Effects of demineralization mode and particle size of allogeneic bone powder on its physical and chemical properties.

At present, the commonly used allogeneic bone powder in the clinic can be divided into nondemineralized bone matrix and demineralized bone matrix (DBM). Commonly used demineralizers include acids and ethylene diamine tetraacetic acid (EDTA). There may be some diversities between them. Also, the size of the bone particle can affects its cell compatibility and osteogenic ability. We produced different particle sizes i.e., < 75, 75-100, 100-315, 315-450, 450-650, and 650-1000 µm, and treated in three ways (nondemineralized, demineralized by EDTA, and demineralized by HCl). Scanning electron microscopy showed that the surface of the samples in each group was relatively smooth without obvious differences. The results of specific surface area and porosity analysis showed that they were significantly higher in demineralized bone powder than in nondemineralized bone powder, however, there was no significant difference between the two decalcification methods. The content of hydroxyproline in nondemineralized bone powder and EDTA-demineralized bone powder had no statistical difference, while HCl-demineralization had statistical significance compared with the former two, and the content increased with the decrease of particle size. The protein and BMP-2 extracted from HCl demineralized bone powder were significantly higher than that from nondemineralized bone powder and EDTA demineralized bone powder, and there were differences among different particle sizes. These results suggested the importance of demineralization mode and particle size of the allogenic bone powder and provided guidance for the choice of the most appropriate particle size and demineralization mode to be used in tissue bioengineering.

Effects of processing and gamma radiation on mechanical properties and organic composition of frozen, freeze-dried and demineralised human cortical bone allograft.

Bone processing and radiation were reported to influence mechanical properties of cortical bones due in part to structural changes and denaturation of collagen composition. This comparative study was to determine effects of bone processing on mechanical properties and organic composition, and to what extent the radiation damaging after each processing. Human femur cortical bones were processed by freezing, freeze-drying and demineralisation and then gamma irradiated at 5, 15, 20, 25 and 50 kGy. In the compression test, freeze drying significantly decreased the Young's Modulus by 15%, while demineralisation reduced further by 90% (P < 0.05) when compared to the freezing. Only demineralisation significantly reduced ultimate strength of bone by 93% (P < 0.05). In the bending test, both freeze drying and demineralisation significantly reduced the ultimate strength and the work to failure. Radiation at 25 kGy showed no effect on compression for ultimate strength in each processing group. However, high dose of 50 kGy significantly reduced bending ultimate strength by 47% in demineralisation group. Alterations in collagen in bones irradiated at 25 and 50 kGy showed by the highest peak of the amide I collagen in the Fourier Transfer Infra-Red spectra indicating more collagen was exposed after calcium was removed in the demineralised bone, however radiation showed no effect on the collagen crosslink. The study confirmed that demineralisation further reduced the ability to resist deformation in response to an applied force in freeze-dried bones due to calcium reduction and collagen composition. Sterilisation dose of 25 kGy has no effect on mechanical properties and collagen composition of the processed human cortical bone.

Minimally invasive treatment of long bone non-unions with bone marrow concentrate, demineralized bone matrix and platelet-rich fibrin in 38 patients.

To determine the efficacy of percutaneous injection of autologous bone marrow concentrated (BMC), demineralized bone matrix (DBM), and platelet rich fibrin (PRF) in the treatment of long bone non-unions. From January 2011 to January 2018 patients with non-union of the lower limbs who were on the waiting list for open grafting with established tibial or femoral non-union and minimal deformity were eligible to participate in this study. Patients were treated with a single percutaneous injection of DBM, BMC and PRF. Our study group comprised 38 patients (26 males and 12 females; mean age 39, range 18 to 65). Non-unions were located in the femur (18 cases) and in the tibia (20 cases). Clinical and imaging follow-up ranged from 4 to 60 months (mean 20 months). Bone union occurred in 30 out of 38 patients (79%) in an average of 7 months (range 3 to 12) and all healed patients had full weight bearing after 9 months on average (range 6 to 12) from injection. In 19 cases the osteosynthesis was removed 12 months on average (range 3 to 36) from surgery. One patient developed infection at the non-union site after treatment. Percutaneous injection of DBM, BMC, and PRF is an effective treatment for long-bone non-unions. This technique allows the bone to heal with a minimally invasive approach and with a hospitalization of 2 days. Key elements of bone regeneration consist of a combination of biological and biomechanical therapeutic approach.

