus: an experimental goat study.

OBJECTIVE: The purpose of this study was to evaluate the effectiveness of demineralized bone matrix (DBM) with and without platelet-rich plasma (PRP) in the treatment of osteochondral defects (OCDs) of the talus. We hypothesized that treatment with DBM would result in more bone formation than no treatment in control OCDs, and that PRP would further enhance the regenerative capacity of DBM. METHOD: A standardized 6-mm OCD was created in each talus of 16 adult goats. According to a randomization scheme, one OCD of each goat was treated with allogeneic DBM hydrated with normal saline (n = 8) or hydrated with autologous PRP (n = 8). The contralateral OCD (n = 16) served as control. After 24 weeks, the animals were euthanized and the tali excised. Various outcome parameters were analyzed with use of macroscopic evaluation, micro-computed tomography (µCT), histology, histomorphometry, and fluorescence microscopy. RESULTS: None of the analyses revealed statistically significant differences between the groups for any of the parameters analyzed in any volume of interest. For example, the mean bone volume fraction (BV/TV) of the defect, as measured by µCT, was 0.56 (95% confidence interval [CI], 0.44-0.68) for DBM hydrated with normal saline and 0.52 (95% CI, 0.40-0.65) for DBM hydrated with PRP, compared to 0.53 (95% CI, 0.45-0.61) and 0.54 (95% CI, 0.44-0.64) for the internal controls, respectively (P > 0.05). CONCLUSION: In contrast to our hypotheses, no beneficial treatment effect of DBM with or without PRP was found for OCDs of the caprine talus.

Considerations in determination of residual moisture in lyophilized demineralized bone matrix: the role of residual moisture analyzers.

The objective of this study is to determine whether a residual moisture analyzer (RMA) can be an acceptable instrument for measuring the residual moisture in lyophilized demineralized bone matrix (DBM). Instruments from two different manufacturers with differing configurations and controls were compared: the Ohaus MB45 and Arizona Instrument MAX4000XL. The effects of various factors such as test temperature, drying profile, end point criteria, lift compensation, chamber configuration, and rehydration on residual moisture (RM) are examined. The performance of the RMAs is based on their ability to reproduce RM results obtained by the current standard gravimetric method. RMAs provide reliable, accurate and reproducible results in a number of industries that rely on the determination of RM. We hypothesize that RMAs are suitable for measuring RM in DBM and provide validation study data with optimized settings for these two instruments. Potentially, such studies will provide justification for allowance of this methodology as an acceptable alternative to the current gravimetric method allowed by American Association of Tissue Banks Standards.

BMP depletion occurs during prolonged acid demineralization of bone: characterization and implications for graft preparation.

Demineralization of allograft bone increases the bioavailability of matrix-associated bone morphogenetic proteins (BMPs), rendering these grafts osteoinductive. While osteoinductivity is related to BMP content, little is known about how the demineralization protocol, in particular, extended demineralization times, affects graft BMP levels. We characterized the BMP-7 content of <710 µm bovine bone powder demineralized under various conditions. Using 1 g of bone per 50 ml of 0.125 N, 0.25 N, or 0.5 N HCl, demineralization was performed at room temperature for 5 min to 24 h. Minimum residual calcium levels were obtained within 90 min and were <1 wt % using the 0.25 N and 0.5 N baths and 17 wt % using the 0.125 N bath. Measured peak BMP-7 levels were also obtained within 90 min and were 161-165 ng g(-1) using the 0.25 N and 0.5 N baths and 55.2 ng g(-1) using the 0.125 N bath. This compares to 5.1 ng g(-1) for undemineralized bone. Further acid bath exposure to 24 h resulted in BMP-7 decline to about 50% of the peak value, which was significant. The BMP-7 half-life was estimated to be 26 h. It is likely that the decline was due to diffusion of BMP-7 from the bone matrix into the acid. These results suggest the importance of not over demineralizing bone grafts and should stimulate further research that can be incorporated into the processing methodology followed by tissue banks.

Comparison of different protocols for demineralization of cortical bone.

Bone is a biological composite material consisting of two main components: collagen and mineral. Collagen is the most abundant protein in vertebrates, which makes it of high clinical and scientific interest. In this paper, we compare the composition and structure of cortical bone demineralized using several protocols: ethylene-diamine-tetraacetic acid (EDTA), formic acid (CH2O2), hydrochloric acid (HCl), and HCl/EDTA mixture. The efficiencies of these four agents were investigated by assessing the remaining mineral quantities and collagen integrity with various experimental techniques. Raman spectroscopy results show that the bone demineralized by the CH2O2 agent has highest collagen quality parameter. The HCl/EDTA mixture removes the most mineral, but it affects the collagen secondary structure as amide II bands are shifted as observed by Fourier transform infrared spectroscopy. Thermogravimetric analysis reveals that HCl and EDTA are most effective in removing the mineral with bulk measurements. In summary, we conclude that HCl best demineralizes bone, leaving the well-preserved collagen structure in the shortest time. These findings guide on the best demineralization protocol to obtain high-quality collagen from bone for clinical and scientific applications.

Comparison of bone demineralisation procedures for DNA recovery from burned remains.

Recovering DNA from modern incinerated bones can be challenging and may require alteration of routine DNA extraction protocols. It has been postulated that incinerated bones share some similarities with ancient bones, including fragmented DNA, surface contamination and highly mineralised structure, all of which can inhibit the successful recovery of genetic material. For this reason, ancient DNA extraction protocols are often used for incinerated modern samples; however, their effectiveness is still somewhat unclear. Much of this uncertainty exists around the demineralisation step of extraction, specifically the length of incubation and retention or removal of supernatant. As obtaining human samples for forensic research can be challenging, porcine models (Sus scrofa domesticus) are often used as substitutes. This study developed real time PCR assays for porcine nuclear DNA in order to investigate the effects of modified demineralisation protocols on DNA yield from femurs exposed to either short (60 min) or prolonged (120 min) burning. Gradient PCR results indicated 56 °C was the ideal amplification temperature for targeted amplicons, with melt curve analysis showing short and long amplicons corresponded to 80.3 °C and 83 °C peaks respectively. Results of altered extraction protocol showed a trend towards higher DNA yields from longer demineralisation periods however this was not significant. By comparison, retaining supernatant post-demineralisation resulted in significantly greater DNA yields compared to discarding it (P < 0.009). Although DNA content yield decreased with burn duration, the demineralisation treatment variations appeared to have the same effect for all burn lengths. These results suggest that for incinerated modern bone retaining the supernatant following demineralisation can dramatically increase DNA yield.

Demineralised bone matrix in veterinary orthopaedics: a review.

Demineralised bone matrix (DBM) is commonly used in human orthopaedics as an allograft prepared from cortical bone. As such, there is a background of literature on the basic science, experimental animal studies and clinical human use of DBM. Because canine DBM is now increasingly available and used in veterinary orthopaedics, this review aims to update the veterinary orthopaedic specialist with the properties and activities of this bone allograft product.

Cell therapy for secondary osteonecrosis of the femoral condyles using the Cellect DBM System: a preliminary report.

We describe a novel treatment of secondary osteonecrosis (ON) of the femoral condyles that is relatively simple, has low morbidity, and does not preclude the patient from other more extensive treatments in the event of failure. Three patients with extensive secondary ON of the femoral condyles were treated with decompression and debridement of the area of ON and grafting with the Cellect DBM System (Depuy Spine, Inc., Raynham, Mass), which provided a graft matrix enriched with a 3-fold to 4-fold increase in osteoprogenitor cells. At 2 years, all 3 patients had no complications and had excellent results with near-normal function and activity levels. Our preliminary results demonstrate that this technique is a viable option, at least in the short term, especially in patients with extensive, multifocal lesions.

Demineralized Bone Matrix Carriers and their Clinical Applications: An Overview.