Fibrous Demineralized Bone Matrix (DBM) Improves Bone Marrow Mononuclear Cell (BMC)-Supported Bone Healing in Large Femoral Bone Defects in Rats.

Regeneration of large bone defects is a major objective in trauma surgery. Bone marrow mononuclear cell (BMC)-supported bone healing was shown to be efficient after immobilization on a scaffold. We hypothesized that fibrous demineralized bone matrix (DBM) in various forms with BMCs is superior to granular DBM. A total of 65 male SD rats were assigned to five treatment groups: syngenic cancellous bone (SCB), fibrous demineralized bone matrix (f-DBM), fibrous demineralized bone matrix densely packed (f-DBM 120%), DBM granules (GDBM) and DBM granules 5% calcium phosphate (GDBM5%Ca2+). BMCs from donor rats were combined with different scaffolds and placed into 5 mm femoral bone defects. After 8 weeks, bone mineral density (BMD), biomechanical stability and histology were assessed. Similar biomechanical properties of f-DBM and SCB defects were observed. Similar bone and cartilage formation was found in all groups, but a significantly bigger residual defect size was found in GDBM. High bone healing scores were found in f-DBM (25) and SCB (25). The application of DBM in fiber form combined with the application of BMCs shows promising results comparable to the gold standard, syngenic cancellous bone. Denser packing of fibers or higher amount of calcium phosphate has no positive effect.

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.

Large fragment demineralization: an alternative pretreatment for forensic DNA typing of bones.

In forensic genetics, the analysis of postmortem bones is one of the most challenging due to the low quantity of degraded endogenous DNA. The most widely used approach for sample preparation, in those cases, is pulverizing the bone. However, processing pulverized bone is extremely delicate, requiring strict laboratory conditions and operating procedures. In fact, several recent publications have focused on non-powder approaches. The objectives of this study were, thus, to validate a non-powder protocol for DNA extraction from forensic bones and an alternative pretreatment, large fragment demineralization (LFD). Thirty human femurs and tibiae received by the Legal Medicine Institute of Brescia, Italy, were included in the study. Bone powder and one transversal section of the diaphysis were sampled from each bone. DNA extraction from the powder was carried out using PrepFiler BTA (BTA), while the transversal section was submitted to the alternative demineralizing pretreatment (LFD) followed by DNA extraction using the QIAamp DNA Investigator. DNA extracts were assessed for human DNA quantity and degradation by means of a validated in-house qPCR assay and amplified with commercial kits. Inhibition assessment was carried out through Quality Sensor analysis using 24plex QS Kit. The differences in quantity, quality of human DNA, and number of alleles detected between both methods were comparable and not statistically significant. We propose the use of the LFD protocol as a complementary approach capable of confirming the genotypes or detect alleles not observed using BTA, without the need for pulverization.

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.

Human placenta-derived matrix with cancellous autograft and demineralized bone matrix for large segmental long-bone defects in two dogs with septic nonunion.

OBJECTIVE: To report the successful treatment of septic nonunion in two dogs with large segmental defects secondary to long-bone fractures by using a novel human placenta-derived matrix (hPM) as adjunct to fixation. ANIMALS: One 3-kg 9-year-old neutered male Yorkshire terrier with a distal antebrachial fracture and one 6-kg 4-year-old spayed female miniature pinscher with a distal humeral fracture. STUDY DESIGN: Short case series. METHODS: Both dogs presented for septic nonunion after internal fixation of Gustilo type II open diaphyseal fractures from dog bite injuries. During revision, debridement of nonviable bone resulted in segmental defects of 32% and 20% of the bone length for the antebrachial and humeral fractures, respectively. The antebrachial fracture was stabilized with a circular external fixator, and the humeral fracture was stabilized with biaxial bone plating. The fracture sites were not collapsed, and full length was maintained with the fixation. Autogenous cancellous bone graft and canine demineralized bone allograft were packed into the defects, and hPM was injected into the graft sites after closure. RESULTS: Radiographic union was documented at 8 weeks and 6 weeks for the antebrachial and humeral fractures, respectively. Both dogs became fully weight bearing on the affected limbs and returned to full activity. CONCLUSION: Augmenting fixation with grafts and hPM led to a relatively rapid union in both dogs reported here.

Chitin from Antarctic krill shell: Eco-preparation, detection, and characterization.