Reconstruction of massive bone defects is challenging for orthopaedic clinicians, especially in cases of severe trauma and resection of tumors in various locales. Autologous iliac crest bone graft (ICBG) is the "gold standard" for bone grafting. However, the limited availability and complications at donor sites resulted in seeking other options like allografts and bone graft substitutes. Demineralized bone matrix (DBM) is a form of allograft using acidic solution to remove mineral components, while leaving much of the proteinaceous components native to bone, with small amounts of calcium-based solids, inorganic phosphates, and some trace cell debris. It is an osteoconductive and osteoinductive biomaterial and is approved as a medical device for use in bone defects and spinal fusion. To pack consistently into the defect sites and stay firmly in the filling parts, DBM products have various forms combined with biocompatible viscous carriers, including sponges, strips, injectable putty, paste, and paste infused with chips. The present review aims to summarize the properties of various kind of viscous carriers and their clinical use combined with DBM in commercially available products. Given DBM'mercially available products. Given DBM;s long clinical track record and commercial accessibility in standard forms, opportunities to further develop and validate DBM as a versatile bone biomaterial in orthopaedic repair and regenerative medicine contexts are attractive.

Immunostaining of Skeletal Tissues.

Immunohistochemistry (IHC) is a routinely used technique in clinical diagnosis of pathological conditions and in basic and translational research. It combines anatomical, immunological, and biochemical methods and relies on the specific binding of an antibody to an antigen. Using the technique with mineralized tissues is more challenging than with soft tissues. Demineralizing the samples allows for embedding in paraffin wax, and also facilitates cryosectioning. This chapter describes methods for IHC on formaldehyde-fixed, demineralized, paraffin-embedded, or frozen sections to detect antigens in skeletal tissues.

Uniform partial dissolution of bone mineral by using fluoride and phosphate ions combination.

Mineral content is one of the main predictors of the mechanical properties of bone tissue. The contribution of the bone mineral phase to the mechanical properties of bone has been investigated by reducing the mineral content of bone with different in vitro treatment techniques such as hydrochloric acid (HCl), ethylenedinitrilo tetraacetic acid (EDTA), and fluoride ion treatment. In this study, we propose a new treatment technique which combines fluoride and phosphate ions. Bovine femur specimens were used to determine the mechanical properties of cortical bone after different fluoride phosphate ion combination treatments. The treatment solutions, which contain different fluoride and phosphate ion concentrations, dissolved part of the bone mineral in a uniform manner throughout the bone samples. Dissolution by products, which precipitated in the bone tissue, contained calcium fluoride with phosphate ions (CaF(2)/P) and fluorapatite/fluorhydroxyapatite-type material (FAp/FHAp) and acted as filler. Depending on the fluoride and phosphate concentration in a treatment solution, the precipitated material's ratio of FAp/FHAp to total fluoride containing phase (FAp/FHAp + CaF(2)/P) in bone tissue also changed. High fluoride ion content in treatment solutions generated more CaF(2)/P type of precipitate, and low fluoride ion concentration generated more FAp/FHAp type precipitates as compared to high fluoride concentration treatments. These experiments show that phosphate ions are another important parameter of a treatment solution, in addition to ionic strength, pH, and the duration of treatment. In vitro, phosphate fluoride combinations partially dissolve bone mineral content in a wider range than fluoride treatment alone in a uniform manner. With this new technique one can control more precisely the partial dissolution of the bone mineral and mineral phase's contribution to mechanical properties of bone tissue.

Effect of hydrazine deproteination on bone mineral phase: a critical view.

Over the last 30 years several techniques have been developed to separate bone matrix and bone mineral, in order to allow for a study of each component independently of the other. Preservation of original characteristics of the phase studied after isolation has always been a great challenge for all such techniques. The hydrazine deproteination procedure, first proposed by Termine, has been one of the processes most widely used for studying bone mineral. It is found to be one of the most effective, notwithstanding controversy over its efficiency in bone deproteination and criticism regarding possible changes it could make to the characteristics of bone mineral. In this work, we have studied the possible chemical and physical alterations caused by the hydrazine deproteination process to bone mineral from rats and to other materials of biological interest. Materials were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA), X-ray diffractometry (XRD), inductive coupled plasma-optical emission spectroscopy (ICP-OES), C-H-N analysis and infrared spectroscopy (FTIR), before and after hydrazine deproteination. Finally, here we present a comprehensive discussion on the criticism of hydrazine deproteination. The experimental results obtained in this work, even when compared to the results in the literature, show that most widespread criticism to the hydrazine deproteination process is not completely justified.