Antarctic krill is a nutrient-rich crustacean that is one of the main species in the Antarctic ecosystem. Antarctic krill shell (AKS) can be used as raw materials to prepare chitin. In this study, lactic acid and dispase were used to prepare Antarctic krill chitin (AKC-1). Amino-monosaccharide contents of chitin samples were detected by pre-column PMP-HPLC method. Analytical instruments were conducted to determine characteristics of chitin samples. Results showed that the amino-monosaccharide content of AKS was 4.62 g/100 g (measured in D-glucosamine). The yield of AKC-1 was 5.49 g/100 g, and the amino-monosaccharide content was 80.90 g/100 g. AKC-1 showed smooth flakes, a porous surface, and α-chitin structural characteristics. The maximum degradation temperature (DTGmax) was 318.3 °C. The yield of deacetylated chitin (AKC-2) was 4.74 g/100 g, with deacetylation degree of 80.8%, viscosity average molecular weight of approximately 145.7 kDa, and amino-monosaccharide content of 97.06 g/100 g. The surface morphology of AKC-2 was similar to that of AKC-1, and the DTGmax was 311.5 °C. A mild, eco-friendly chitin preparation method and an amino-monosaccharide content detection method of raw material before chitin preparation are described in this study, which can provide technical support for comprehensive utilization of Antarctic krill resources.

An Ion-exchange Bone Demineralization Method for Improved Time, Expense, and Tissue Preservation.

Here, we describe an ethylenediaminetetraacetic acid (EDTA)-based bone demineralization procedure that uses cation-exchange resin and dialysis tubing. This method does not require solution changes or special equipment, is faster than EDTA alone, is cost-effective, and is environmentally friendly. Like other EDTA-based methods, this procedure yields superior tissue preservation than formic acid demineralization. Greater protein antigenicity using EDTA as opposed to formic acid has been described, but we also find significant improvements in carbohydrate-based histological staining. Histological staining using this method reveals cartilage layers that are not distinguishable with formic acid demineralization. Carbohydrate preservation is relevant to many applications of bone demineralization, including the assessment of osteoarthritis from bone biopsies and the use of demineralized bone powder for tissue culture and surgical implants. The improvements in time, expense, and tissue quality indicate this method is a practical and often superior alternative to formic acid demineralization.

Induction of osteogenic differentiation by demineralized and decellularized bovine extracellular matrix derived hydrogels associated with barium titanate.

Bone tissue-derive biomaterials have become of great interest to treat diseases of the skeletal system. Biological scaffolds of demineralized and decellularized extracellular matrices (ECM) have been developed and one of these options are ECM hydrogels derived from bovine bone. Nanomaterials may be able to regulate stem cell differentiation due to their unique physical-chemical properties. The present work aimed to evaluate the osteoinductive effects of ECM hydrogels associated with barium titanate nanoparticles (BTNP) on dental pulp cells derived from exfoliated teeth. The addition of BTNP in the ECM derived hydrogel did not affect cell proliferation and the formation of bone nodules. Furthermore, it increased the expression of bone alkaline phosphatase. The results demonstrated that the nanobiocomposites were able to promote the osteogenic differentiation, even in the absence of chemical inducing factors for osteogenic differentiation. In conclusion, bovine bone ECM hydrogel combined with BTNP presented and increased expression of markers of osteogenic differentiation in the absence of chemical inducing factors.

In vitro analysis of the influence of mineralized and EDTA-demineralized allogenous bone on the viability and differentiation of osteoblasts and dental pulp stem cells.

Grafting based on both autogenous and allogenous human bone is widely used to replace areas of critical loss to induce bone regeneration. Allogenous bones have the advantage of unlimited availability from tissue banks. However, their integration into the remaining bone is limited because they lack osteoinduction and osteogenic properties. Here, we propose to induce the demineralization of the allografts to improve these properties by exposing the organic components. Allografts fragments were demineralized in 10% EDTA at pH 7.2 solution. The influence of the EDTA-DAB and MAB fragments was evaluated with respect to the adhesion, growth and differentiation of MC3'T3-E1 osteoblasts, primary osteoblasts and dental pulp stem cells (DPSC). Histomorphological analyses showed that EDTA-demineralized fragments (EDTA-DAB) maintained a bone architecture and porosity similar to those of the mineralized (MAB) samples. BMP4, osteopontin, and collagen III were also preserved. All the cell types adhered, grew and colonized both the MAB and EDTA-DAB biomaterials after 7, 14 and 21 days. However, the osteoblastic cell lines showed higher viability indexes when they were cultivated on the EDTA-DAB fragments, while the MAB fragments induced higher DPSC viability. The improved osteoinductive potential of the EDTA-DAB bone was confirmed by alkaline phosphatase activity and calcium deposition analyses. This work provides guidance for the choice of the most appropriate allograft to be used in tissue bioengineering and for the transport of specific cell lineages to the surgical site.

Demineralized bone matrix paste formulated with biomimetic PLGA microcarriers for the vancomycin hydrochloride controlled delivery: Release profile, citotoxicity and efficacy against S. aureus.

Infection and resulting bone defects caused by Staphylococcus aureus is one of the major issues in orthopaedic surgeries. Vancomycin hydrochloride (VaH) is largely used to manage these events. Here, a human derived bone paste supplemented with biopolymer microcarriers for VaH sustained delivery to merge osteoinductive and antimicrobial actions is described. In detail, different emulsion formulations were tested to fabricate micro-carriers of poly-lactic-co-glycolic acid (PLGA) and hydroxyapatite (HA) by a proprietary technology (named Supercritical Emulsion Extraction). These carriers (mean size 827 ± 68 µm; loading 47 mgVaH/gPLGA) were assembled with human demineralized bone matrix (DBM) to obtain an antimicrobial bone paste system (250 mg/0.5 cm3 w/v, carrier/DBM). Release profiles in PBS indicated a daily drug average release of about 4 µg/mL over two weeks. This concentration was close to the minimum inhibitory concentration and able to effectively inhibit the S. aureus growth in our experimental sets. Carriers cytotoxicity tests showed absence of adverse effects on cell viability at the concentrations used for paste assembly. This approach points toward the potential of the DBM-carrier-antibiotic system in hampering the bacterial growth with accurately controlled antibiotic release and opens perspectives on functional bone paste with PLGA carriers for the controlled release of bioactive molecules.

Injectable porcine bone demineralized and digested extracellular matrix-PEGDA hydrogel blend for bone regeneration.

Extracellular matrix (ECM) has a major role in the structural support and cellular processes of organs and tissues. Proteins extracted from the ECM have been used to fabricate different scaffolds for tissue engineering applications. The aims of the present study were to extract, characterize and fabricate a new class of hydrogel with proteins isolated from pig bone ECM and combine them with a synthetic polymer so it could be used to promote bone regeneration. Porcine bone demineralized and digested extracellular matrix (pddECM) containing collagen type I was produced, optimized and sterilized with high pressurized CO2 method. The pddECM was further blended with 20% w/v polyethylene glycol diacrylate (PEGDA) to create an injectable semi interpenetrating polymer network (SIPN) scaffold with enhanced physicochemical properties. The blend tackled the shortfall of natural polymers, such as lack of structural stability and fast degradation, preserving its structure in more than 90% after 30 days of incubation; thus, increasing the material endurance in a simulated physiological environment. The manufactured injectable hydrogel showed high cytocompatibility with hOb and SaOs-2 cells, promoting osteogenic proliferation within 21 days of culture. The hydrogel had a high compression modulus of 520 kPa, low swelling (5.3 mg/mg) and millimetric volume expansion (19.5%), all of which are favorable characteristics for bone regeneration applications.

Cytoskeletal and extracellular matrix proteins resist the burning of bones.

Due the proteins from bone remains are highly resistant to pass of time and environmental conditions, they could tell us about the events that probably happened in the past. In the forensic and physical anthropology context, burnt bone remains are one of the most common pieces of recovered evidence and, generally, they are associated with funerary practices, criminal scenes or massive catastrophic events. In the present study, bone pieces of pigs were calcined at different calcination temperatures, and proteins were searched using biochemical, immunochemical and ultrastructure visualization under these experimentally conditions. For this purpose, it was successfully developed a non-demineralizing protein extraction method from burnt bone remains and the use of specific antibodies permitted the identification of different extracellular matrix and intracellular proteins. While collagen proteins type I and IV were identified and detected under middle and high calcination temperatures (300°C and 600°C); cytoskeletal proteins as actin, tubulin and, the microtubule associated protein Tau, were found under calcination process, even up high calcination temperatures. Under ultrastructural analysis, fibrous materials with a classical disposition of collagens were observed even at high calcination temperatures of the burnt bone remains. The protein identification and characterization in burnt bones as performed in present studies, is clearly demonstrating that using specific strategies for protein characterizations it is possible to found protein biomarkers in burnt bone remains and this strategy could be useful for forensic and anthropological purposes.

Laboratory Monitoring of Bone Tissue Remodeling after Augmentation of Impression Intraarticular Fracture with Different Types of Bone Graft.

The effects of bone graft materials on the inflammatory response and biochemical markers of bone remodeling were studied on a rabbit model of fracture augmentation with the following grafts: ß-tricalcium phosphate, demineralized bone matrix, nanostructured carbon implant, and porous titanium implant made by additive 3D printing. The markers of bone remodeling and the blood system response in the postoperative period were studied. It was found that porous titanium implant and ß-tricalcium phosphate induced osteogenesis and minimized osteoclastic resorption. Augmentation with nanostructured carbon implant and demineralized bone matrix stimulated the processes of osteoclastic resorption.

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.

Cretaceous dinosaur bone contains recent organic material and provides an environment conducive to microbial communities.

Fossils were thought to lack original organic molecules, but chemical analyses show that some can survive. Dinosaur bone has been proposed to preserve collagen, osteocytes, and blood vessels. However, proteins and labile lipids are diagenetically unstable, and bone is a porous open system, allowing microbial/molecular flux. These 'soft tissues' have been reinterpreted as biofilms. Organic preservation versus contamination of dinosaur bone was examined by freshly excavating, with aseptic protocols, fossils and sedimentary matrix, and chemically/biologically analyzing them. Fossil 'soft tissues' differed from collagen chemically and structurally; while degradation would be expected, the patterns observed did not support this. 16S rRNA amplicon sequencing revealed that dinosaur bone hosted an abundant microbial community different from lesser abundant communities of surrounding sediment. Subsurface dinosaur bone is a relatively fertile habitat, attracting microbes that likely utilize inorganic nutrients and complicate identification of original organic material. There exists potential post-burial taphonomic roles for subsurface microorganisms.

The chances of establishing a real-world Jurassic Park are slim. During the fossilization process, biological tissues degrade over millions of years, with some types of molecules breaking down faster than others. However, traces of biological material have been found inside some fossils. While some researchers believe these could be the remains of ancient proteins, blood vessels, and cells, traditionally thought to be among the least stable components of bone, others think that they have more recent sources. One hypothesis is that they are in fact biofilms formed by bacteria. To investigate the source of the biological material in fossil bone, Saitta et al. performed a range of analyses on the fossilized bones of a horned dinosaur called Centrosaurus. The bones were carefully excavated in a manner to reduce contamination, and the sediment the bones had been embedded in was also tested for comparison. Saitta et al. found no evidence of ancient dinosaur proteins. However, the fossils contained more organic carbon, DNA, and certain amino acids than the sediment surrounding them. Most of these appeared to have a very recent source. Sequencing the genetic material revealed that the fossil had become a habitat for an unusual community of microbes that is not found in the surrounding sediment or above ground. These buried microbes may have evolved unique ways to thrive inside fossils. Future work could investigate how these unusual organisms live and whether the communities vary in different parts of the world.

Effect of different concentration of demineralized bone powder with gellan gum porous scaffold for the application of bone tissue regeneration.

The prevalence of bone-related diseases has increased, the population growth as a result of the aging phenomenon requires more effective treatments for regeneration of bone defect. Although an autogenous bone graft was used in traditional operation method, they are very inefficient in current bone defect surgery and very difficult to gather the required amount of bone for operation. It is becoming a gradually growing disease, hence there is a need for developing a new method for preparing biomimetic scaffolds. DBP (demineralized bone powder), a potent bone regeneration material, has a trace amount of ions and bone mineral component. Especially, GD (Gallus gallus var domesticus) DBP has a unique property, which has melanin, for strengthening bones, increasing ALP activity and bone mineralization, compared to other available biomaterials. For that reason, GD DBP was combined with GG (gellan gum). The material was characterized in vitro and in vivo rat model. The first priority in this work was given to assessing the attachment and proliferation rates of BMSCs following the in vivo experiment in rats. The results of 1% sample showed better osteogenic effects that can be used in clinical application after studying in larger animals for better bone regeneration and tissue engineering.